(EXS 87) Riccardo A. A. Muzzarelli (Auth.), Prof. Dr. P. Jolles, Prof. R. A. A. Muzzarelli (Eds.) - Chitin and Chitinases-Birkhauser Basel (1999)

EXS87
Chitin and Chitinases

Edited by P. Jolles and R.A.A. Muzzarelli
Springer Basel AG
Editors
Prof. Dr. P. Jolles
Laboratoire de Chimie
des Substances Naturelles
URA C.N.R.S. No. 401
Museum National d'Histoire Naturelle
63, rue Buffon
F-75005 Paris
France
Prof. R.A.A. Muzzarelli

Center for Innovative Biomaterials
Faculty of Medicine, University
Via Ranieri 67
1-60100 Ancona
Italy
Library of Congress Cataloging-in-Publication Data

Cbitin and Chitanases / edited by P. Iolles and R. A. A. Muzzarelli.
p. cm. -- (EXS ; 87)
Includes bibliographical references and index.
ISBN 978-3-0348-9760-0
ISBN 978-3-0348-8757-1 (eBook)
DOI 10.1007/978-3-0348-8757-1
1. Chitin. 2. Chitinase. 3. Chitosan. 1. Iolles, Pierre, 1927-.
II. Muzzarelli, Riccardo A.A., 1937III. Series.
QP702.C5C47 1999
573.7'74--dc21
Deutsche Bibliothek Cataloging-in-Publication Data
Cbitin and chitinases / ed. by P. Jolles and R. A. A. Muzzarelli. Basel ; Boston; Berlin: Birkhuser, 1999
(EXS; 87)
87. Chitin and chitinases. - 1999
EXS. - Basel ; Boston; Berlin: Birkhuser
Friiher Schriftenreihe
Fortlaufende BeiI. zu: Experientia
The publisher and editor can give no guarantee for the inforrnation on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other
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The use of registered names, trademarks etc. in this publication, even if not identified as such, does
not imply that they are exempt from the relevant protective laws and regulations or free for general
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This work is subject to copyright. All rights are reserved, whether the whole or part of the material
is concemed, specifically the rights of translation, reprinting, re-use of illustrations, recitation,
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ofuse, permission ofthe copyright owner must be obtained.
1999 Springer Basel AG
Originally published by Birkhauser Verlag in 1999
Softcover reprint of the hardcover Ist edition 1999
987654321
Contents
List of Contributors .
VII
Preface . . . . . . . .
XIII
Riccardo A. A. Muzzarelli
Native, industrial and fossil chitins.
Chitin synthesis
Regula A. Merz, Markus Horsch, Lars E. Nyhten and Dora M Rast
Biochemistry of chitin synthase . . . . . . . . . . . . . . . . . . ..
Jose Ruiz-Herrera andAlfredo D. Martinez-Espinoza

Chitin biosynthesis and structural organization in vivo .
39
M Henar Valdivieso, Angel Duran and Cesar Roncero

Chitin synthases in yeast and fungi . . . . . . . . . . .
55
Jeroen Bakkers, Jan W. Kijne and Herman P. Spaink

Function of chitin oligosaccharides in plant and animal development
71
Subba Reddy Palli and Arthur Retnakaran

Molecular and biochemical aspects of chitin synthesis inhibition
85
Hildgund Schrempj
Characteristics of chitin-binding proteins from streptomycetes. .
99
Chitinases
Daizo Koga, Masaru Mitsutomi, Michiko Kono and
Masahiro Matsumiya
Biochemistry of chitinases . . . . . . . . . . . . .
111
Jon D. Robertus and Arthur E Monzingo

The structure and action of chitinases .
125
Bernard Henrissat
Classification of chitinase modules. .
137
Graham W. Gooday
Aggressive and defensive roles for chitinases .
157
Alfredo Herrera-Estrella and Ilan Chet

Chitinases in biological control . . . . . . . .
171
VI
Contents
Lee A. Hadwiger
Host-parasite interactions: elicitation of defense responses
in plants with chitosan . . . . . . . . . . . . . . . . . .
185
Klaus-Dieter Spindler and Margarethe Spindler-Barth

Inhibitors of chitinases . . . . . . . . . . . . . . . . .
201
Gilles Bleau, FrMeric Massicotte, Yannick Merlen and

Chantale Boisvert
Mammalian chitinase-like proteins . . . . . . . . .. . . . . . . . 211
Mohammed Shahabuddin and Joseph M Vinetz
Chitinases ofhuman parasites and their implications
as antiparasitic targets. . . . . . . . . . . . . . . . . . . . . . . . . 223
Riccardo A.A. Muzzarelli
Analytical biochemistry and dinical significance of
N-acetyl--D-glucosaminidase and related enzymes. . . . . . . . . 235
Exogenous chitosans
Riccardo A. A. Muzzarelli, Monica Mattioli-Belmonte,
Armanda Pugnaloni and Graziella Biagini
Biochemistry, histology and dinieal uses of chitins and chitosans
in wound healing . . . . . . . . . . . . . . . . . . . . . .
Saburo Minami, Yoshiharu Okamoto, Koji Hamada, Yukio
Fukumoto and Yoshihiro Shigemasa
Veterinary practice with chitin and chitosan . . . . . .
Seiichi Tokura, Hiroshi Tamura and Ichiro Azuma
Immunologieal aspects of chitin and chitin derivatives
administered to animals. . . . . . . . . . . . . ..
. 251
. . . . . 265
. . . . . . . 279
Clinical and biochemieal evaluation of chitosan for
hypercholesterolemia and overweight control. . . .
. . . 293
Ida Genta, Paola Perugini, Franca Pavanetto, Tiziana Modena,

Bice Conti and Riccardo A. A. Muzzarelli
Microparticulate drug delivery systems. . . . . . . . . . . . .
305
Raul G. Cuero
Antimicrobial action of exogenous chitosan
315
Subject index . . . . . . . . . . . . . . . .
335
List of Contributors
Ichiro Azuma, Institute for the Immunological Sciences,
Hokkaido University, Sapporo 060, Japan
Jeroen Bakkers, Leiden University, Institute of Molecular Plant Sciences,
Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands;
e-mail: BAKKERS@rulbim.leidenuniv.nl
Graziella Biagini, Center for Innovative Biomaterials, Faculty ofMedicine,
University, Via Ranieri 67, 1-60100 Ancona, Italy
Gilles Bleau, Departement d'Obstetrique-Gynecologie,
Centre Hospitalier de l'Universite de Montreal, Hpital Saint-Luc, 264,
boul. Rene-Levesque est, Montreal, Quebec, Canada H2X lPl;
e-mail: bleaug@ere.umontreal.ca
Chantale Boisvert, Departement d'Obstetrique-Gynecologie,
boul. Rene-Levesque est, Montreal, Quebec, Canada H2X IP1;
e-mail: boisvertc@ere.umontreal.ca
Ilan Chet, The Hebrew University of Jerusalem,
Otto Warburg Center for Agricultural Biotechnology,
Faculty of Agriculture, P.O. Box 12, Rehovot 76100, Israel
Bice Conti, Department ofPharmaceutical Chemistry, University ofPavia,
Viale Taramelli 12,1-27100 Pavia, Italy
Raul G. Cuero, Prairie View A & M University,
Texas A & M University Systems, CARC, P. O. Box 4079,
Prairie View, TX 77446, USA; e-mail: Olimpa@aal.com
Angel Duran, Instituto de Microbiologia Bioquimica,
Consejo Superior de Investigaciones Cientificas/
Universidad de Salamanca, Edificio Departamental, Room 219, Avda.
Campo Charro s/n, E-37007 Salamanca, Spain
Yukio Fukumoto, Asa Zoological Park, Asa-Kita,
Hiroshima 731-3355, Japan
Ida Genta, Department of Pharmaceutical Chemistry, University of Pavia,
Viale Taramelli 12,1-27100 Pavia, Italy
VIII
Graham W Gooday, Department of Molecular and Cell Biology,

University of Aberdeen, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, UK; e-mail: g.w.gooday@abdn.ac.uk
Lee A. Hadwiger, Dept. ofPlant Pathology, Washington State University,
Pullman, WA 99164-6430, USA; e-mail: chitosan@mail.wsu.edu
Koji Hamada, Ohmu Agricultural Mutual Aid Association, Omu,
Monbetsu 098-1702, Japan
Bemard Henrissat, Architecture et Fonction des Macromolecules
Biologiques CNRS, 31 Chemin Joseph Aiguier,
F-13402 Marseille Cedex 20, France; e-mail: bemie@afmb.cnrs-mrs.fr
Alfredo Herrera-Estrella, Centro de Investigaci6n y Estudios Avanzados,
Unidad Irapuato, A.p. 629, 36500 Irapuato, Gto. Mexico;
e-mail: aherrera@irapuato.ira.cinvestav.mx
Markus Horsch, Department ofPlant Biology, University of Zurich,
Zollikerstrasse 107, CH-8008 Zrich, Switzerland
Jan W Kijne, Leiden University, Institute of Molecular Plant Sciences,
Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands;
e-mail: KlJNE@rulbim.leidenuniv.nl
Daizo Koga, Laboratory of Biochemistry,
Department of Biological Science, Faculty of Agriculture,
Yamaguchi University, Yamaguchi 753-8515, Japan;
e-mail: koga@agr.yamaguchi-u.ac.jp
Michiko Kono, Fisheries Research Laboratory, Faculty of Agriculture,
The University ofTokyo, Maisaka, Shizuoka 431-0211, Japan;
e-mail: amkono@honga.ecc.u-tokyo.ac.jp
Alfredo D. Martinez-Espinoza, Departamento de Ingenieria Genetica,
Unidad Irapuato, Centro de Investigaci6n y de Estudios Avanzados deI
Instituto Politecnico Nacional, Apartado Posta1629, Irapuato 36500,
Gto. Mexico; e-mail: amartinez@irapuato.ira.cinvestav.mx
Frederic Massicotte, Departement d'Obstetrique-Gynecologie,
boul. Rene-Levesque est, Montreal, Quebec, Canada H2X IP1;
e-mail: massicottef@ere.umontreal.ca
IX
Masahiro Matsumiya, Laboratory ofMarine Products Uzilization,

Department ofMarine Science and Resources, College ofBioresource
Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan;
email: matsumiya@brs.nihon-u.ac.jp
Monica Mattioli-Belmonte, Center for Innovative Biomaterials,
Faculty ofMedicine, University, Via Ranieri 67, 1-60100 Ancona, Italy
Yannick Merlen, Departement d'Obstetrique-Gynecologie,
boul. Rene-Uvesque est, Montreal, Quebec, Canada H2X IP1;
e-mail: merleny@ere.umontreal.ca
Regula A. Merz, Department of Plant Biology, University of Zurich,
Zollikerstrasse 107, CH-8008 Zrich, Switzerland;
e-mail: remerz@botinst.unizh.ch
Saburo Minami, Department ofVeterinary Surgery, Faculty of Agriculture,
Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan;
e-mail: minami@pear.agr.tottori-u.ac.jp
Masaru Mitsutomi, Laboratory of Food Chemistry,
Department of Applied Biological Sciences, Faculty of Agriculture,
Saga University, Saga 840-8502, Japan; e-mail: mitsutom@cc.saga-u.ac.jp
Tiziana Modena, Department of Pharmaceutical Chemistry,
University ofPavia, Viale Taramelli 12,1-27100 Pavia, Italy
Arthur F. Monzingo, Institute of Cellular and Molecular Biology,
Department of Chemistry and Biochemistry, University of Texas, Austin,
TX 78712, USA; e-mail: amonzingo@mail.utexas.edu
Riccardo A. A. Muzzarelli, Center for Innovative Biomaterials,
Faculty ofMedicine, University, Via Ranieri 67, 1-60100 Ancona, Italy;
e-mail: muzzarelli@popcsi.unian.it
Lars E. Nyhlen, Department of Plant Biology, University of Zurich,
Zollikerstrasse 107, CH-8008 Zrich, Switzerland
Yoshiharu Okamoto, Department ofVeterinary Surgery,
Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami,
Tottori 680-8553, Japan; e-mail: okamoto@pear.agr.tottori-u.ac.jp
Subba Reddy Palli, Rohm and Haas Research Laboratories,
727 Norristown road, P.O. Box 904, Spring House, PA 19477, USA
F. Pavanerto, Department of Pharmaceutical Chemistry,

Paola Perugini, Department of Pharmaceutical Chemistry,

Armanda Pugnaloni, Center for Innovative Biomaterials,
Faculty ofMedicine, University, Via Ranieri 67, 1-60100 Ancona, Italy
Dora M. Rast, Department of Plant Biology, University of Zurich,
Zollikerstrasse 107, CH-8008 Zrich, Switzerland;
e-mail: rast@botinst.unizh.ch
Arthur Retnakaran, Natural Resources Canada, Canadian Forest Service,
Great Lakes Forestry Centre, 1219 Queen Street East, P.O. Box 490,
Sault Ste. Marie, Ontario P6A 5M7, Canada; e-mail: aretnak@nrcan.gc.ca
Jon D. Robertus, Institute of Cellular and Molecular Biology,
Department of Chemistry and Biochemistry, University of Texas,
Austin, TX 78712, USA; e-mail: jrobertus@mail.utexas.edu
Cesar Roncero, Instituto de Microbiologia Bioquimica,
Consejo Superior de Investigaciones Cientificas/
Universidad de Salamanca, Edificio Departamental, Room 219,
Avda. Campo Charro s/n, E-37007 Salamanca, Spain;
e-mail: crm@gugu.usal.es
Jose Ruiz-Herrera, Departamento de Ingenieria Genetica,
Unidad Irapuato, Centro de Investigaci6n y de Estudios Avanzados deI
Instituto Politecnico Nacional, Apartado Postal 629, lrapuato 36500,
Gto. Mexico; e-mail: jruiz@irapuato.ira.cinvestav.mx
Hildgund Schrempf, FB Biologie/Chemie, Universitt Osnabrck,
Barbarastrasse 11, D-49069 Osnabrck, Germany;
e-mail: schrempf@mail.biologie.uni-osnabrueck.de
Mohammed Shahabuddin, Medical Entomology Section,
Laboratory ofParasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health,
4 Center Drive MSC 0425, Bethesda, MD 20892-0425, USA;
e-mail: mshahabudd@nih.gov
Yoshihiro Shigemasa, Department of Materials Science,
Faculty ofEngineering, Tortori University, 4-101 Koyama-Minami,
Tottori 680-8552, Japan
XI
Herman P. Spaink, Leiden University, Institute of Molecular Plant

Sciences, Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands;
e-mail: SPAINK@rulbim.1eidenuniv.nl
Klaus-Dieter Spindler, Universitt Ulm, Abteilung Allgemeine Zoologie,
Albert-Einstein-Allee 11-13, D-89069 Ulm, Germany;
e-mail: klaus-dieter.spindler@biologie.uni-ulm.de
Margarethe Spindler-Barth, Heinrich-Heine-Universitt Dsseldorf,
Lehrstuhl fiir Hormon- und Entwicklungsphysiologie, Universittsstr. 1,
D-40225 Dsseldorf, Germany; e-mail: spindler@rz.uni-duesse1dorf.de
Hiroshi Tamura, Faculty of Engineering, Kansai University and HRC,
Suita, Osaka 564-8680, Japan
Seiichi Tokura, Faculty of Engineering, Kansai University and HRC,
Suita, Osaka 564-8680, Japan
M. Henar Valdivieso, Instituto de Microbiologia Bioquimica,
Consejo Superior de Investigaciones Cientificasl
Universidad de Salamanca, Edificio Departamental, Room 219,
Avda. Campo Charro s/n, E-37007 Salamanca, Spain
Joseph M. Vinetz, WHO Collaborating Center for Tropical Diseases,
Department of Pathology and Division of Infectious Diseases,
University ofTexas Medical Branch, Keiller 2.138,
301 University Blvd., Galveston, TX 77555-0609, USA
Preface
Chitin, the insoluble polymer of N-acetylglucosamine, is the most
abundant nitrogen-bearing organic compound found in nature, present in
insect exoskeletons, crustacean shells and fungal cell walls.
We have selected the most recent and sophisticated chitin-related advances in life sciences, and approached chitin from an original standpoint:
the prompt and enthusiastic response of the colleagues invited to collaborate is gratefully acknowledged.
The first part of this book, after a short presentation of chitin in the
environment, is devoted to chitin biosynthesis. Successive1y, we discuss
the biochemistry of chitin synthase and the state of knowledge of chitin
synthesis in vitra, chitin biosynthesis and structural organization in viva,
and the chitin synthases of yeasts and fungi. The role of chitin oligosaccharides in plant morphogenesis, and biochemical aspects of inhibitors of
chitin synthesis, are also approached. Some chitin-binding proteins are
reviewed.
The second part is devoted to chitinases, which split the -I ,4-glucosidic
bonds of chitin as, in a less pronounced manner, lysozymes also do. Biochemical, structural and evolutionary aspects conceming chitinases are
discussed. Chitin-containing organisms produce chitinases, but some
organisms deprived of chitin, such as a wide variety ofbacteria and higher
plants, also produce chitinases for their defence. These aspects are reviewed in aseries of chapters. Some enzyme inhibitors are also mentioned.
Newly characterized mammalian chitinase-like proteins are presented.
Aspects concerning N-acetyl--D-glucosaminidases, enzymes releasing
N-acetylglucosamine monomers from chitin, are also discussed in relation
with their growing medical importance.
The third part ofthe book is devoted to chitosan, a family of deacetylated
chitins. The agricultural, food, cosmetic and pharmaceutical industries
more and more frequently use this polysaccharide in the form of threads,
fibers, films, gels, microspheres and liposomes. Exciting applications are
discussed in aseries of chapters that emphasize the fact that chitosan applications based on its biological significance often depend on its biodegradability.
We are confident that this book will provide a stimulating background
for further fruitful research on chitin in the biochemical and biological
area.
January 1999
Pierre Jolles and


ed. by P. Jolles and R.A.A. Muzzarelli
@ 1999 Birkhuser Verlag BasellSwitzerland
Native, industrial and fossil chitins

Center for Innovative Biomaterials, Faculty ofMedicine, University, Via Ranieri 67,
I-60100Ancona, Italy
Summary. Countless living organisms continuously synthesize and degrade chitin enzymatically, for nutritional, morphogenetic and defensive or aggressive purposes. Chemically modified
chitins are important in the light of their biochemical significance in medicine and crop
protection; their environmentally friendly behaviour permits industrial exploitation ofthe huge
chitinous biomasses generated by fishing activities and biotechnology. Chitin is promptly
metabolized in sediments, and fossil chitin is not frequently encountered.
Being convinced that this book will attract many readers not fully acquainted with chitin, I deern it appropriate to provide abrief introduction.
The reader is referred to a large body of information on chitin available
in a number ofbooks: a selection is listed below [1-20].
Chitin, (1-4)-linked 2-acetamido-2-deoxy--D-glucan, is widely distributed among invertebrates. At least 10 gigatons (1.10 13 kg) of chitin are
synthesized and degraded each year in the biosphere. It is found as a-chitin
in the calyces of hydrozoa, the eggshells of nematodes and rotifers, the
radulae of mollusks and the cutieies of arthropods, and as -chitin in the
shells of brachiopods and mollusks, cuttlefish bone, squid pen, and pogonophora tubes. Chitin is found in exoskeletons, peritrophic membranes and
the cocoons of insects. Chitin is ubiquitous in fungi: the chitin in fungal
walls varies in crystallinity, degree of covalent bonding to other wall components, mainly glucans, and degree of acetylation.
The polymorphie forms of chitin differ in packing and polarities of
adjacent chains in successive sheets; in the -form all chains are aligned in
parallel manner, whereas in a-chitin they are antiparallel. The molecular
order of chitin explains its physiologieal role and tissue characteristics, for
instance in the insect cutiele and tendon (a-chitin) and in the pen ofCephalopoda (-chitin). The grasping spines of Sagitta are made ofpure a-chitin
because they should be suitably hard to hold a prey. Also, solubility and
reactivity are different.
In the areas of fisheries, textiles, food and ecology, scientists and industry people were urged to upgrade chitin in order to exploit renewable
resourees and to alleviate waste problems. Today chitins and chitosans
from different animals are eommercially available.
Chitin isolates differ from eaeh other in many respeets, among whieh are
degree of aeetylation, typieally elose to 0.90; elemental analysis, with
R.A.A. Muzzarelli
nitrogen content typically c10se to 7%, and N/C ratio 0.146 for fully
acetylated chitin; molecular size; and polydispersity. The average molecular weight of chitin in vivo is probably in the order of the MDa, but chitin
isolates have lower values due to partial random depolymerization occurring during chemical treatment and depigmentation steps. Polydispersity
may vary depending on such treatments as powder milling and blending of
various chitin batches.
Isolated chitin is a highly ordered copolymer of2-acetamido-2-deoxY-D-glucose, the major component, and 2-amino-2-deoxy--D-glucose. As a
point of difference from other abundant polysaccharides, chitin contains
nitrogen. Chitobiose, O-(2-amino-2-deoxy--D-glucopyranosyl)-(1-4)-2amino-2-deoxy-D-glucose, is the structural unit of native chitin. Bound
water is also apart of the structure.
Chitin is easily hydrolyzed by acids, but is stable to dilute alkali; in warm
concentrated alkali it is oxidized by air. Chitin hydrolysates can be prepared
byadding chitin to concentrated HCl at 4C and stirring at 40C. Excess
acid is then removed with ion-exchange resin, and the product is resuspended to prepare the so-called colloidal chitin, which remains stable for
several weeks when stored at 4C. In the wet state it is degraded by a number of microorganisms, which produce chitinolytic enzymes or other enzymes with unspecific activity towards chitin. Colloidal chitin is being
used since the 1950s for the study of chitinases.
The solubility of chitin is remarkably poorer than that of cellulose because of the high crystallinity of chitin, supported by hydrogen bonds
mainly through the acetamido group. Ethanol-containing calcium chloride,
dimethylacetamide containing 5-9% LiCl (DMAclLiCl) and N-methyl-2pyrrolidinonelLiCl are systems where chitin can be dissolved up to 5%.
The main chain of chitin is rigid at room temperature, so that mesomorphic
properties may be expected at a sufficiently high concentration ofpolymer.
Circular dichroism of Congo red bound to the chitin films, obtained by
moisture uptake from DMAclLiCl solutions, reveals a cholesteric structure, having an organization similar to that naturally occurring in the
chitin cutic1e.
Chitosans
Chitosan indicates a family of deacetylated chitins. In general, chitosans

have a nitrogen content higher than 7% and a degree of acetylation lower
than 0.40. The removal of the acetyl group is a harsh treatment usually
performed with concentrated NaOH. Protection from oxygen, with a
nitrogen purge or by addition of sodium borohydride to the alkali solution,
is necessary in order to avoid undesirable reactions such as depolymerization and generation of reactive species. Commercial chitosans may contain
insoluble highly acetylated fractions that come from the core of the
granules submitted to heterogeneous deacetylation. The acetyl groups in

the acid-soluble fractions are randomly distributed, whilst the insoluble
fractions contain relatively long sequences of acetylated units.
The presence of a prevailing number of2-amino-2-deoxyglucose units in
a chitosan facilitates bringing the polymer into solution by salt formation.
Chitosan is a primary aliphatic amine that can be protonated by selected
acids, the pK ofthe chitosan amine being 6.3. The following salts, among
others, are water-soluble: formate, acetate, lactate, malate, citrate, glyoxylate, pyruvate, glycolate and ascorbate.
Despite the alteration due to deacetylation, chitosan from crab tendon
possesses a crystal structure showing an orthorhombic unit cello The unit
cell comprises four glucosamine units; two chains pass through the unit cell
with an antiparallel packing arrangement. Main hydrogen bonds are
0305 (intramolecular) and N2 06 (intermolecular). The crystal
structures of salts and derivatives have also been determined, for instance
for chitosan ascorbate.
Chitosan can be obtained from fungi, easily cultured on simple nutrients.
Chitosan is present in the cell wall ofMucorales and can be isolated from the
accompanying glucans by extraction with either acetic acid or alkali, the latter being preferred when glucans are to be dissolved. The final molecular
weight is in the order of 500 kDa, and the degree of acetylation is around 0.10.
Chitin and chitosan derivatives
In the past, chitin has been often considered as an intractable biopolymer

due to the difficulties encountered in dissolving and reacting it. As soon as
the molecular association is prevented or depressed, chitin lends itself to
many reactions, affording a wide choice of modified chitins. On chitosan,
the reactions of the primary amino group and primary and secondary
hydroxyl groups can be easily performed. The chemical modifications of
chitin and chitosan, carried out under mild conditions in order to protect
glycoside and acetamido linkages, yield more soluble polymers. The latter
have higher biodegradability in animal bodies and physical properties of
interest for applications in the solid state or in solution.
Chitin treated with NaOH yields alkali chitin, a widely used unstable
intermediate which reacts with 2-chloroethanol to yield 0-(2-hydroxyethyl) chitin, known as glycol chitin: this material was probably the first
derivative to find practical use and to be recommended as a substrate for
lysozyme. The reaction of alkali chitin with sodium monochloroacetate
gives O-carboxymethyl chitin sodium salt, soluble in water. The latter compound has been and still is one of the protagonists of chitin chemistry,
together with N-carboxymethyl chitosan; both have found applications in a
variety of fields. Chitosan can be reacetylated with acetic anhydride to
obtain water-soluble partially reacetylated chitin. Countless chitin deri-
R.A.A. Muzzarelli
vatives have so far been prepared; the present trend is to exert control over
the modification reactions in order to achieve the best performance or to
enhance the biological significance. For example, upon regiospecific
oxidation, chitin yields 6-oxychitin, a fully water-soluble compound [21];
regioselective synthesis affords chitin sulfates endowed with anti-HIV-l
activity [22].
Fossil chitin
Whilst chitinous materials are relatively resistant to degradation under certain conditions, for instance suspension in seawater, and desert sand, they
are promptly degraded in other environments such as ocean sediments.
A huge number of mineralized skeletal structures containing chitinprotein complexes settle into deep-sea sediments: there, extraction of
organic matter is performed by fungi, algae and bacteria, which use it as a
nutrient. Extracellularly secreted enzymes hydrolyze the organic polymers
of the skeletal remains colonized by the microorganisms. Because those
chitinases seem to be particularly stable and effective under deep-sea conditions, sediments contain little chitin.
Detection of chitin in fossils is not frequent: there are reports of fossil
chitin in pogonophora, and insect wings from amber, but fossils of
crustacea were found to contain only traces of chitin, and no chitinlike
microfibrils were detected by electron microscopy.
Terrestrial arthropods have a fossil record that reaches back to 420
megayears ago (Upper Silurian): their remains are preserved as cuticle
fragments. Arachnids were found preserved by precipitation of silica; millipeds occur as calcified remains; coleoptera fossils were recovered from
buried peat. The fossil cuticles revealed alkanes and alkenes, indicating
substitution of chitin by more resistant organic compounds [23-25].
Chitin is not generally preserved as such, but some of the most spectacular examples of soft part preservation involve replication in calcium
phosphate: apatite minerals inhibit the decay of organic compounds. AIthough proteins have a short survival time even within CaC03 crystals, it is
possible that phosphate salts provide protection from degradation. Replication of soft tissues is more rapid in calcium phosphate than in any other
mineral, and therefore preserves the highest fidelity in detail, such as in the
case of insect eyes. Interestingly, chitin associations with calcium phosphate are being studied today for bone regeneration purposes.
Conclusion
Chitin is amply distributed in the biosphere where it constitutes the

exoskeletons of many organisms, but also provides delicate structures and
tissues such as the flying apparatus of insects, and tendons of crustaceans.

Chitin nitrogen is recyded by biodegradation operated by microbial genera
which exist in just about every conceivable environment. Chitin therefore
represents a widely distributed organic compound of nitrogen.
The nitrogen present in chitin imparts unique properties to this dass of
polysaccharides. Goods manufactured from chitin are environmentally
important items; chitosan solutions are used to recover proteins and to
preserve cereal seeds, or to prepare biomaterials such as wound dressings
and biodegradable packaging.
Acknowledgements
This chapter was prepared with the financial contribution of the Italian National Research
Council, "Progetto Finalizzato Materiali Speciali Tecnologie Avanzate 11", Rome (contract no.
98.00032.PF34).
References
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3
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5
6
7
8
9
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15
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19
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Chapman D, Haris PI (eds) (1998) New Biomedical Materials -Applied and Basic Studies
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Domard A, Jeuniaux C, Muzzarelli RAA, Roberts G (eds) (1996) Advances in Chitin
Sciences, Jacques Andre, Lyon
Coosen MFA (ed) (1996) Applications of Chitin. Technomic, Lancaster, USA
Japanese Society for Chitin and Chitosan (1995) Chitin and Chitosan Handbook. Gihodo
Shuppan Co, Tokyo
Jeuniaux C (1963) Chitine et chitinolyse. Masson, Paris
Jeuniaux C, Voss-Foucart MF (1991) Chitin biomass and production in the marine environment. Biochem Syst Eco119: 347-356
Muzzarelli RAA (1977) Chitin, Pergamon, Oxford
Muzzarelli RAA (1996) Chitin Chemistry. In: JC Salamone (ed) The Polymerie Materials
Encyclopedia. eRC Press, Boca Raton FL, p 312-314
Muzzarelli RAA (ed) (1996) Chitin Enzymology, Vol. 2, Atec, Grottammare
Muzzarelli RAA, Muzzarelli BB (1998) Structural and functional versatility of chitins. In:
S Dumitriu (ed) Structural Diversity and Functional Versatility ofPolysaccharides. Marcel
Dekker, NewYork, 569-594
Muzzarelli RAA, Pariser ER (eds) (1978) Proceedings ofthe First International Conference
ofChitin/Chitosan. Massachusetts Institute ofTechnology Press, Cambridge, MA
Muzzarelli RAA, Peter MG (eds) (1997) Chitin Handbook, European Chitin Society, Atec,
Grottammare
Muzzarelli RAA, Stanic V; Ramos V (1998) Enzymatic depolymerization of chitins and
chitosans. In: C Bucke (ed) Methods in Biotechnology. Humana Press, London
Muzzarelli RAA (1985) In: GO Aspinall (ed) The Polysaccharides; Academic Press, New
York, vol. 3
Muzzarelli RAA, Jeuniaux C, Gooday GW (eds) (1986) Chitin in Nature and Technology;
Plenum, New York
Neville AC (1975) Biology of the arthropod cuticle. Springer, Berlin
Richards AG (1951) The Integument ofArthropods. Univ Minnesota Press, St Paul
Stevens WF, Rao MS, Chandrkrachang S (1996) Chitin and Chitosan. AlT, Bangkok
Wood WA, Kellogg ST (eds) (1988) Methods in Enzymology, Val. 161: Lignin, Pectin and
Chitin. Academic Press, San Diego
Zikakis JP (1984) Chitin, chitosan and related enzymes. Academic press, London
Muzzarelli RAA, Muzzarelli C, Cosani A, Terbojevich M (1999) 6-0xychitin, novel
hyaluronan-like regiospecifically carboxylated chitin. Carbohyd Polym 39: 361-367
R.A.A. Muzzarelli
22 Nishimura SI, Kai H, Shinada K, Yoshida T, Tokura S, Kurita K, Nakashima H, Yamamoto

N, Uryu T (1998) Regioselective synthesis of sulfated polysaccharides: specific anti-HIV-l
activity of novel chitin sulfates. Carbohydr Res 306: 427 -433
23 StankiewiczAB, Mastalerz M, HofCHJ, BierstedtA, Flannery MB, Briggs DEG, Evershed
RP (1998) Biodegradation of the chitin-protein complex in crustacean cuticle. Org Geoehem 28: 67-76
24 Stankiewicz AB (ed) (1998) Nitrogen Containing Maeromoleeules in Biosphere and Geosphere, ACS Symp. Series 707. American Chemical Society, Philadelphia
25 BierstedtA, Stankiewicz BA, Briggs DEG, Evershed RP (1998) Quantitative and qualitative
analysis of chitin in fossil arthropods. Analyst 123: 139-145
Chitin synthesis

ed. by P. JollE" and R. A. A. Muzzarelli
1999 Birkhuser Verlag Basel/Switzerland
Biochemistry of chitin synthase

Regula A. Merz, Markus Horsch, Lars E. Nyhlen and Dora M. Rast
Department ojPlant Biology, University ojZrich, Zollikerstrasse 107,
eH-8008 Zrich, Switzerland
Summary. This article compiles the papers dealing with the biochemistry of chitin synthase
(CS) published during the last decade, provides up-to-date information on the state ofknowledge and understanding of chitin synthesis in vitra, and points out some firmly entrenched
ideas and tenets of CS biochemistry that have become of age without hardly ever having been
critically re-evaluated. The subject is dealt with under the headings "Components of the CS
reaction" (educt, cation requiremement and intermediates; product), "Regulation of CS" (cooperativity and allostery; non-allosteric activation or priming of CS; latency), "Concerted
action of CS and enzymes of chitinolysis", "Inhibition of CS", "Multiplicity of CSs", and
"Structure ofCS" (the putative UDPGlcNAc-binding domain ofCS; identification ofCS polypeptides; glycoconjugation). The prospects are outlined of obtaining a refined three-dimensional (3D) model ofthe catalytic site of CS for biotechnological applications.
Introduction
The pivotal role chitin plays in the life cyc1es of arthropods as well as fungi
and some other microorganisms (for recendy described examples, see
[1-3]) and the vast array of technological applications of this polymer have
been known for a very long time and been dealt with in many books
[4-8]. Indeed, ever since the first description ofthe enzyme synthesizing
chitin as an N-acetylglucosaminyltransferase using the uridine diphosphate
(UDP)-activated monomer as the sugar donor ([9]; Fig. 1), chitin synthase
(CS) has remained both an attractive and an intriguing enzyme for biochemists, molecular cell biologists, entomologists, mycologists, parasitologists and scientists representing other disciplines as well- for researchers
in academia and industry alike. Despite this broad interest and the copious
bibliography dealing with CS obtained through the four decades ofinvestigations, only a comparatively modest insight has been gained into the basic
biochemical characteristics of the enzyme. And even with these, there is
overall agreement only as to phenomenology, but often not as to interpretation (for examples, see [10, 11]; earlier papers cited in these publications are, with some exceptions, not referred to anymore in this review).
10
R. A. Merz et al.
C~H
HO~
UDP
uridine-5'-diphosphoN-acetyl-a-glucosamine
(UDPGlcNAc)
[chitin1n
chitin synthase (eS)

UDP
[chitinln+1
Figure 1. Sum equation for the reaction catalyzed by chitin synthase (CS; UDP-N-acetyl-Dglucosamine: chitin 4--N-acetylglucosaminyltransferase; EC 2.4.1.16). R 1 = H [9], CS (= R 2 ;
[12,13]), protein (;eCS; [14,15]), or priming UDPGlcNAc-transferase (;eCS; this article).
No inference is made by the scheme as to the mechanism of chain elongation (cf. [16, 17]).
Components of the CS reaction

Educt, cation requirement and intermediates
The obligate substrate for the enzyme is UDP-N-acetylglucosamine
(UDPGlcNAc). The presence ofMi+, Mn2+ or C02+ (depending on enzyme
species) is indispensable for CS activity [1, 18-22]. The significance of
this requirement has not been elucidated, but presumably reflects an essential role of CS for a divalent metal cation in substrate binding_
Chain assembly occurs without the formation of free chito-oligomers
[23]_ Also, in contrast to the original view (see Fig_ 1, R=H), there is no
need for a soluble GlcNAc acceptor, such as a chito-oligomer with n ~ ca.
10 (higher analogues are insoluble), exogenous chitin does not act as a
'primer' either (for a discussion ofthe term, see [24]), and a lipid intermediate or a lipid acceptor is generally held not to be involved_ Nevertheless,
there are reports of the formation of GlcNAc-glycolipids acting as intermediates of chitin synthesis, namely GlcNAc-glucosyldiacylglycerol [25]
and dolicholdiphosphate-linked chito-oligomers with a maximum chain
length of n = 8 [26,27]. The latter appear to be transferred to a polypeptide
acceptor and, thence, to act as GlcNAc-acceptor substrates for CS ([14, 15];
11
Fig. 1, R j = priming polypeptide). In any case, the experimental evidence

implies that CS acts on a covalently-bound primer, which, according to the
definition ofthe enzyme, is chitin (Fig. 1; R 1 = CS) - a situation turning the
systematic name of the enzyme into a definitional oddity. It is not known
whether the presumptive primed-CS educt and the UDPGlcNAc binding
site for the iterating events of chitin chain elongation concern distinct
regions of the same polypeptide or are situated in two different, physically
c10sely associated polypeptides [CS multienzyme (ME) polypeptide vs. CS
ME complex; for definitions, refer to [28]; for an hypothesis, see "Nonallosteric activation or priming of CS" and "Structure of CS"]. As is also
evident from Fig. 1, there is, additionaIly, a requirement for an enzyme that
catalyzes the removal of (part ot) the polymer chain to produce pure
chitin. Hence, the mode of action of CS cannot be assessed adequately
without a consideration of co-occurring chitinolytic activity (see section
"Concerted action of CS and enzymes of chitinolysis").
Amphiphiles, such as phospholipids [1, 29-32] and some strongly amphipathic lipid-linked sugars [32-37] stimulate CS activity (for earlier ref's
for the effect of lipids on CS, see [38)). The generally low specificity of
glycerophosphatides and the vastly different responses of CSs of different
origin to individual representatives of these cast doubt, however, on their
direct involvement in the CS reaction, beyond the fact that, for good activity, the enzyme apparently needs to be bound to a membrane, either a natural
or a suitable artificial one. With the 3-sterol glycoside digitonin (DIG) the
situation appears to be different, inasmuch as it effects solubilization ofthe
enzyme, the process being preceded by a stimulatory action of the saponin,
as quite generally occurs with other detergentlenzyme combinations [39].
However, DIG interacts with CS also in a more specific manner, producing
a pattern of stimulation/inhibition, as do other, structurally c10sely related
spirostanol glycosides [40]. The CS-inhibitory potency ofthese is particularly pronounced in the case of the respective glycoforms not carrying a
terminal glucose at the side-chain residue. The ability of DIG not only to
bind to genuinely membrane-bound CS efficiently and quite specificaIly,
yet producing a low degree of inhibition, as weIl as to harbour the enzyme
protein in a liposome that obviously mimics natural conditions (see [41)),
might be the main reason, why DIG has remained the agent of choice for
solubilizing CS.
Product
To reiterate what is known of the chemical structure of chitin lies outside

the scope ofthis artic1e, but the supramolecular organization ofthe product
deserves comment. The chitin synthesized by DIG-solubilized CS (16S)
has been described as consisting of lozenge-shaped particles about 10 nm
wide and some 50-100 nm long [12], as lateral aggregates of spindlelike
segments of similar dimensions [30, 33, 42] or as extremely slender (width
12
R.A. Merz et a1.
of 1-2 nm; corresponding to one or two mo1ecu1ar chains) and very 10ng
(severa1 hundred nm, up to 2 f.llll) fibri1s emanating from the 16 S partic1es
[35]. From a mechanistic point ofview, it is highly unlikely that CS catalyses the synthesis of a fibrillar product; on Ockham 's razor, the reaction
would, rather, yield single, monomolecular chitin chains. Thus, the formation of thick fibrils observed in some studies represents probably an artefactual postsynthesis event, which could be caused by the long incubation
times used. Indeed, whereas only very thin long fibrils were present after
reacting CS with substrate and activators for short times, long-time incubation yielded configurations of thick fibres consisting of twisted 5 -1 O-nmfibrils, while thin fibrils were no longer discemible (Fig. 2; L.E. Nyhlen and
D. M. Rast, unpublished). Therefore, the notion that the generation and
crystallization of nascent chitin occur simultaneously and are very tightly
coupled [43], should be discounted (see also [30], and section "Inhibition of
CS"). Besides, evidence for a temporal gap between the CS-polymerization
reaction and the assembly of the polymer chains into microfibrils has been
obtained also by another experimental approach [44].
Obviously, the extent to which crystallization of chitin occurs to form
fibrils depends largely on the physicochemical composition of the enzyme's microenvironment and the presence of other enzymes, particularly
chitin-modifying entities. On the other hand, the possibility cannot be
exc1uded that the extreme lengths observed of chitin fibrils synthesized in
vitra after short-time incubation in the absence of chitinase (Fig. 2a; for
further examples, see [35]) are artefactual, too, as in situ the chain length
of newly synthesized chitin is likely to be restricted by co-occurring
chitinase (see Fig. 3 and text). Indeed, purified 16 S-CS ex chitosomes display virtually no chitinase activity (tested with chromogenic and fluorogenic chitotetraoside analogues as the substrates; C. Mayer, M. Horsch,
D.M. Rast, unpublished results).
Termination of the chain in polysaccharide synthesis can occur in principle by transfer of a hetero-group to its growing end and, thus, prec1ude
further addition by the specific polymerase concemed [24]. Depending on
the structure ofthe residue transferred, this may result in a simple capping
ofthe growing end or in a cross-linking reaction. Altematively, co-occurring specific lytic enzymes could restrict chain length. In either case,
prevention of further chain elongation must be an extemally determined,
controlled event [24]. Reports are not available of heterotransferases specifically acting at the nonreducing end of nascent chitin, and the enzymes
effecting cross-linking between chitin and co-occurring polymers in situ,
for example -glucan [45,46], have not been investigated to any extent.
Transglycosylating chitinases, hexosaminidases as well as -glucosidase
and -glucanases are probably involved [47]. An experimentally based
model that encompasses restriction of the chain 1ength and a remodelling
of chitin chains in a controlled manner is presented under section "Concerted action of CS with the enzymes of chitinolysis".
13
Figure 2. Influence of the duration of incubation of CS with activators and substrate on the
supramolecular structure of the product synthesized in vitra. Sampies were negatively stained
after reaction times of (a) 6 min and (b) 25 min. The enzyme preparation (from Agaricus
bisparus mycelium) consisted of 16 S-CS obtained by dissociation of gradient-purified chitosomes (peak fraction) with DIG. For methodological details, refer to [35].
R. A. Merz et al.
14
Regulation of es
Co-operativity and allostery
Homotropic-heterotropic regulation with GlcNAc acting as an allosteric

activator is a firmly established tenet of the biochemistry of CS; its validity
has recently been revisited [23,48]. Thus, irrespective ofthe type of enzyme
preparation used (105 S chitosomal CS, 16 S-CS ex chitosomes or 16 S-CS
ex walls) and regardless ofwhether GlcNAc was present, substrate kinetic
curves could reasonably well be parameterized on the basis of the Monod
mathematical model for co-operative ligand binding. The stereochemical
requirements of the allosteric site of CS for ligand binding are: (i) an
aminoglucopyranose skeleton with the amino function acetylated, and
(ii) a single-bonded oxo-function present at C(1), which is preferentially a
hydrogen bond donor, that may be equatorially spaced off but must not
be a-anomeric. In the absence of any evidence for more than one type
of allosteric site of CS, the notion is to be discounted, therefore, that
GlcNAc would be but a poor substitute at an effector site for UDPGlcNAc
[49,50]. The same holds for the suggestion [51] that the CS inhibitory action ofnikkomycin (for essential structural features, see [52]) results from
its competition with GlcNAc or UDPGlcNAc at the effector-binding site.
Since the intracellular concentrations of free GlcNAc appear to be extremely low, it is a reasonable hypothesis that its source is situated at the
cell surface, which is also the locus operandi ofCS, where GlcNAc is likely to be generated through the action of enzymes effecting lysis of chitin,
namely, chitinase and -N-acetylhexosaminidase (HexNAc'ase; [53,47]).
In view of the additional evidence provided by Horsch et al. [23] for the
existence of a genuine allosteric site of CS for GlcNAc as well as of the
observation that the activation constant
(GlcNAc) is lower with 16 S-CS
than with 105 S-CS [23,35], the almost complete loss ofGlcNAc stimulation of CS reported by Kang et al. [13] upon isolation of the enzyme by
gel filtration of a microsomal fraction, entrapment of CS into its reaction
product, digestion of this with chitinase and extraction of the enzyme into
buffer must be considered an experimental artefact. This may in part be due
to contaminating chitinase and HexNAc'ase acting on firmly bound traces
of chitin from the entrapment step, since neither of the chitinolytic enzymes
would be separated from CS under the purification scheme followed in that
study (see [53,54]).
The generally accepted view whereupon the substrate kinetics of CS are
govemed solely by features ofhomotropic-heterotropic regulation calls for
a critical reevaluation, however, since the sigmoidicity of the CS activity
vs. [UDPGlcNAc] plots has, in fact, never been observed to become completely abolished in the presence of saturating [GlcNAc] [23,35,48-50,
55] - in contrast to what is required by the theory of allostery. Accumulation at low substrate concentrations of soluble products (which would not
K:
15
be detected by the filtration assay) as the cause ofthis phenomenon can be

excluded, since free chito-oligomers were not found as reaction intermediates (tested using a variety of purified enzyme, substrate and effector
concentrations as weH as the most sensitive analytical method presently
available [23, 56]). The next section presents an hypothesis for this hitherto
unexplained kinetic idiosyncrasy of the CS reaction.
Non-allosteric activation or priming o/es

In the absence of GlcNAc and at low concentrations of UDPGlcNAc the
CS overall reaction is absolutely dependent on proteolytic activation and
kinetically displays two distinct phases ([23,48]; M. Horsch and D.M. Rast,
unpublished data). The first, nonlinear phase of the reaction is characterized by a gradual increase in the rate of product accumulation with time,
the curve on the one side extrapolating towards a steady rate of synthesis at
the start of the incubation and towards a steady rate of synthesis on the
other, whence the second, linear phase of the reaction is reached. The
approach to the steady-state rate is slow. These kinetics are independent of
the length of the enzyme 's preexposure to the protease and also not an artefact that would be due to the formation of soluble chitooligomers [23].
Hence, areaction proceeds under these conditions that enables CS to
change from a truly latent into a fully active state even in the absence of
GlcNAc. Considering the nature ofthe reactants, CS and UDPGlcNAc only,
the conclusion thus appears unavoidable that there occurs an autoglycosylation prior to chitin chain formation of the CS system. That the frequency of
enzyme activation shows a linear dependence on [UDPGlcNAc] is in line
with this conclusion. Deceleration of the activation rate with decreasing
enzyme concentration and vice versa, furthermore, indicates that this selfglycosylation might be intermolecular. Therefore, it may be assumed that
two different UDPGlcNAc-binding sites/two different enzymes are involved
in de novo chitin synthesis.
Since knowledge of the molecular structure of CS is quite lirnited (see
"Structure of CS"}, the type of GlcNAc linkage generated by this putative
activation or priming reaction, the site at which it takes place and the
mechanism whereby it causes chitin chain formation can presently only be
speculated upon. It is useful, therefore, to turn to the synthesis of glycogen
as a paradigm, since this represents the only polysaccharide synthase for
which more information is available regarding the mechanism of reaction
(for reviews, see [57,58]). Thus, the efficient synthesis of glycogen requires the sequential action of two UDPGlc-transferases, namely, a glycogen
initiator synthase termed glycogenin (GG; EC 2.4.l.186) and glycogen
synthase proper (GS; EC 2.4.1.11), both ofwhich are subunits ofthe glycogen synthase complex (ca. 15S; [59]). GG is a self-glucosylating protein
that primes glycogen synthesis. It effects auto-glucosylation by first gen-
16
R. A. Merz et al.
erating a glucosyl-tyrosine linkage [Glc(I-O)TyrI94; see [60] for amino

acid sequence] and then successively adding further glucose units to finalIy generate an enzyme-bound oligomer of a-I,4-linked Glc units (n averaging 8; [61]). The self-glucosylation appears to be intermolecular, as the
rate departs from constancy and falls away upon lowering the enzyme concentration [62,63]. It is upon the maltosaccharide acceptor structure that
GS performs chain elongation to afford the polysaccharide. The GG reaction has certain other properties that clearly distinguish it from that catalysed by GS [57,62,64-67]: (i) GG activity is only displayed in the presence of high concentrations of various salts (GS is not active under such
conditions); (ii) the pH optimum ofGG is higher than that ofGS (ca. 8.5
vs. 7.0); (iii) the first glucosyl transfer is Mn2+-dependent, whereas the
following elongation events proceed with Mg 2+ (GS is not activated by
Mn2+ and is, in fact, essentially unaffected by either cation); (iv) GG is not
inactivated by UDP-pyridoxal (a specific affinity label for GS); (v) GG is
not activated by glucose-6-phosphate, a potent effector of GS; (vi) the Km
(UDPGlc) of GG is ca. 103 X smaller (this means that at low substrate concentrations only the GG component of the GS complex is operative and,
thence, generates a storage pool ofprimed GS substrate that is available for
glycogen synthesis proper as conditions become favourable for this (that is
high [UDPGlc], and effector); (vii) the time-course approach of the GS
overall reaction to constant rate is sigmoidal if the primer has to be generated first, and hyperbolic when exogenous primer is provided.
Four lines of argument may lead one to suggest that a situation corresponding to that of the GGIGS system could exist, in an analogous manner,
for the chitin synthesizing system: (I) the particular kinetics of the CSreaction that proceeds in the absence of GlcNAc (as stated above); (2) some
of the differential properties of GG vs. GS, particularlY those concerning
items (ii), (iii) and (v)-(vii) have similarly been described to occur between
different CS-preparations; (3) an analogous priming function ofthe DG42
protein, catalyzing the synthesis of an array of chito-oligosaccharides [68],
in the formation of hyaluronic acid (containing a chito-oligosaccharide
residue at its reducing end [69]); and (4) the evidence accumulating in
recent years that a priming protein may be a fundamental property not only
ofthe GS system, but of polysaccharide synthesis in general (for references, see [70]). The as yet hypothetical enzyme that would generate covalently-bound chito-oligosaccharide as a primer for es sensu stricto is
hereafter referred to as 'chitogenin' (Fig. I, R j =t:. es).
Latency
Limited proteolysis generally greatly stimulates es activity [32,37,7175]. This indicates that most of a cell's es pool may be present in a 'classical' proenzyme form prior to product deposition at the proper time and
17
site [76]. Recent papers do, however, attest to the still puzzling nature of the
phenomenon, and its significance remains elusive [21,22, 77, 78].
The most telling example in the present context may be CSIII. This is
generally held not to be zymogenic and displays a preference of C0 2+ over
Mg2+ as the activating cation [78-80]. Nevertheless, its activity becomes
greatly increased if the (detergent-extracted) membrane preparation harbouring it is treated with trypsin in the presence of UDPGlcNAc prior to
allosteric stimulation of chitin synthesis by GlcNAc. This protease treatment also has a bearing on the metal requirement of the CS-system concerneel, inasmuch as the trypsin-dependent activity is favoured by an
increase in [Mg 2+] and as there seemingly occurs areversal in the metal preference ofthe re action [21]. In view ofreasons (i)-(vi) below, the possibility has to be considered that the trypsin- and Mg 2+-mediated stimulation of
CSIII does not directly concern csm proper, but a tightly associateel, truly latent UDPGlcNAc-transferase species generating a product that efficiently enhances the overall rate of chitin synthesis; this enzyme is hypothesized here to be 'chitogenin' (see section "Non-allosteric activation or
priming of CS"):
(i)
(ii)
(iii)
(iv)
(v)
(vi)
There exist some suggestive analogies between the chitin and the glycogen synthesis systems.
CSIII is presumably a multi enzyme complex [21,78].
Some ofthe UDPGlcNAc provided at the trypsin pre-incubation step
(in the absence of GlcNAc) undoubtedly becomes reacted upon anel,
thence, incorporated into acid-insoluble product (representing phase
1 of the CS overall reaction).
The deduced AA sequences of chito-oligosaccharide synthases of
various origins show high similarities to those offungal CSs [69].
Phenomenologically, 'chitogenin' would simply be perceived as an
activator of CSIII, as holds, for example, for the gene product of
CHS4 (= CAL2/CSD4/SKT5; for new nomenc1ature, see [81]), which
is an essential component ofthe CSIII-complex.
The CHS4-product has no homology with any known protease, is
zymogenic and appears to be involved in substrate processing [78].
Cooperation in efficient chitin synthesis of two CSs differentially regulated with respect to limited proteolysis and cation requirement woulel, finally, also obviate the need to entre at a change in the cation specificity of
CSIII proper upon preincubation of the enzyme preparation with protease
in the presence ofUDPGlcNAc [21].
Concerted action of CS and enzymes of chitinolysis
Although manipulation of CS activity by allosteric and non-allosteric
activation, inc1uding limited proteolysis, is sufficient to allow some control
ofthe enzyme's activity in vitra, additional mechanisms must exist that pro-
18
R. A. Merz et al.
vide for a tight regulation of chitin synthesis in vivo, at the transcriptional

as well as at the posttranslational level [77]. Examples are phosphorylation/dephosphorylation as mediated by the Ca2+-calmodulin system [82, 83],
interaction of CS-secretory vesicles with components of the cytoskeleton
(for references, see [84]), and controlled action of CS with respect to cooccurring enzymes at the site of product deposition. Concerning these, the
correct balance between the activity of an appropriately regulated chitinolytic system and that ofthe CS system appears paramount in vivo, not only
in filamentous as weIl as in yeast growth offungi [47,53,85], but probably
also in other biological systems effecting a quasi-simultaneous synthesis
and degradation of chitin [86].
A speculative scheme for the controlled synthesis and lysis of chitin during
hyphal growth through the concerted action ofCS, chitinase and HexNAc'ase
is depicted in Figure 3. The hypothesis is based on premises (i)-(viii):
(i)
(ii)
(iii)
(iv)
(v)
The topology ofthe site ofaction ofthe chitin synthesizing system at

the cell surface, encompassing the plasma membrane, the periplasmic
space as weIl as the wall fabric itself(references in [23,41]).
The co-occurrence in log-phase hyphae of genuinely wall-associated
CS, chitinase and HexNAc'ase, any ofwhich being held in the wall
compartment by two types of interaction, namely hydrophobic bonding in an amphiphilic environment and very tight, probably covalent
binding to wall components [47, 54].
The regulatory properties of CS, encompassing mechanisms of nonallosteric activation (including partiallatency) as well as allosteric stimulation (see the respective sections ofthis review).
The presence in some CS species (100 S- as well as 16 S-size forms)
of a chito-oligosaccharide residue, besides an N-glycoconjugated partial structure [41,87-89], which could conceivably serve as a region
of complexation for the non-catalytic high-affinity chitin binding
domain of chitinase [90].
The characteristics ofthe constitutive complex chitinolytic system of
growinglbranching hyphae, which consists of several HexNAc'ases
~
Figure 3. A biochemical model for the controlled synthesis and catabolism of chitin in situ. An
indication is given in the graph of the approximate spatial arrangement of the enzymes at the
cell surface. Part (a) depicts an integrated tripie enzyme system consisting ofCS (EC 2.4.1.16),
chitinase (EC 3.2.1.14) and -N-acetylhexosaminidase (HexNAc'ase; EC 3.2.1.52). Part (b) illustrates the transglycosylating activity and the tandem action of chitinase and HexNAc 'ase in the
hydro lysis of chitin. The chitinolytic enzyme species ofparts (a) and (b) are isozymes differing
in substrate chain length preference and some other properties [53]. The integrated action of (a)
and (b) encompasses four steps: (1) de novo synthcsis of chitin by activated CS; (2) remodelling of nascent as weil as of preformed, partly crystalline chitin through the combined transglycosylatinglhydrolysing activity of chitinase and HexNAc'ase; (3) progressive lysis of chitin,
with increasing amounts of shorter chito-oligomers and N,N' -diacety1chitobiose ('chitobiose')
becoming available for chitinase and HexNAc'ase associated with CS; and (4) HexNAc'asemediated c1eavage of the glycosidic bond of chitobiose resulting in the generation of G1cNAc
available for CS-activation and, simultaneously, potentially providing a donor for transglycosylation reactions (adapted from [47]).
19
~
../1
- ...........~-7
(O)-{!~-<:.>- -0
"'~ .' "..t/~
/~
cf
(a)
o ,GlcNAc; l>-O, UDPGlcNAc; 00, chitobiose; the sign
- -0 marks the nonreducing end and - 7 points to the reducing end 01 the polymer; reducing GlcNAc
units generated through the action 01 chitinase and HexNAc'ase are shown as
0---;:. . The active sites 01 the enzymes are marked as ~ (chitin synthase),
~ (chitinase), ~ (HexNAc'ase),~ , allosteric site 01 ChS
lor GlcNAc; - , site of hydrolytic attack of chitinase. To better iIIustrate transglycosylation, the GlcNAc residues of two chains between which the event is depicted to
take place are tagged differently ( and -0-0-0-).
Figure 3. Legend see p. 18
20
R. A. Merz et al.
and two types of chitinases (A, B) differing in pH optimum, affinity

to polysaccharide gels, ability to degrade preformed chitin and response to treatment with proteases [53,54].
(vi) The tandem action of the binary chitinolytic system in effecting the
breakdown of chitin to chiobiose and GlcNAc [47,91].
(vii) The transg1ycosy1ating activity of genuinely wall-associated as well
as of soluble fungal chitinases and HexNAc'ases ([47, 54, 56, 92];
C. Mayer, U. Sennhauser, D.M. Rast, unpublished data).
(viii) The observation that in each size-type preparation of chitinase -large
and small-particu1ate as well as soluble ones - CS, chitinase and
HexNAc' ase co-occur [53].
Part (a) of the model concerns the synthesis of chitin, and part (b) both the
remodelling of chitin through the combined hydrolysing/transglycosylating action of chitinase and the final breakdown of the polymer. Thus, the
controlled deposition of chitin in a growing wall can be hypothesized to
proceed in four steps. These are, however, not meant to be single, separate
events, but to occur quasi-simultaneously, the time-Iapse of the preceding
step overlapping with the onset ofthe following:
(1) Polymer synthesis by CS (presumed to be a multi enzyme complex
with a chitin initiator synthase as a subunit; Fig. 1, R j =I:- CS) upon activation
by limited proteolysis and allostery. (2) Restriction as weH as extension of
the chain length of chitin synthesized de novo by the action of chitinase;
according to the substrate chain length specificity of chitinase (n ~ 4; as
opposed to HexNAc'ase; see [47]), this can use as alternative acceptors
-1,4-linked GlcNAc residues of the required length that are covalently
bound to other polymers, for example, chito-oligosaccharides conjugated
with protein or lipid, and thus effect cross-linking. (3) Rearrangement by
chitinase (type B) also ofpreformed chitin, the attack gradually resulting
in an increased production of chito-oligomers as the preferred substrates of
a proteolyticaHy activatable chitinase (type A), postulated to be (loose1y)
associated with CS by van der Waals forces and carbohydrate recognition.
(4) Hydrolysis ofthe end product of chitinase action by HexNAc'ases, one
of which is fairly strongly associated with CS (16 S forms; ex 100 S-CS as
well as ex walls), thus generating the CS effector GlcNAc to provide for
full capacity binding of UDPGlcNAc and, thence, de novo synthesis of
chitin.
There are three major implications inherent in the biochemical scheme
presented for the concerted action of CS and chitinolytic enzymes in logphase hyphae:
(i)
The -l,4-linked partial structures present in individual chitin chains

can be of a threefold origin, namely, a moiety generated by a chitin
initiator synthase (Fig. 1, R j ; hypothesized to be 'chitogenin'; see
"Non-allosteric activation of CS") that encompasses the reducing end
21
of the polymer, stretches generated de novo by es sensu stricto, and

residues attached by transglycosylating chitinase at the nonreducing,
that is the acceptor end of a growing chain.
(ii) Entrapment of es into its product [13,50] reduces its activity, as
undoubtedly must also the displacement of the enzyme from the
source of its substrate at the cell surface to the adjacent area of the
developing wall (for potential mechanisms ofUDPGlcNAc release on
to the protoplast, see [41]). It is this situation, where the mural nonzymogenic chitinolytic system [part (b) of Fig. 3] has its main function, besides that of providing for a dynamic remodelling of preformed chitin, inasmuch as reduction of the length of eS-attached
chitin to that ofa short oligomer restores some ofthe catalytic potential of the enzyme molecule concemed and as the concomittant
breakdown ofpreformed chitin yields an ample supply ofthe effector
GlcNAc to temporarily maintain some further polymer synthesis at
this site.
(iii) eonceming the mediator function between the lysis and the synthesis
of chitin assigned to the HexNAc'ase of the tripie enzyme system
represented by part (a) ofthe scheme, the transfer ofGlcNAc to es
would be particularly effective if the association between the two
enzymes were such that the active site ofthe former was positioned in
dose proximity to the allosteric site of the latter. The mediator role of
HexNAc' ase could be even more sophisticated, if the enzyme, provided with its substrate by a nearby chitinase, were also to chemically modify es and, thus, to generate a covalently linked acceptor structure. With its generally high leaving group/acceptor tolerance (for
reaction mechanism, see [47]), HexNAc' ase appears intrinsically well
befitted for such a function.
For a discussion of the biochemical model of the controlled chitin metabolism in hyphal growth in the physiological context, refer to [23,47, 53].
Inhibition of es
Some nudeoside-peptides (NPs), namely, nikkomycins and polyoxins,
kinetically behave as excellent competitive inhibitors of es; reported
Ki-values are 2:: 0.1 J.1M [52, 71, 93-98]. Despite the wide use ofNPs as a
tool in chitin biochemistry, the complex structure-activity relationships observed with numerous analogues, the large differences exhibited by them
in inhibitory potency with es species having a different cation preference
(Mg 2 + vs. e0 2 +) as well as the non-competitive behaviour of nikkomycin
with a mutant es [51] have remained unexplained mechanistically. With
chito-oligomer synthases, however, the inhibitory potency ofNPs appears
to be quite moderate. Thus, the DG42 protein has K/s of ca. 50 J.1M [68],
22
R.A. Merz et a1.
whereas with CS systems values are usually in the 1-10 JlM range. A similar situation has been reported for the Mn 2 +-dependent chito-oligosaccharide synthase presumably catalysing the priming reaction for CS [14, 15],
which was weakly inhibited, while co-occurring chitin deposition was
strongly affected [27]. Pentachloronitrobenzene (PCNB) is another agent
that inhibits CS specifically (other chlorobenzenes, such as hexachlorophenol and pentachlorophenol, are almost ineffectual), potently (K j values
~ 1 J.1M; best with highly purified CS preparations, for example, reconstituted chitosomes), and competitively [99]. Its inhibitory efficacy is highest
in the absence of GlcNAc, at low substrate concentrations and with short
incubation times. PCNB does not interfere with the proteolytic activation
of the CS system [99].
In view of these observations and the finding that the CS reaction is
biphasic (see "Non-allosteric activation or priming of CS"), the question
arises whether glycosylation of the putative CS-primer protein or chitin
chain elongation represents the primary target of PCNB and NPs. No definite answer can be given yet because most of the data reported were based
on CS preparations that were highly active (achieved either by high enzyme
and/or high substrate concentrations and in the presence of saturating
GlcNAc, whence steady-state conditions are reached quickly). Nevertheless, the available experimental evidence indicates that PCNB interferes
with the first priming rather than an iterating chain elongation reaction. The
complete lack of structural similarity between PCNB and UDPGlcNAc,
however, makes it highly improbable that it could 'compete' as a monomerlc substrate in the polymerization reaction. Considerlng the chemical
reactivity of PCNB (cf. [100]) and further experimental data (the inhibitor/CS complex is not disrupted upon gel filtration or density gradient centrifugation in high gravitational fields, even in the presence of solvent for
the ligand; [99]), competition of PCNB with UDPGlcNAc for a non-carbohydrate reactant is therefore an obvious alternative. In analogy to the
situation with glycogenin (see [58]), this could conceivably be the predicted invariant tyrosine of the catalytic site (see Figs. 4 and 5; Tyr26 1). In contrast to this, inhibition of chitin synthesis by NPs might, indeed, result by
interference at a chain elongation step.
Certain compounds that are structurally largely different from NPs and
PCNB and also from each other influence the activity of CS according to a
stimulation/inhibition pattern [see examples (i) and (ii) below]. The CSmodulatory activity of these agents depends not only on their concentration
but also on the presence of organic solvents as weH as the type oftest enzyme
preparation used. This situation evidently prec1udes a reliable assessment of
inhibitor constants and a truly mechanistic interpretation of the data.
(i) With the stilbene derivative Calcofluor White M2R (CFW), there was no
inhibition ofthe activity of 100 S-CS exA. bisporus (up to 20 J.1M CFW,
whereafter there occurred a sharp dec1ine to nearly zero residual activity
23
at 100 J.1M), 16 S-CS ex chitosomes was slightly stimulated (some 30%

at 1 J.1M) and then gradually inhibited (with 150 ca. 8 J.1M), while 16 S-CS
ex walls displayed only inhibition (with Kapp 5 J.1M) [99]. The latter
response occurred also with 16 S-CS ex walls from Mucor rouxii [30],
although in this case the CS-inhibitory efficacy ofCFW was more than
10 times lower. CFW is, hence, unlikely to act by a single mode, namely
intercalation between polysaccharide chains, as generally assumed
when claiming crystallization of the polymer into fibres to be a biochemical, rather than a biophysical event clearly separated from the
polymerization step (see also section "Product").
(ii) In the case ofthe polyene macrolide antibiotics (PMAs), the complexity
of the interaction with CS that underlies their modulatory effect on the
activity ofthe enzyme [40, 99] is intrinsically even greater than in the case
of CFW, since aqueous solutions of PMAs consist of a variety of potentially reactive species, ranging from single molecules at ::;; ca. 10-7 M to
micelIes and aggregates therefrom above the critical micellar concentration [101, 102]. Thus, nystatin and mepartricine B (MEB), sonicated in
buffer, exhibited a pattern of very slight stimulation followed by net inhibition with chitosomes from M rouxii; Iso-values were in the 100-J.1M
range [40]. Under the same conditions (this means applied at 100 J.1M in
the absence of solvent), but using 100 S-CS exA. bisporus, amphotericin
B (AMB) stimulated enzyme activity (byabout 150-170%) and caused
no inhibition at all, whereas in the presence of solvent (2 % dimethylformamide, itself producing some stimulation), there was 50% net
inhibition at 1 J.1M with the same enzyme isolate [99].
The CS-modulatory activity of these polyenes is undoubtedly a consequence oftheir strongly binding to the enzyme (the complexes generated upon incubation of 100 S-CS with AMB, [14C]AMB methyl ester or
[14C]MEB did not become disrupted by gel filtration or density gradient
centrifugation). Since all polyenes bind to membrane sterol, among
which particularly weH with ergosterol [101, 102], but only the heptaene
AMB and ring-size analogues therefrom produce a sizable response
[34, 40], the CS/polyene interaction concerned has probably not
the character of a simple membrane perturbance. A superposition of
mimicking a lipid intermediate (see "Educt, cation requirement and
intermediates"), when present in mixed micelIes together with CS (that
is as liposomes; cf. [41]), as the cause of the stimulatory action of
heptaenic polyenes and specific binding of the monomeric form at
'the active site ofCS as the reason for inhibition, appears to be an hypothesis worthy of consideration.
24
RA. Merz et al.
Multiplicity of CSs
The unequivocal assignment of an enzyme to CS EC 3.2.1.16 requires critical
identification of the product by direct means, for example, a combination of
assessment of soluble products afforded by chemical or enzymic hydro lysis
and X-ray di:ffraction analysis of the presumptive chitin. Whereas this holds
for the CSs that were described a long time ago, for example those of Saccharomyces cerevisiae [12], Schizophyllum commune [45] andM rouxii [30, 33],
and recently also that of Saprolegnia monoica [103], other CSs have often
been named such simply by the behaviour of the product generated under
standard assay conditions, namely, measurement of the incorporation of the
glycosyl residue ofUDPGlcNAc into filter-retainable material that is insoluble in dilute acid or alkali and sometimes additionally by identification of
chitobiose upon digestion of the deposit with chitinase. On this and similar
CS assays alone, however, other UDPGlcNAc-transferases, such as those
performing elongation at the nonreducing ends of the glycan side chains of
N-glycoproteins, producing hyaluron-like polysaccharides ([69,104], see
also [105]), synthesizing the backbone of complex lipo-chito-oligosaccharides [106,107], or possibly priming the CS system (see Fig. I, R j "# CS),
would eventually qualify as CSs as weIl (if studied with marginally purified
enzyme preparations). Indeed, the NodC N-acetylglucosaminyltransferase
has been explicitly referred to as CS [108].
Conversely, the available experimental evidence is insufficient to rule
out the possibility that the carbohydrate skeleton of Nod factors primarily
originates from the catalytic action of the bacterial analogue of CS, namely a
UDPGlcNAc-transferase involved in the synthesis of peptidoglycan that is
probably also present in the crude membrane fractions serving as the source
of the NodC enzyme, followed by a subsequent modification of the product
through the combined hydrolytic and transglycosylating activity of co-occurring lysozyme [47,54,56,92]. A similar caveat as to the concept of de novo
synthesis ofthe backbone ofNod factors by NodC has been presented earlier,
likewise because of the insufficient purity of the enzyme preparation used to
identify the presumptive NodC UDPGlcNAc-transferase [107].
Although more critical for experiments in vivo, another source of error
may come from assessing the purported chitin product with CFW, a fluorocbromic agent intercalating between polysaccharide chains (for references,
see [43, 109]). The CFW test is unspecific, since it affords positive staining
not only with chitin but also with other glycans, inc1uding various -glucans and mannoproteins as weIl as bacterial exopolysaccharides [110].
Moreover, unequivocal interpretation of results is complicated by the fact
that the dye also has an effect on the activity of CS itself, inasmuch as it can
inhibit or stimulate CS (see "Inhibition ofCS").
The limited specificity of the indirect CS assays is of a particular concem,
when enzyme preparations used to identify the enzyme and to assess its 'typical' characteristics are crude microsomal or mixed-membrane fractions, or
25
extracts obtained from the membrane-bound species simply by solubilization

with DIG. This is frequently the case. Therefore, some of the apparent discrepancies reported for the properties of CS can reflect genuine differences
between enzyme species of different ceHular or organismic origins, but could
as weH derive from a misidentification of the UDPGlcNAc-transferase in
question. It may explain the bewildering multiplicity of CSs reported hitherto, representing five different classes [111-117], some of which, however,
have been found to be nonessential for chitin synthesis in vivo.
Structure of es
Despite the wealth of articles on CS published during the last decade,

knowledge of the structural properties of the enzyme is surprisingly smaH
and mainly concems amino acid (AA) sequences deduced from cloned
chitin synthesis genes (CHSs; cf., [81]), some ofwhich, however, code for
UDPG1cNAc-transferases not catalysing the defining reaction ofCS (see
Fig. 1, and "Multiplicity ofCSs"). Figure 4 represents a summary ofthese reports. Based on this as weH as some additional information from other
-glycosyltransferases, a three-dimensional model of the catalytic site of
CS has been established (Fig. 5). Further, there are various papers reporting the isolation of CS proteins (see below for references). However, the
evidence for a particular one to, indeed, represent a CS polypeptide is less
than compelling, and the few AA sequence data obtained by direct analysis of one of these are, therefore, of doubtful value. Figure 6 displays the
key evidence for the unequivocal identification of a genuine CS polypeptide.
The putative UDPGlcNAc-binding domain oles
A large number of derived AA sequences (ca. 70) for CS are known from
fungal genes (cf. [112]; formore recentexamples, see [116,118,119]). In
cases where the fuH gene has been sequenced, this codes for up to 1500
AA residues. However, either for CS, or for homologous proteins, there
does not exist structural information that goes beyond the primary level.
The assignment of gene sections that are highly likely to represent protein
domains with catalytic function, therefore, relies fuHy on predictive analyses. A first clue as to the catalytic domain was found through multiply
aligning the derived CS sequences and singling out a section of about 450
residues that display high consensus among aH genes known so far [120].
Furthermore, structure prediction studies for CS and related sequences
using hydrophobic cluster analysis [17] and knowledge-based homology
modeHing [120] independently concluded that the protein fold is of the
altemating I a type and gave a selection of AA residues that are invariant
26
R. A. Merz et al.
ClI' 51'
.re t
CR.;'
~"cr Cliit
I'bUo !lode
.IDol. dlO"~
.tnw
h&.A
cblatl exoW
21>gU.
Con.ln.I'U1
a~
:a.'WI
21>gU.
~bgu
-210 . ---220 -- ---
-. ----- - -110. ----- - - -200. --- - -
W.
Wh
--no. ------300.-
W.
CHI p
p t.
etLI~
"\101' CII
rbUo nod.C
Mal. 4g4a
n;rpy ha ...
rbiae .ICO.
2bau
Co"' ....."'.
2bau ....
::iIbgu
.3b9u ...
-----uo...9
Wh
210.---
. ..
-270.---- 210..,0.
",0
Pli
WM
PIZ
CHI . . p
,.. t CM.a
uucr CHI'
:rhilo Q04C
.10.1.. dgt:a
It.rpy hall.
rtllM .xoM
2bau
Con alu.
:iIIbgy. D\Ia
::Ib9\a ..
lbgu: . . .
' 00.--
...
- -
-----110.----ull
'20 .... -----"0.-- . . .

all
--
---"0. -ul3
Figure 4. Multiple alignment of deduced amino acid sequences for largely different -glycosyltransferases with the UOPGlc-binding subdomain of a phage T4 ONA-modifying -glucosyltransferase (POB-code 2bgu) as the structural and functional homology template. The
sequence tags used are: CRS ssp, consensus secondary structure prediction for chitin synthases
(
, a-helix; VWIv, -strand; T, turn; [120]; yeast CRS2, chitin synthase 2 from Saccharomyces cerevisiae [125]; neuer CHS4, chitin synthase 4 from Neurospora crassa [126], rhilo
nodC, rhizobial nodulation factor from Rhizobium loti [127]; xenla DG42, hyaluronan synthase
from Xenopus laevis [128]; strpy hasA, hyaluronan synthase from Streptococcus pyogenes [129];
rhime exoW, succinoglycan synthase from Rhizobium meliloti [130]; 2bgu, -glucosyltransferase from the bacteriophage T4 [122]; consensus, consensus colouring as in Figure 5; 2bgu
num, sequence nurnbering of 2bgu; 2bgu ss, secondary structure of 2bgu (
, a-helix;
WM, -strand); 2bgu sse, secondary structure elements of2bgu as assigned by [124]. Amino
acids are coloured within similarity groups, that is white, Gly, Pro, Ala; grey, Ser, Thr; light red,
Asn, Gin; red, Asp, Glu; blue, Lys, Arg; black, Phe, Tyr, Trp, His; yellow, Cys; green, Ile, Leu,
Val, Met.
27
Figure 5. An hypothesis for the 3D structure of the catalytic site of CS, based on the data
presented in Fig. 4 and a space-filling model ofthe UDPG1c binding subdomain ofthe DNAmodifying -glucosyltransferase from the phage T4 (residues 179- 342, inc\uding the uridinediphosphoryl part of its ligand UDPG1c; [143]). Code 2bgu of the Brookhaven National
Laboratory PDB, Upton, NY, USA; reproduced using the program RasMol by R. Sayle, Glaxo
Research & Development, UK). The amino acids are coloured according to the degree of
consensus within the alignment of 2bgu with various other -glycosyltransferases listed in
Figure 4, that is red, strictly conserved in a11 sequences; light red, conserved in at least four
sequences inc\uding 2bgu; green, conserved in at least three sequences, but dissimilar in 2bgu.
in aH CRS genes and in other genes coding for -glycosyltransferases. The

latter study has proposed either a (/akbarrel fold or a structure similar
to the nucleotide-binding (Rossmann) fold for CS, using QSLAVE, an
expert system ofprotein fold prediction [121]. A fold similar in topology
to the Rossmann fold, separated by a deep central eIeft that forms the
binding site for the DDP-sugar substrate, was indeed found for a glycosyltransferase from the phage T4 [122]. This structure was classified together with glycogen phosphorylase into a glycosyltransferase
superfamily of protein folds on purely 3D-structural analogy criteria
[123, 124].
The functional analogy as weH as the good agreement of the phage glucosyltransferase (2bgu) fold with the structure predictions for CS and
other -glycosyltransferases lead us to establish the working hypothesis
that these proteins all belong to this same fold superfamily. Thus, a match
for one ofthe phosphate binding loops l1/alO of2bgu [124] can be local-
28
R. A. Merz et al.
ized in the genes of all classes of CRSs and of related -glycosyltransferases (Fig. 4). Furthermore, the alignment can be extended to the whole
2bgu half-domain that is responsible for the binding of the UDP-sugar
moiety by matching the secondary structure elements (Fig. 4, 2bgu sse)
with the secondary structure prediction for the CS sequences (Fig. 4, CRS
ssp). Within the alignment of the UDP-sugar binding domain of 2bgu,
pairwise homologies are in the 24-30%AA identity range for such different enzyme sequences as those for a S. cerevisiae class I CS (Fig. 4; for
sequence originator citations, see caption to the figure), a N. crassa c1ass
IV CS, a nodulation factor from R. loti, and two hyaluronan synthases,
fromX laevis and from S. pyogenes. Sequence homologies ofthe R. meliloti succinoglycan synthase (rhime exoW) and 2bgu with any ofthe other
sequences are, however, not evident on a residue-to-residue basis (8-12 %
id. range).
A localization in the 2bgu subdomain structure (Fig. 5) of the 9 residues
that are conserved in all aligned sequences (Figs. 4, 5; deep red consensus
colouring) leaves 4 which make direct contact with the UDP-sugar ligand
moiety, namely Asn215 (Asp in all others), Tyr261, Arg269 (or Lys) and
Glu272 (or GIn). Also, Trp341 is strictly conserved and may playa key
structural role in the hinge region between the two subdomains of the fold.
Furthermore, the ligand-binding surface ofthe subdomain hosts many residues that are conserved in at least 3 ofthe prediction sequences (Figs. 4, 5;
green consensus colouring) and those that additionally are similar in 2bgu
(Figs. 4, 5; light red consensus colouring). The N-terminal extensions of
the prediction sequences represent 150-200 residues that are, in terms of
length and predicted secondary structure, in perfect accord with the N-terminal subdomain of 2bgu. Since this appears not to make c10se contacts to
the substrate ligands in the model case and - therefore not unexpectedly since sequence homology in this region is less pronounced among the
various -glycosyltransferase sequences, a full alignment with 2bgu is not
suggested at this juncture.
Identification 0/ es polypeptides
A recently developed new procedure for the purification of CS [88,89],

relying on the selective adsorption of chitosomes to concanavalin A
(ConA)-gel and selective desorption of the enzyme from this, yields the
purest active CS preparation described hitherto, with components identified separately as a genuine CS polypeptide vs. contaminant proteins (Fig. 6;
[41, 89]) - regardless of whether obtained from a mixture of membranes
and wall fragments [13], from unspecified microsomes [22, 131, 132], a
mixed-membrane fraction [32] or chitosomes [37, 99, 133-135]. Thus,
there exists a 60-kDa polypeptide representing a UDPGlcNAc-transferase
with the defining properties of CS, as assessed by the standard test (Fig. 6,
29
Mr
[kDa]
200
116
97
66
45
31
21
Figure 6. Identification of a 60-kDa CS polypeptide: SDS-PAGE patterns (silver staining) of
three different preparations of active CS obtained by subjecting chitosomal CS to affinity
chromatography (AC) procedures. A Gradient-purified chitosomes (peak fraction; isolated
accordingto [136]; D, asA, purified by heparin AC (as described by [89], B, asA, obtained upon
removal of contaminating proteins by conventional ConA-AC and desorption ofthe CS that had
remained tighly bound to the column packing by a mixture of methyl-a-mannopyranoside and
NaCI (each 0.5 M) applied under batch conditions (for experimental details, refer to [41, 88]);
C, as B, but desorption effected with NaCI only (0.5 M). Band C: the protein bands at 32 kDa
and below are due to ConA leakage from the AC colurnn.
lane C). The identity of this with a CS polypeptide was eonfirmed further
by eomparison ofthe SDS polyacrylamide gel eleetrophoresis (PAGE) pattern ofthe isolate with that ofan aetive CS preparation eolleeted upon lipose1eetive affinity ehromatography (AC) of ehitosomes on heparin (Fig. 6,
lane D), whieh also displays the 60-kDa band. The relevanee for chitin synthesis of the additional 57 -kDa band in the latter preparation ean only be
speeulated upon. Sinee desorption ofCS from ehitosome-loaded ConA-gel
with MeMan/DIG, instead of MeMan/NaCl (Fig. 6, lane B), yields the
same sharp 57-kDa band, besides the 60-kDa polypeptide [41, 89], and
sinee the 57-kDa band is also present in the SDSIPAGE pattern of a CS
isolate obtained by AC of ehitosomes on wheat germ agglutinin (WGA,
[89]), the polypeptide concerned might represent a more hydrophobie
subunit ofthe (as yet hypothetical) CS multienzyme complex (see sections
"Components of the CS reaction" and "Non-allosteric activation or priming of CS").
The fact that ConA affinity columns display leakage of various species
of ConA, of which the 32-kDa component is the most prominent (Fig. 6,
lanes B,C; [41, 89]; see also [137, 138]), casts doubt on the claim by
30
R. A. Merz et al.
Machida and Saito [132] to have purified CS to homogeneity said to be

represented by a single 30-kDa polypeptide, mainly because their isolation
protocol involved ConA-AC. Indeed, the amino-terminal sequence (12
amino acids) ofthe putative 30-kDa CS protein was reported to be identical with that of the corresponding stretch of ConA [139] and displays
homology to none of the corresponding AA sequences derived from cloned
CRS genes.
Glycoconjugation of es
Based not only on indirect but also on direct evidence, both 100 S- and
16 S CS exist as glycoforms [41, 87-89, 99]. Thus, there occurs a specific
adsorption ofthe enzyme to the saccharide binding site ofConA - signifying N-glycosylation of CS, AC on lentillectin (LL) affords a binding fraction (accounting for some 60 % oftotal activity) - indicating fucosylation
of the enzyme species concemed at the C-6 position of the N-proximal
GlcNAc moiety, and AC of the same enzyme isolate on WGA likewise
yields a binding fraction (about 50% oftotal), the lectin affinity ofwhich
is annihilated in the presence of N,N',N" '-triacetylchitotriose - pointing to
the presence of a chito-oligomer residue in the glycan side chain of CS.
Moreover, the 60-kDa polypeptide present in the SDS-PAGE electropherograms of CS purified by fast protein liquid chromatography stains weIl
with the periodic acid-Schiff reagent. Quantitatively, the results of the
analysis by high performance anion exchange chromatography/pulsed
amperometric detection of the carbohydrate monomer constituents generated
by trifluoroacetic acid-hydrolysis ofthe LL- and WGA-fractions were somewhat variable; qualitatively, however, they do demonstrate a complex glycoconjugation ofpart ofthe ceIl's CS pool.
Outlook
The list ofthe major gaps in present-day knowledge ofthe biochemistry of

CS, summarized under items (i)-(ix) below, may appear discouragingly
impressive, but would by no means be shorter for any other polysaccharide synthase catalysing a cell surface-Iocated product: (i) the identity of
those polypeptides of the chitin synthesizing system that unequivocally
represent UDPGlcNAc-transferases effecting the formation of chitin de
novo, that is catalyse the generation of the GlcNAc-acceptor/priming substrate and perform chitin chain elongation; (ii) the distinctive physicochemical features of CS polypeptides; (iii) the cause of the absolute, but
differential requirement of CSs for a divalent cation as a cofactor; (iv) the
possible involvement of a lipid intermediate; (v) the origin of the bound
chitin (or chito-oligomer) serving as the primer substrate for CS EC
31
2.4.1.16; (vi) the mechanism of chain elongation (processing at the nonreducing end vs. insertion at the reducing, enzyme-bound end of the
growing chain); (vii) es - a polypeptide containing two catalytic domains
or a multi enzyme complex?; (viii) the types of enzymes responsible for
chain-Iength restriction of chitin by simple capping or cross-linking the
nascent product with co-occurring polymers to which this becomes bound
in situ; and (ix) the significance of (partial) latency of es.
The very limited data available on the structure ofthe chitin-synthesizing
system has undoubtedly represented the decisive obstacle to a speedy progress ofthe research into the reaction mechanism ofeS and the interaction
of this with enzymes catalysing the concerted remodelling and breakdown
of chitin in vivo, as required for understanding growth of any organism
relying on chitin as a building block ofits exoskeleton (refer to "Introduction" for examples). The dearth of structural knowledge of es has been a
barrier also to an adequate streamlining of the enormous efforts invested
during the last decade into unravelling the molecular biology of chitin synthesis amI, therefore, also an impediment to efficiently exploit the biotechnological potential of the enzyme. eonsidering some very recent advances
in es biochemistry pointed out in this review and using as the strategy a
combination of protein and enzyme technology as well as molecular biology methods, besides various types of spectroscopy performed with pure
es entities or relevant fragments thereof, the prospect of thus gaining a
refined 3D model of the catalytic site( s) of es is good. The availability of
this information would evidently benefit also the investigations into any of
the other problem areas listed above [(iii)-(ix)]. Besides, it would allow a
truly rational design of es inhibitors as potential specific biocidal agents
for use in agriculture and medicine [140, 141]. The ideal 3D model of es,
of course, would be that of a glycoform, since glycoconjugation undoubtedly has a bearing on the activity and the cellular integration of es, as it
has on all proteins that enter the secretory pathway (see [142, 143]). Although this is clearly a goal that realistically cannot be reached in the near
future, the chances appear fair of finally attaining even this, since efficient
automated techniques for the carbohydrate analysis of glycoproteins are
becoming routine [143], and mo1ecular modelling can now be performed
also with glycosylated enzymes (fr examples, see [144]).
Acknowledgements
The senior author's research is supported by project no. 3064.1 of the Swiss Commission for
Techno10gy and Innovation (joint program with Novartis) and the Swiss National Science Foundation (grant no. 31-39699.93). It is also a p1easure to thank Prof. G.H.N. Towers, University of
British Co1umbia, Vancouver, for reading the manuscript and Mr. U. Jauch, University of
Zrich, for photographic work.
32
R. A. Merz et al.
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141 Kurtz MB (1998) New antifungal drug targets: avision for the future. ASM News 64:
31-39
142 Rudd PM, Dwek RA (1997) Glycosylation: heterogeneity and the 3D structure of proteins.
Crit Rev Biochem Mol Bio132: 1-100
143 Rudd PM, Dwek RA (1997) Rapid, sensitive sequencing ofoligosaccharides from glycoproteins. Curr Opin Biotechnol8: 488-497
144 Rudd PM, Woods RJ, Wormald MR, Opdenakker G, Downing AK, Campbell ID, Dwek
RA (1995) The effect of variable glycosylation on the functional activities of ribonuclease,
plasminogen and tissues plasminogen activator. Biochim Biophys Acta 1248: 1-10

Chitin biosynthesis and structural organization

in vivo
Jose Ruiz-Herrera and Alfredo D. Martinez-Espinoza
Departamento de Ingenier{a Genetica, Unidad lrapuato, Centro de Investigacion
y de Estudios Avanzados deI Instituto Politecnico Nacional, Apartado Postal 629,

lrapuato 36500, Gto. Mexico
Summary. Many organisms utilize chitin as a structural component of the protective cell walls
or exoskeletons which surround them. These structures are light and resistant composites with
specific structural and mechanical properties which allow them to fulfill their protective role.
Chitin, in the form of microfibrils, is immersed in a matrix of proteins and other polysaccharides. Chitin microfibrils provide the high strength which allows them to resist tensions and
modulus. The cementing compounds protect chitin from chemical attack; keep the microfibrils
separate, preventing fracture; and provide support to tensions. The resulting structures adopt
specific forms which are conserved during growth and are transmitted in a hereditary fashion.
Synthesis of these complex structures involves the following steps: (i) synthesis of chitin either
intracellularly or at the interphase with the extracellular medium; (ii) transport of the chitin
molecules to the extracellular space; (iii) chemical modification of part of the noncrystallized
chitin and association with other molecules; (iv) crystallization ofthe unmodified chitin which
is covered by the rest of the components. The resulting supramolecular structure acquires
viscoelastic mechanical properties; (v) maturation of the composite through formation of
secondary covalent bonds among its components, and deposition of different substances.
Introduction
Throughout evolution, many organisms have utilized chitin as a structural

component. In all the systems studied so far, synthesis of chitin occurs as
the result of a transglycosylation reaction catalyzed by membrane-bound
enzymes collectively called chitin synthetases. These utilize the nuc1eotide
uridine diphosphate N-acetylglucosamine (UDPGlcNAc) as the corresponding sugar donor, the basic biochemical mechanisms appearing to
be rather uniform. Nevertheless, the specific mechanism by which the
polymer is synthesized in vivo by the different organisms appears to have
selective characteristics. In the following pages we will describe the current
general ideas on the mechanisms involved in chitin biosynthesis in vivo, its
transport to the exocellular space where it crystallizes in the form of microfibrils, its most common modifications and its association with other molecules in order to give rise to the protective structures which surround the
organisms.
40
J. Ruiz-Herrera and A. D. Martinez-Espinoza
Cytological basis of chitin biosynthesis
Because of its insolubility it has been generally accepted that chitin is

deployed in the extracellular space. Nevertheless, soluble precursors and
the polymerizing enzyme chitin synthetase itself are synthesized intracellularly. Moreover, bona fide reports demonstrate intracellular synthesis
of chitin in fungi, protozoa and invertebrates [1-3], and that UDPGlcNAc
is not compartmentalized, but accessible to the enzyme in the cytosol [4].
Chitin synthetase activity may be detected in almost all membranous
fractions from fungal homogenates, but not in the cytosol. Comparison of
the predicted amino acid sequences of all chitin synthetase genes cloned
thus far demonstrates the existence of a hydrophobie domain loeated
towards their C-termini [5], which agrees with a membranous location of
the enzymes. Nevertheless, location of enzymatic activity in the different
membranous fraetions may be considered to be due to an artifaetual aggregation of enzyme-carrying membranes with other particulate fraetions,
including the wall.
Evidence for this has been described in the fungus Mucor rouxii, where
separation of cell-free extracts by isopycnic sedimentation revealed that the
low-density membranes containing chitin synthetase aetivity eould beeome
aggregated with membranous fraetions of higher density depending on the
buffer used [6]. In this sense, the description of contradictory results on the
loeation of chitin synthetase in homogenates from the yeast Saccharomyces cerevisiae, either plasmalemma [7] or mierovesicles [8,9], depending
on the method of eell breakage, is relevant. Further results have settled this
issue in adefinite way, demonstrating the intraeellular location of chitin
synthetase in fungi. These include (i) findings from electron-mieroscopie
autoradiography that chitin synthesis by permeabilized cells of M. rouxii
oceurs in the cytoplasm, and not in the plasmalemma [10]; (ii) studies
which reveal, by eleetron-microseopie immunoassays, the loealization of
chitin synthetase in apical vesicles (ehitosomes) of Neurospora crassa
hyphae [11]; and (iii) the immunolocalization of chitin synthetase 3 in
whole cells of S. cerevisiae in a fraetion eorresponding probably to the
Golgi, and not in the plasmalemma [12].
In arthropods, eonelusive evidence supports the location of the enzyme
in the Golgi complex, from where it is transferred to the eell surface in the
form of vesicles [1], whereas in the protozoan Eufolliculina uhligi chitin
synthetase is aceumulated in cortieal vesicles. Aecording to all these data,
it may be concluded that, as already suggested several years aga [13, 14],
chitin synthetase follows the exocytie route of membrane-bound proteins,
accumulating in eytoplasmie vesicles during its transit to the eell surface.
Its relative accumulation in a vesieular form and in its target membrane
may explain part of the eonflieting data existing in the early literature.
Chitin biosynthesis and structural organization in vivo
41
Cellular location of chitin synthetases and their transport to the sites

of chitin biosynthesis: the fungal chitosome
As described above, in arthropods and protozoa chitin synthetase is accumulated in the Golgi apparatus and specialized vesicles. In fungi, massive data have accumulated indicating that the structures where chitin
synthetase is accumulated in the cytosol are a kind of specialized microvesicles-denominated chitosome [3, 13, 14]. Chitosomes have been purified to a high degree from fungi representatives from the main taxa,
demonstrating that they contain over 80% of the total chitin synthetase
from the cells. These yields, the fact that harsh or mild cell breakage
procedures all gave rise to identical structures, and their chemical and
enzymatic uniqueness (see below) all silenced initial criticism suggesting
that chitosomes might be artifacts ofthe vesiculation oflarger membranes.
Purified chitosomes appear in the form of spheroidal structures
measuring 40-70 nm in diameter (Fig. lA, B). In sections they appear surrounded by a thin membrane 6.5-7.0 nm thick [15] (Fig. 1 C). Chitosomes
are made of protein and lipids in a ratio of 1.4-2.0, polar lipids being more
abundant than neutraiones [16, 17]. Among polar lipids, phosphatidylcholine and phosphatidylserine were the most prominent ones, whereas
sterols were the most abundant neutral lipids. This composition was quantitative and qualitatively different from the gross of the cell membranes.
The most relevant differences were the abundance of ergosterol and glycolipids, and the absence of phosphatidylserine in chitosomal membranes
[16-18]. Protein composition of chitosomes is also unique [19]. Purified
chitosomes incubated with UDPGlcNAc and activators synthesize very
fine microfibrils which crystallize in their interior, appearing either coiled,
kinked or straight, and eventually breaking apart the chitosomal membrane
[15] (Fig. ID, E). Similar images were obtained during electron microscopic studies of chitin microfibril synthesis by homogenates from the flour
beetle Tribolium castaneum [20], extending the fungal results to arthropods. The results reveal that chitosomes synthesize chitin microfibrils
through an asymmetrie mechanism, that is accepting GlcNAc residues
from UDPGlcNAc at the cytosolic face, and de1ivering chitin moleeules at
the inner face (see Fig. 2).
Treatment of chitosomes with digitonin gave rise to their dissociation in the
form of 16S subunits with an M r ca. 500 kDa, which conserved enzymatic
activity [21]. Chitosomal subunits are enriched in a few polypeptides, suggesting that the catalytic polypeptide is active in the form of a multiproteic
complex [22]. 16S chitosomal subunits synthesize extreme1y fine chitin
microfibrils, whose width has been calculated to correspond to the association
of two GlcNAc chains [23]. These microfibrils associate at later periods of the
synthetic reaction in the form ofvery thick and short microfibrils [24].
All these data helped to identify chitosomes as the conveyors responsible
for the transport of chitin synthetase to the cell surface. Nevertheless,
42
1. Ruiz-Herrera and A. O. Martinez-Espinoza
Figure I. Fungal chitosomes, and chitin synthesis in vitro. (A) Negatively stained (uranyl
acetate) chitosomes isolated from Mucor rouxii. (B) Negatively stained chitosomes from
Saccharomyces cerevisiae. (C) Ultrathin section of chitosomes from M rouxii. (0) Microfibrils
made after incubation of S. cerevisiae chitosomes with substrate and activators. (E) same as
(0), showing a microfibril bundle crystallized inside a chitosome. Magnification bars: A, B, E,
100 nm; C, 50 nm; 0, 25 nm.
whether the chitin biosynthetic process in whole cell occurs during the
transit of the chitosomes to the surface, or once the chitosomes fuse with
the plasmalemma, is unknown (Fig. 2). In this sense it is important to indicate that in crustaceans and protozoa evidence exists that synthesis of
chito-oligomers associated with proteins occurs intracellularly, these complexes being secreted in a postsynthetic reaction [1, 2].
Although the origin of chitosomes is uncertain, it has been reported that
chitin synthetase is made in the endoplasmic reticulum (ER) and follows
the normal exocytic route to the outer surface of the cell in S. cerevisiae
43
+ Protease, Mg'+, GIcNAc, UOPGlcNAc
UDP
E~
0-
~
1
n.....
.......Q
0
0
--=<i
CH,OH
NHCOCH,
CH,oH
H~O~O---------- ----------------- ----O~OH

NHCOCH, CH,OH
NHCOCH,
NHCOCH,
H~
b~o
CH,OH
NHCOCH,
OH
0 _____ - - - - - - - - - - - - - - - - - - - - - - - - - - . 0
NHCOCH,
-~
'O~H
CH,oH
Figure 2. Scheme representing chitin synthesis by chitosomes and other chitin-synthesizing

microvesic\es in vivo and in vitro. (A) Schematic representation of transmembrane chitin
synthetase in the microvesic\es. (B) Intrusion of chitin chains into the microvesicle. (C) Representation of the vectorial mechanism involved in (B). (D, E) Alternate pathways of chitin
synthesis in vivo. In (D), chitin chains are made inside microvesic\es. In (E), chains are
synthesized and extruded at the surface of the plasmalemma. Notice that chains contain
reducing and nonreducing ends represented by open rectangles or filled circ\es. (F) Arrangement of chains in antiparallel fashion. (0) Crystallization of chitin microfibrils in the -configuration.
44
1. Ruiz-Herrera and A. D. Martinez-Espinoza
[12]. Regarding the mechanisms involved in chitin synthetase traffic and

delivery, it is probable that we may apply the same basic mechanisms
described for the vesicle transit in general [24]. Nevertheless, some
reasonable doubts exist for these generalizations. First, chitin synthetase
amino acid sequences deduced from cloned chs genes lack a canonical
signal peptide [5]; second, chitosomes have no adenosinetriphosphatase
(ATPase) activity, a universal plasmalemmal enzyme; third, chitosomes
have no additional enzymatic activities besides chitin synthetase [6]; and
fourth, the polypeptidic composition of chitosomes is unique [19].
Chitin crystallization and physicochemical characteristics
Although the mechanisms for the deployment of chitin in the exocellular

structures appears to be different in the several organisms analyzed, all of
them finally lead to its crystallization in the form of microfibrils. In fungi,
chitin microfibrils are usually made of 20-400 sugar chains associated
through hydrogen bonding, whereas in other organisms the size and
packing of the chains is more variable. Also variable is the association of
chitin with other components. In fungi, no evidence for an association of
chitin with proteins during the biosynthetic process has been described, and
it is assumed that sugar chains are extruded to the cell wall space. In
animals and protozoa, different mechanisms appear to operate. In insects
chito-oligomers are made intracellularly, and then transported across the
plasma membrane to the extracellular space where they become associated
with lectin-type proteins [25, 26]. In crustaceans, chitin appears to be
synthesized in Golgi cistemae associated with proteins which are secreted
further on [1]; whereas in ciliates similar chitoproteins are secreted through
an exocytic process involving cortical vesicles [2].
In contrast to cellulose, where two different crystalline forms exist, only
one being present in nature, three different crystalline polymorphie forms
of chitin exist under natural conditions. These forms differ in the packing
and polarity of the GlcNAc chains. The most abundant, a chitin, where
chains are antiparallel, is present in arthropods, fungi and the cysts of
Entamoeba (review in [27, 28]); chitin, made ofparallel chains, has been
identified in the pen of the squid Loligo, chaetae from Aphrodite, tubes of
pogonophores, loricae from some protozoans and the spines of several
diatoms. In the third form, y chitin, two chains would run in one direction,
and another, in the opposite direction. This form has been reported in
cocoon fibers ofthe beetle Ptinus and the stornach of Loligo, and is not fullyknown.
According to data from Blackwell et al. [29], both a and chitins consist of arrays of sheets or chains stabilized by C = o .. H - N bonds, as weIl
as by nonbonding interactions between the pyranose rings, except for the
absence of bonding between the sheets in chitin. This characteristic
45
explains the introduction of water molecules in the crystals of chitin,

leading to its swelling. Unit cell type and dimensions of both chitins are
very different, leading to their distinct X-ray difIraction patterns. The
reason why chitin can crystallize in three different forms remains unknown,
but it is possible that the process is affected by the presence of some
compounds during crystallization of the polymer, since solvation of or y
chitins in HCI or lithium thiocyanate solutions, respectively, reverts them
to a chitin [30]. Nevertheless the phenomenon has several consequences.
First, the mechanical properties of the three forms are different, probably
coadjuvating to their different roles. Second, the fact that in two forms the
sugar chains are antiparallel indicates that the processes of synthesis and
assembly into microfibrils cannot be simultaneous and must be separated
in time (Fig. 2E-G).
Analysis ofthe tensile mechanical properties ofthe three forms of chitin
revealed that all of them behaved as viscoe1astic polymers, but varied in
stiffness, strength and extensibility by several orders of magnitude, their
properties being sensitive to wetting [30]. a Chitin was the stiffest and
displayed the highest maximum tensile strength. Oue to its insolubility
chitin hardly yields to the normal procedures for molecular weight
determination. Through determination of the intrinsic viscosity values of
solutions of chitin in N,N-dimethylacetamide-5 % LiCI, molecular weights
ranging from 0.8 to 1.8 x 10 6 were calculated for different crustacean
chitins [31]. U sing a different approach, that is differential labelling of
chitin and their terminal residues in a synthetic reaction, we calculated that
chitin molecules of M rouxii are made of over 2000 sugar residues [32].
The idea that synthesis and crystallization of chitin are nonsimultaneous
processes rests on different pieces of evidence. In vivo crystallization, but
not synthesis of chitin microfibrils, is interfered by Calcofluor white and
Congo red in algae [33], ciliates [34] and fungi [35, 36]. The polymer thus
synthesized is very sensitive to chitinase and not crystalline, but becomes
resistant and crystalline when it is dried [37]. The nascent chitin, but not
the preformed one, is very sensitive to chitinase [38, 39] and chitin
deacetylase [32, 40]. All these data reveal the existence of a gap between
chitin polymerization and essembly.
Chemical modifications of chitin in the extraceUular space

Chitin crystallization in the form of microfibrils, its modifications and its
association with other compounds occurs outside the permeability barrier
of the cell, posing the problem of how the energy for the reactions involved
is made available to the enzymes responsible for the processes. Nascent
chitin can be exposed to several chemical modifications. Some of them
change different and important properties ofthe polysaccharide, and therefore the skeletal structures, such as viscoelasticity, reactivity and resistance
46
to the harmful conditions of the medium [3]. One of these modifications,

chitinolysis, is of vital importance, since it is necessary for the expansion
of the cell walls and exoskeletons; otherwise the organisms would remain
encased into rigid structures. Accordingly, this important modification
involves the partial and careful degradation of chitin by chitinases [41, 42],
which weaken chitin in an ordered way and facilitate expansion. An
example is Mucor spp. where spore germination is blocked by allosamidin,
a specific inhibitor of chitinases [42]. In fungi, cell wall expansion utilizes
the turgor pressure exerted by the protoplasm as the driving force [43]. It
has been also shown that chitinases have a transglycosidase activity, which
may playa role in the linkage of chitin with glucan, thus participating in
the macromolecular arrangement of the wall [44]. Another role of
chitinases is cell division; in S. cerevisiae it has been suggested that
chitinases playa role in the separation of daughter and mother cells after
budding [45].
Chitosan has been found in several insect species such as Oecophila
longinoda, Anax inmaculifrons and Apis cerana indica; crustaceans including Lepas sp., Sacculina rotundata and Neptunes sanguinolentus; and
some Arachnida and Myriapoda [46]. Chitosan is also present in a variety
of fungi. It can account for up to 30% of the vegetative cell wall of some
zygomycetes, constituting a characteristic component of the cell walls of
this fungal group. Chitosan was originally reported in the walls of the
sporangiophores and mycelium of Phycomyces blakesleeanus [47], and
later on in the cell walls ofthe yeast and mycelial forms of M. rouxii [48].
It is also present in the ascospore walls of S. cerevisiae [49], and in
vegetative walls of Schizosaccharomyces pombe [50].
Substantial evidence exists indicating that chitosan biosynthesis occurs
by the deacetylation of chitin, rather than by de novo biosynthesis involving
the action of a specific polymerase. No possible nucleotide precursors
made of glucosamine, but only UDPGlcNAc has been detected in the
cytosol of fungi [40]. Deacetylation of chitin probably occurs in its nascent
state. Presumably due to the insolubility of preformed chitin, the latter is
a poor substrate of chitin deacetylase. An enzyme able to deacetylate
soluble derivatives ofchitin was found by Araki and Ito [51] in Mucor. This
enzyme was later on found to lead to the synthesis of chitosan when cellfree extracts of M. rouxii containing a mixture of chitin synthetase and
chitin deacetylase were incubated with UDPGlcNAc [40]. Evidence for the
participation in the process of a particulate, rather than a soluble form of
the deacetylase, has been obtained [32]. Chitin deacetylases with M r
ranging in the order of 35-42 kDa have also been described in Arthropoda
[46], indicating the universality of the mechanism for chitosan biosynthesis. The gene coding for chitin deacetylase from M. rouxii has been
cloned [52], and its sequence revealed strong similarities to several
rhizobial nodB genes. It was suggested that the gene encodes for a
peptidoglycan deacetylase.
47
That deacetylation of chitin is the process for chitosan synthesis in viva

is supported by two lines of evidence. Disruptants in the gene coding
for chitin synthetase 3 from S. cerevisiae lack chitosan, indicating that
the compound is indeed made through the deacetylation of chitin [53]. In
P. blakesleeanus it was observed that fluorescent wheat germ lectin
(specific for GlcNAc) bound only to the tip of stage I sporangiophores.
However, when they were treated with a substance that degrades chitosan
(HN0 2 ), the lectin bound to the whole sporangiophore. This results suggests that nascent chitin synthesized at the apex becomes deacetylated to
chitosan as it moves to the lateral walls of the sporangiophore, where it
covers the chitin microfibrils [54]. Due to its physical properties (easy
hydration), it is thought that chitosan does not playa structural role, but
nevertheless it has significant biological importance. Removal ofN acetyl
groups makes a polysaccharide resistant to chitinases. In this way covering
ofthe chitin microfibrils with chitosan protects the integrity ofthe cell wall
[32, 55]. Chitosanases are less abundant than chitinases in normal habitats.
Also important are the mechanical properties of chitosan, which make it
appropriate as a cementing compound; its ability to neutralize anionic
charges; and perhaps its capacity to accumulate important ions within the
wall. In arthropods, chitosan formation allows stretching of the cutic1e,
inc1uding extreme cases, as in the physogastric queens oftermites [46].
Association of chitin with other components
Once in the extracellular space, chitin may associate chemically with other
components of the cell walls or exoskeletons, thus acquiring different
properties. Association of chitin with proteins has been described in a large
number of invertebrates (reviewed in [56]). Proteins form covalent links
with chitin, giving rise to structures with higher strength, but pliable and
flexible. In insects these properties allow movement and limit expansion
[41]. Proteins then "harden" or sc1erotize. This reaction is very important; normal functions of insect integument depend on sc1erotization.
Sc1erotization involves the formation of adducts of chitin and proteins with
the oxidation products of diphenolic substrates. Of these, the most important ones appear to be N-acetyldopamine and N--alanyldopamine
(Fig.3). Through oxidation, these products form reactive a-quinone or
p-quinonemethide derivatives which cross-link proteins and chitin via
Michael-type conjugate addition and Schiff's base formation with free
amino or OH- groups. Neverthe1ess in viva and in vitra data obtained by
double label revealed that the most important associations between
phenolic compounds and chitin occur through noncovalent bonds, which in
the case ofinsects stabilize the sc1erotized cutic1e [56a].
The resulting chitin-protein complexes gain stability, providing hardness
and rigidity, and can associate with additional substances such as lipo-
48
1. Ruiz-Herrera and A. D. Martinez-Espinoza
0
11
HO
'-'::::
HO
NH-C-CH 3
0
11
HO
'-'::::
HO
NH-C-CH 2-CH 2-NH 2
Figure 3. Two phenolic compounds involved in sc1erotization of insect integument. (A) Nacetyldopamine. (B) N--alanyldopamine.
proteins and waxes, which provide impermeability properties to the

exoskeletons. These associations can also be found in the cyst walls of
Rhizopoda and Ciliata [57], the shells of inarticulate Brachiopoda and
Mollusca, as well as the envelopes of eggs, cysts and masticatory organs of
different invertebrates [41]. Chitin can also associate with the proteins
called arthropodins or sclerotins to create stable glycoprotein conjugates,
probably through aspartyl or histidyl residues [57]. Chitin-protein aggregates provide a substrate for calcium and silica deposition. This
mineralization process seems to be common in crustaceans and mollusks,
where it provides rigidity and ensures stability to the exoeskeletons [41,
58]. Chitin also forms conjugates with carotenoids, giving color to tissues
in insects and crustaceans [41].
In fungi, evidence exists that chitin also associates with glycoproteins. A
substantial number of wall proteins are resistant to extraction with
detergents and can be rendered soluble only after treatment with chitinase
[59]. In Ustilago maydis, 30% ofthe protein was extracted by the action of
hydrolases [60]. It has been suggested that mannoproteins regulate the
formation of cross-links between polymers [61]. Reports exist which
document the association of chitin with other components inc1uding polysaccharides, such as galactomannan [41] and glucans [62]. These associations occur through different covalent linkages, for example with the
involvement of basic amino acids [62]; a linkage involving the glucosereducing end of -l,6 glucans and the C6 of GlcNAc [63]; and a -l,4
linkage between the reducing end of GlcNAc and the nonreducing end of
-l,3-glucan [64]. These associations transform formerly soluble glucans
into insoluble derivatives [65]. In Zygomycetes which do not contain
49
glucans in their cell walls (except for the spores), chitin associates with
chitosan, and forms interactions with other polymers such as polyuronides
[55,65].
Organization of chitin in the form of coherent composites
Cell walls and exoskeletons are not made by the simple and disordered
aggregation of different chemical compounds. They are in fact coherent
structures, true composites, light and resistant with specific structural and
mechanical properties which allow them to fulfill their protective role [66].
They adopt specific forms, some of them extreme1y elaborate, which are
conserved during the growth ofthe organism and are transmitted in a hereditary fashion. Elaboration of the supramolecular structure of these composites involves the association of their components in an orderly fashion
with the formation of a three-dimensional complex structure. Since chitin
is the main skeletal component of these structures, it is easy to understand
that its association with other chemical components is of great importance.
As described, chitin in the form of microfibrils is immersed in a matrix of
proteins and other polysaccharides [3, 59]. The resulting structures behave
like composites, the chitin microfibrils providing them with high strength
to resist tensions and modulus (Fig. 4). The cementing compounds protect
chitin from chemical attack; keep the microfibrils separate, preventing
fracture; and provide support to tensions.
Although chitin is not the most abundant component in the cell walls and
exoskeletons, removal of the rest of the chemical components leaves a
ghost that retains the original shape, whereas chitin digestion originates the
complete destruction of the structures. Accordingly, it may be suggested
that chitin serves as the organizer or the anchor, around which protective
structures are made. In fungal protoplasts, regeneration starts with the
deposition of chitin microfibrils around the cell, upon which proteins start
to be deposited [59]. Similarly, lorica formation by ciliates involves primarily the association of chitin microfibrils into a structure over which other
components are deposited later on.
We can summarize the steps giving rise to either cell walls or exoskeletons as follows: (i) Chitin molecules are synthesized either intracellularly
or at the interphase with the extracellular medium. (ii) Chitin molecules are
transported to the extracellular space. (iii) In its noncrystalline stage, part
of the chitin is the substrate for important modifications, and associates
with other molecules. The resulting supramolecular structure acquires
viscoelastic mechanical properties. (iv) The unmodified chitin crystallizes
and is covered by the rest of the components. The resulting structure
matures into a composite which provides protection to the organism.
50
1. Ruiz-Herrera and A. O. Martinez-Espinoza
Figure 4. Chitin in the cell walls of fungi and cyst wall of amoeba. (A) Section of a gerrninating
spore of M . rouxii. Chitin was stained with colloidal gold-tagged wheat germ lectin. (8) Tangential section of a germinating spore of M . rouxii showing the microfibrillar arrangement of
chitin in the wall. (C) Positive replica ofthe cyst wall from Entamoeba invadens. Alkali-treated
wall shows the presence ofrandomly-oriented chitin microfibrils on the outer surface. (0) Fragment of isolated Neurospora crassa cell wall stained with ferritin-labelled wheat germ lectin.
Magnification bar, 100 nm.
Acknowledgments
Original work from the authors was partially supported by Consejo Nacional de Ciencia y
Tecnologia and CONCYTEG, Mexico. 1. R. H. and A. o. M. E. are National Investigators. 1. R. H.
is member ofthe Centro lnternacional de Ciencias, Cuernavaca, Mexico.
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Cambridge, I-li

ed. by r Jolles and R... Muzzarelli
Chitin synthases in yeast and fungi

M. Henar Valdivieso, Angel Dunin and Cesar Roncero
Instituto de Microbiologia Bioquimica, Consejo Superior de Investigaciones Cientificasl
Universidad de Salamanca and Departamento de Microbiologia y Gem!!tica,
Universidad de Salamanca, E-37007 Salamanca, Spain
Summary. The polysaccharide chitin is an important structural component of the cell walls of
many fungi. Chitin synthesis is directly governed by an enzymatic activity called chitin synthase
(CS). The use ofthe budding yeast Saccharomyces cerevisiae as a biological model allowed the
identification of three distinct chitin synthase activities: CSI, involved in repair functions at the
end of cytokinesis; CSII, responsible for the synthesis of the primary septum that separates
mother and daughter cells; and CSIII, responsible for the formation ofthe ring (bud scar) where
most ofthe cell wall chitin is located. These chitin synthases differ not only in functions but also
in catalytic properties. The catalytic subunit of each of these activities is encoded by separated
genes, CHSI, CHS2 and CHS3, respectively, although it has been shown in S. cerevisiae that
CSIII activity also depends on the products of other genes. To date, several chitin synthase
(CHS) genes have been also identified in other fungi; most of them are similar to ScCHS 1 and
ScCHS2 genes and are c1assified in chitin synthases c1asses I, 11 and III in terms of sequence
similarity. The rest are defined as two CHS classes, IV and V, highly similar to ScCHS3. While
CHS c1ass V genes have been only identified in filamentous fungi and their functions are
unknown, class IV genes, which inc1udes ScCHS3, are involvcd in the synthesis ofmost chitin
in yeast cells.
General aspects of fungal chitin

Chitin synthesis has been described in several systems, but the fungal cell
wall - particularly the yeast cell wall - is the most extensively studied.
Chitin is a minor but essential structural polymer of the wall of Saccharomyces cerevisiae vegetative cells. It is mainly located in a ring that
constitutes the neck between the mother and the growing daughter budo At
cytokinesis, a new deposition of chitin occurs in a centripetal fashion in the
furrow of the invaginating plasma membrane. In this way, a thin disklike
structure, the so-called primary septum, is formed, and this closes the gap
between mother and daughter cells. Finally, the two cells separate in an
asymmetrie al fashion so that most of the chitin remains in the mother cell
in a structure called the bud scar, while a less conspicuous structure - the
birth scar - remains on the surface of the daughter cell (for a review see
[1]). Cell separation requires partial degradation ofthe chitin remaining in
the mother cell by the action of a periplasmic acidic chitinase. Chitin, although in a very small percentage of its total amount, is also randomly
located around the wall ofthe mother cell, forming part of (1,3)glucanchitin [2] and (1,6)glucan-chitin [2] covalently bound complexes, which
are important for the maintenance of wall integrity.
56
M. H. Valdivieso et al.
Chitin synthesis also takes place in other events of the S. cerevisiae life
cyc1e, that is mating and sporulation. During conjugation, haploid cells of
complementary mating types form projections (shmoos) toward each other
under the stimulus of the corresponding sexual pheromone. During this
process, chitin is synthesised and is deposited mainly in the subapical
portion of the shmoo [3]. Finally, during sporulation a layer of chitosan is
formed in the ascospore walls [4]; this forms a complex with a dityrosinerich layer that is responsible for the resistance of the ascospore.
Chitin synthases
Chitin synthases use uridine-diphospho-N-acetylglucosamine (UDPG1cNAc)
as substrate and catalyse the reaction 2n UDPG1cNAc ~ [G1cNAc--l,4G1cNAc]n + 2n UDP. The in vitro activity of chitin synthase from many
fungi is stimulated by controlled pretreatment of the enzyme preparation
with a protease, even after its solubilisation with detergents. This is why
this activity has been traditionally considered as zymogenic [1]. Chitin
synthase activity is located at the plasma membrane [5] where chitin synthesis occurs, although it is also present in intracellular vesic1es called "chitosomes" that have been proposed to deliver chitin synthase to the plasma
membrane [6]. Chitin synthesis occurs as a transmembrane process in a
vectorial way in which the substrate is located on the intracellular side and
the product is extruded to the outside of the membrane, where the polysaccharide is located. The chitin synthesised in vivo and in vitro is quite
uniform in size, between 120 and 170 G1cNAc units in length [7].
Until 1986, it was thought that only one chitin synthase activity exists in
yeast. The work ofBulawa et al. [8] described a mutant defective in in vitro
zymogenic chitin synthase activity that had neither a growth defect nor a
reduced rate of chitin synthesis. Obviously, another chitin synthase activity
had to be responsible for in vivo chitin synthesis. In cell extracts from chsl
strains two different chitin snythase activities called "chitin synthase 2" [9]
and "chitin synthase 11" [7], respectively, were detected independently.
Later on (see below), the existence of two different genes - CHS2 and
CHS3 - that were indeed responsible for these two new activities was
reported: chitin synthase 11 (CSII) [10] and chitin synthase III (CSIII) [11,
12]. For more extensive reviews on yeast chitin synthesis see [13, 14].
The three CSs described so far in S. cerevisiae differ significantly with
respect to their zymogenic behaviour, cation dependence and optimum pH.
In addition, differential inhibition of these CSs by polyoxin D and nikkomycins, very specific chitin synthase competitive inhibitors, has been
described (see below for further details).
57
Chitin synthase I
The S. cerevisiae CHSI gene encodes a protein of 130 kD and constitutes
the structural gene for chitin synthase I (CSI) (Tab. 1). After the gene
(CHSI) had been cloned, its disruption demonstrated that it was not involved in chitin synthesis in vivo and that it was not an essential gene.
When grown on complex media, the growth rates of the wild-type and
isogenic chsl were indistinguishable. However, when chsl cells were
grown in minimal medium (more precisely, in poorly buffered media),
small refractile buds were produced [8]. When observed under electron
microscopy, these lysed buds were seen to have a hole located in the centre
of the birth scar [15]. This phenotype could be prevented by buffering the
growth medium, or by adding sorbitol as an osmotic stabiliser. Addition to
the medium ofthe chitinase inhibitor allosamidin or disruption ofthe CTSI
gene, which codes for an acidic chitinase [16], also al1eviated the phenotype. These data point to the notion that Chs 1p counterbalances the
physiological lytic effect that chitinase exerts at the time of cytokinesis to
separate mother and daughter cells. However, the possibility that CSI might
be activated by acidic culture conditions cannot be ruled out, and hence in
its absence septum formation may be impaired during cell separation.
Some ehs1strains do not show lysis in acidic medium. Therefore, in addition to CSTI the product of at least another unknown gene (named SCSI
for suppressor of chitin synthesis) must be involved in this phenotype [16].
Treatment of S. cerevisiae .!! cells with the complementary pheromone
(a-factor) promotes a three- to fourfold increase in chitin content [3].
CHSI has four pheromone response elements in the upstream portion of
the gene, which confer inducibility on CHSI transcription. Indeed, upon
a-factor treatment the levels of CHSI messenger RNA (mRNA) rise
rapidly and considerably although transiently [17, 18]. However, CHSI is
not responsible for chitin synthesis during mating [19]. The CHSI gene
may playa role in mating, perhaps as arepair enzyme during the wall lysis
that must occur for the fusion of mating cells. However, the efficiency of
zygote formation in crosses in which the two complementary strains were
chsl was identical to that found in the control experiment [20].
CSI is the major CS activity detected in vitra (it accounts for more than
90%). CSI levels and the product of CHSI (Chslp) remain constant during
vegetative growth in synchronised cells [18,21]. It therefore seems that this
activity is not regulated by synthesis and degradation. Chs 1p resides partly
in the plasma membrane and partly in the chitosomes. It has been shown
that chitosome production is blocked in an endocytosis mutant (end4-I).
This and other results raise the possibility that the temporal and spatial
regulation of chitin synthesis carried out by CSI may be mediated by the
mobilisation of an endosomal pool of CHSI enzyme [21].
Budding neck ring and lateral wall Glycoprotein; posttranslationally

in vegetative cells; in mating;
regulated
ascospore chitosan layer
Transcription induced during sporulation [22]

Required for Chs3p localization;
required for fusion in mating
As above but not the ascospore

chitosan layer
Ascospore chitosan layer?
As in CHS3 but not the
ascospore chitosan layer
Not determined
Asin CHS3
Catalytic component
of chitin synthase III
Required for chitin synthase III
Related to CHS4
Required for chitin synthase III?
No
No
No
No
No
No
CHS4
SHCl
CHS5
CHS6
CHS7
Glycoprotein; transcription activated in

mating, sporulation and by calcofluor
Posttranslational activator of chitin

synthase III; required for proper Chs3p
localization
[lA. Trilla, unp.]
[22]
[20,55]
[22, 54, 56]
[12,18,21,22,27,
28]
[10, 18,25,28]
CHS3
Regulated transcriptionally and

posttranslationally
Structural gene for chitin synthase 11 Primary septum
No
[8,15,17,18,21]
Transcription activated by mating factor
CHS2
In cytokinesis (repair function)
References
Other comments
Structural gene for chitin synthase I
Time or place of chitin synthesis
No
Essential Proposed enzymatic function
CHSI
Gene
Table 1. Genes involved in yeast cell wall chitin synthesis
v.
g.
~
'"o
f:
;:r:
00
59
Chitin synthase 2
A chitin synthase II (CSII) activity, whose levels are approximately 5% of
those of CSI, was identified in chsl.t1 mutants [9]. CSII shares some
properties with CSI: it is located at the plasma membrane and it is
stimulated in vitro by N-acetylglucosamine and by partial proteolysis.
However, CSI and CSII differ in cation specificity and in their optimum pH
values. CSII proved to be more sensitive to polyoxin D and NaCI than CSI,
which permitted detection of this new activity in extracts from wild-type
cells[9].
Since no chs2 mutant was available, the CHS2 gene was cloned by
detecting an increase in the CS activity of chsl.t1 cells transformed with a
multicopy-plasmid genomic library [10]. As was the case for CHSI, overexpression of CHS2 in Schizosaccharomyces pombe led to the expression
of a CS activity with characteristics similar to those of the activity observed
in Saccharomyces cerevisiae.
The CHS2 gene was sequenced and compared with CHSI. The predicted
amino acid sequence of Chs2 was very similar to that of Chs 1 (42%
identity in the carboxyl two-thirds of the protein, reviewed in [22]). It has
been suggested that the similar regions are related to common cata1ytic
functions, whi1e the amino-terminal regions may be invo1ved in regulation
or localisation of the respective enzymes. However, for both synthases
most or all ofthe nonhomologous region can be deleted with little effect on
their enzymatic activity in vitro or on their function in vivo, indicating that
regulation of these truncated proteins has been properly achieved. On the
other hand, small deletions in the homologous regions are deleterious to
enzymatic activity and function [23, 24]. Thus, it appears that all the information required for membrane localisation, enzymatic activity and
function resides in the homologous regions of CSI and CSII.
CHS2 was originally reported to be an essential gene [10]. However,
subsequent work demonstrated that the viability of chs2L1 spores ranges
from 0 to 90%, depending on the strain background and on the germination media used [11, 25, 26]. Disruption of CHS2 produces several
interesting phenotypes. Initial observations indicated that no defined
primary septa could be observed in strains lacking a functional CHS2 gene
[10]. Instead, thick amorphous septa are formed at the neck region between
mother and daughter cells, and chitin is overproduced in up to twofold
amounts in comparison with the wild-type level [11,25]. In addition, chs2.t1
cells aggregate in clumps, are misshapen and abnormally large, contain
large vacuoles and in some cases are multinucleated [25]. The conclusion
is that the CHS2 gene must be involved in primary septum formation and,
perhaps, also in some related final step of cytokinesis.
The fact that CSI and CSII perform the same biochemical reaction in
vitro but seem to have different roles in vivo suggests that these enzymes
must be strict1y regulated. As mentioned above, CSII is involved in the
60
formation of the primary septum (Tab. 1); therefore, it must be active immediately before cytokinesis. This temporal regulation seems to be
achieved by transcriptional and posttranscriptional mechanisms. CHS2
mRNA, Chs2 protein and the level of CSII activity are cell cycle-regulated, peaking at the time of septation [18, 27, 28]. Furthermore, CHS2
transcription and CSII activity have been shown to be abolished in pheromone-treated, sporulating and stationary-phase cells [18, 22]. It is therefore
concluded that CSII is only present in actively growing cells. Furthermore,
Chs2 protein has a very rapid turnover rate [18, 28], in agreement with a
cyclic synthesis-degradation mechanism of control. Chs2p degradation
seems to occur at the vacuole [28].
As well as this temporal regulation, CSII must be spatially regulated so
that it will only be functional at the mother-bud neck. Chs2p localises to the
neck only at the end of mitosis [28]. It is not known what mechanism
restricts the localisation of Chs2p at this part of the membrane, although it
has been suggested that the 10-nm neck filaments could be involved in this
function, since mutants in neck filaments are able to form septumlike
structures at abnormallocations [29].
All the above results argue against the previously proposed hypothesis
that suggested that chitin synthases would be uniformly distributed throughout the membrane and would be proteolytically activated when and where
required [1]. It is important to keep in mind that for CSII no physiological
activator has yet been identified and that there is no evidence suggesting
the conversion from a zymogenic precursor to the active form in vivo.
CRS1 and CHS2 homologues in other fungi
Using degenerate oligonucleotides corresponding to conserved sequences

of CHSl, CHS2 and Candida albicans CHSI genes, polymerase chain
reaction (PCR) techniques were employed to analyse genomic DNA from
taxonomically different fungal species. Most species had several CS gene
homologues, which were included in three different classes according to
the presence of some signature domains [30]. It has been suggested that
these classes are taxonomically relevant. However, whether the genes of
each group have similar functions in vivo remains to be elucidated. Two
short conserved sequences (Gln-Asp-Arg and Gln-Arg-Arg-Arg-Trp) are
present in all chitin synthases. These two domains have been shown to be
essential for CS activity in the case ofthe Chs2 enzyme [24, 31].
The C. albicans CaCHSl gene was cloned by complementation of the
S. cerevisiae chsl mutant [32], but its sequence was seen to be more similar
to that ofthe SeCHS2 gene, and it has been proposed to be essential [33];
therefore, it is likely to be responsible for septum formation [34]. In fact, a
recently cloned isoform ofthis enzyme is able to complement the morphologic defect of an S. cerevisiae mutant [35]. The CaCHS2 gene, which was
61
eloned by PCR and whose sequenee is very elose to that of the S. cerevisiae
CHSl gene, is indueed at the yeast-mycelium transition [36]. The Cachs2A.
strain grows normally, has a 20-50% reduction in chitin synthesis in
hyphae, shows a Calcofluor staining pattern similar to that ofthe wild-type
strain [33] and does not have reduced virulence [34]. In Ustilago maydis,
also a dimorphie fungus, two CS genes (Umchsl and Umchs2) were eloned
by PCR. Both genes are expressed in the yeast and the mycelium phases.
Their deletion leads to a reduction in growth rates, CS activity levels and
the chitin content of the mycelial cells. However, mating, virulence and
dimorphie behaviour are not affected in the deletant strains [37, 38].
Several CS genes have been eloned in the filamentous fungus Aspergillus nidulans. chsAA. mutants grow as weIl as the wild-type [39], but chsAA.
chsDA. double mutants displaya remarkable deerease in the effieiency of
conidia formation [40], implicating these genes in this process. The
AnCHSB gene encodes a chitin synthase activity that requires Mg ++, is
inhibited by polyoxin D and is activated after trypsin treatment when overexpressed in S. cerevisiae [41]. Deletion of this gene produces severe
growth defects that eannot be remedied by the presence of sorbitol [39].
Colonies are minute, do not conidiate, and display a strong degree of
branching and disorganised lateral walls. However, the mycelia are not
deficient in chitin synthesis. Therefore, the AnCHSB-encoded enzyme
does not contribute to the rigidity of the cell wall, but it is necessary for
normal hyphal growth and organisation [42]. These data made AnCHSB a
eandidate for the functional homologue of SeCHS2. The AnCHSC gene is
expressed in the mycelium, but its absence has no effeet on morphology or
growth rates in comparison with those ofthe wild-type [43]. Similar results
have been obtained in A. fumigatus, although the gene nomenclature used
is slightly different [44-46].
In other filamentous fungi, several chitin synthase genes have been isolated,
but only in a few cases has some in vivo function been assigned. Neurospora
crassa chs-l A. mutants grow slowly, have aberrant hyphae and reduced CS
activity [47]. However, chs-2A. mutants have normal morphology even though
they have redueed levels of CS activity and increased sensitivity to Edifenphos
[48]. Rhizopus oligosporus chsl and chs2 play some role in the hyphal stage
of growth but not in the late stage of spore formation [49].
CS homologue genes have also been found in organisms that do not have
crystalline chitin in their cell walls, as is the case for some Oomycetes [50]
and for the fission yeast Sehizosaccharomyces pombe [30]. However, so far
no physiological role has been assigned to these genes.
Chitin synthase 3
Since S. cerevisiae mutants defective in CSI or CSII aetivities turned out to
be able to synthesise chitin in vivo, it was evident that at least one more CS
62
activity had to be present in S. cerevisiae cells. To study chitin synthesis

rather than chitin synthases, two different approaches were used to isolate
chitin-deficient mutants. The first consisted ofthe isolation of Calcofluorresistant mutants [19]. Calcofluor is a fluoro chrome that exerts an antifungal action on S. cerevisiae and other fungi by interfering with the
process of chitin synthesis [51]. These mutants had no apparent defect in
growth or morphology and had wild-type levels ofCSI and CSII activities.
However, they displayed a 10-fold decrease in the amount of chitin present
in their cell walls during vegetative growth and mating. The second screening was designed to isolate mutants defective in the incorporation of exogenous 3H-glucosamine into 3H-chitin [22]. Five complementation groups
were isolated in these screenings: CHS3ICALlICSD2, CHS4ICAL2ICSD4,
CHS5ICAL3, CHS61CSD3 and CHS7 (see below). dUlOl and kti2 mutants,
isolated in screenings searching for mutants defective in ascospore cell
wall synthesis and in a search for mutants resistant to the Kluyveromyces
lactis killer toxin, respectively, turned out to belong to the CHS3 group
[13].
The CHS3 gene (Tab. 1) was initially cloned by complementation of the
Calcofluor resistance phenotype ofthe chs3 mutant [12]. CHS3 deletion is
not lethaI and prornotes a phenotype identical to that of the original chs3
mutant. The CHS3 sequence shares limited but significant homology with
the carboxyl portion of CHSl and CHS2. It was shown that chs3 mutants
are defective in basal CS activity when assayed in the presence of Mg ++.
Transformation with a multicopy plasmid carrying the CHS3 gene restored
the wild-type level of this new activity [12]. These results confirmed that
Chs3 is the structural gene for CSIII; the activity responsible for chitin
synthesis in vivo.
There are some differences in the characteristics ofthe CS enzymes. First,
the cation dependence of the three chitin synthases is different. While CSI
and CSIII have a preference for Mg ++, CSII shows the highest activity when
assayed with Co++. Additionally, the presence ofNi++ cations inhibits CSI
and CSII but does not affect CSIII [52]. Second, the zymogenic property of
CSIII is not simple. Several proteases, including trypsin and chemotrypsin,
activate CSI as well as CSII in vitro [1, 9], while simple treatment ofCSIII
with trypsin inactivates the enzyme [7, 11, 12]. CSIII requires the presence
of substrate during protease treatment in order to be activated to some
extent [53]. Third, overexpression of either CHSl or CHS2 in Saccharomyces cerevisiae or Schizosaccharomyces pombe cells results in
increased activity [8, 10], whereas that of CHS3 does not ([12], T. Cos,
unpublished). This result implies that Chs3 must interact with some
regulatory components indispensable for activity.
By the use of appropriate disruptants or mutants that permitted the analysis of the function of each of the chitin synthases in the absence of the
other two, a specific function was attributed to CSI, CSII and CSIII. Thus,
it was confirmed that CSII catalyses the formation of the primary septum
63
A
~'
Figure I. Chitin synthesis is localized during cell cycIe. (A, B) Chitin synthesis takes place at
the base ofthe new bud during vegetative growth (A) or at the base ofthe shrnoo during a-factor
treatment (B). Chitin is shown under fluorescence microscopy after staining with calcofluor
during 90 min. (C) Chs3p is highly polarized to the base ofnew buds during vegetative growth,
where chitin synthesis takes place. Chs3p is observed after immunofluorescence staining.
[25]. eSIII proved to be responsible for the synthesis of a chitin ring that
forms at the base of an emerging bud (see Fig. 1) and the chitin interspersed
in the cell wall [25]. eSIII is also responsible for the synthesis of the chitin
that forms at the base ofthe shmoo projection (see Fig.1) during mating
[19] and is required for the synthesis ofthe chitosan layer ofthe ascospore
cell wall [27]. As indicated above, eSI has arepair function that facilitates
separation between mother and daughter cells [15] (see Tab. 1 for a summary).
Regulation of eSIII in Saccharomyces cerevisiae
The strategies used to obtain mutants deficient in chitin synthesis in vivo

[19, 22] allowed the isolation of several other mutants in addition to the
already described chs3. Further work with these mutants led to the isolation
of four new genes: CHS4 (SKT5 = CSD4 = CAL2), CHS5 (CAL3), CHS6
(CSD3) and eHS7. Strains defective in any of these genes have severely
reduced levels of chitin in their cell walls (see [13] and Tab. 1).
64
Since CHS3 encodes the catalytic subunit ofCSIII [12], the firstworking
hypothesis was that these genes would act as regulators of this activity.
None ofthem seems to be involved in the expression of CHS3 [20,54], and
all the available evidence indicates that they act as regulatory subunits of
CSIII activity. This hypothesis is in c1ear agreement with the evidence
indicating that CSIII activity is regulated at posttranslationallevel [28],
T. Cos, unpublished data) despite the transcriptional regulation of CHS3
expression observed during the cell cyc1e [27]. Beyond these general ideas,
currently, a fair amount of information about the role of CHS4 and CHS5
gene products in the regulation of CSIII activity is available.
CHS5 encodes a protein with a fibronectin type III domain that is involved in the polarised transport of Chs3p to the division site with the
participation ofMy02p [20, 55]. Apparently, correct positioning ofChs3p
at the septum site is required for CSIII to function properly, as seen in the
c1ear defect in this activity observed in chs5 null mutants [20]. In addition,
Chs5p is also involved in mating, specifically, in the process of cellular
fusion. These data point to a general role of Chs5p in the polarised transport ofproteins during the Saccharomyces life cyc1e. In the absence ofthis
protein, this transport is impaired, and consequently chitin synthesis is
defective.
The CHS4 gene product, Chs4p, has been also recently implicated in
Chs3p positioning [56]; however, its function is significantly different from
that of Chs5p. The model evolving from these studies suggests that Chs5p
acts in the polarised transport of secretory vesic1es, inc1uding Chs3p, while
Chs4p is specifically involved in the localisation ofChs3p at the base ofthe
mother-bud neck. This restricted position of Chs3p also depends on
septins, which mark the localisation for Chs3p through an intermediate
protein, Bni4p [56], which interacts physically with Chs4p and septins.
Interestingly, Chs4p has another important function in CSIII activity.
This protein is required for functional CSIII activity in vitro, but its absence
can be compensated by proteolytic treatment of membrane extracts [53,
54]. This suggests that Chs4p can act as a direct regulator of CSIII activity
(recall that Chs4p interacts physically with Chs3p); indeed, overexpression
of Chs4p results in a significant increase in CSIII activity [22, 54]. The
exact way in which Chs4p acts in chitin synthase activity is not yet known,
although the evidence obtained to date suggests a role ofthis protein in the
correct folding ofChs3p in the membrane [54]. Recent findings also suggest that there are different regions of Chs4p involved in the localisation or
activation of CSIII [56]. It has recently been shown that the C. albicans
genome contains a CHS4 homologue (EMBL, Ace. AB0033I 0), but its
function remains to be tested.
Chs4p also has an important role during mating, specifically in the
formation of mating pairs, but lacks any role in the synthesis of the chitosan
ascospore layer. It is tempting to speculate that the role of Chs4p in
chitin synthesis during vegetative growth would be assumed during sporu-
65
lation by Shc1p, a highly homologous protein (43.4% idendity) that is

induced during sporulation [22]. Confirmation of this hypothesis awaits
further work.
In comparison with these genes, very little is known about CHS6 and its
gene product. Null chs6 mutants lack chitin, but their CSIII activity is not
significantly affected [22, 62]. The reason for this is unknown, but very
preliminary evidence hints at a role for Chs6p in Chs3p localisation [62].
However, its function must be significantly different from that Chs4p or
Chs5p, as can be concluded from the characteristic phenotype observed in
chs6 mutants. CHS6 has a very conserved (43.3% identity) homologue,
YKR027, but this gene is not involved in chitin biosynthesis [62]. The
function of CHS7 in chitin synthesis has not yet been determined.
CHS3 homologues and function of CSIII activity in other fungi
Bowen's c1assification of fungal chitin synthase genes [30] defines three

different CHS classes (see above) that exclude the ScCHS3 gene and its
possible homologues. The cloning and characterisation of CaCHS3 [57]
andNcCHS4 [58], two ScCHS3 homologues, allowed the design of specific
primers for the amplification ofthis gene in other organisms. This strategy
led to the isolation of the ScCHS3 homologues shown in Figure 2 (see
figure legend for references). This figure was obtained using the Clustal
algorithm and comparing equivalent regions of all the proteins (from amino
acids 1002 to 1107 of ScCHS3). This philogenetic alignment clearly identified two perfectly separated clusters; the upper one included ScCHS3 and
all the genes with homologies greater than 50% in this region. All these
genes are assumed to code for class IV chitin synthases. The lower part of
the tree represents a new class of chitin synthases, class V, clearly related to
class IV [59] and so far described only in filamentous fungi. The tree also
separates yeast class IV chitin synthases from those of filamentous fungi,
probably indicating the evolutionary separation of these two groups of
organisms and that in agreement with the different functionality of class IV
chitin synthases between yeast and fungi (see below).
It is noteworthy that CHS homologues have also been identified in
beings ranging from bacteria (nodC genes) to higher organisms (DG42 in
Xenopus) [22]. This clearly indicates that the catalytic domain of CS (the
most conserved part) is very ancient, and since these genes are more related
to class IV CS than to any other CS classes, it is probable that all fungal CS
genes would have derived from a class IV CS ancestor (see J. Bakkers
et al., this volume).
What is the function of class IV CS in fungi? Unfortunately, there is no
clear answer to this question, partly because only four class IV CS genes
have been characterised so far. In yeast, either Saccharomyces or Candida,
the CHS3 gene product is not essential but is involved in the synthesis of
66
-r-t
C.CHS3
SoCHS3
YlCHS3
AgCHS3
ScCHS3
KlCHS3
HpCHS3
MgCHS3
NcCHS4
ThCHS3
AnCHSE
CnCHS3
McCHS3
AfCHSE
AnCHSD
PbCHS3-1
PbCHS3-2
30
25
20
15
10
Figure 2. Philogenetic alignment of dass IV and V chitin synthases. The origin of the sequences
is as folIows: SoCHS3 (Swaniomyces occidentalis), YlCHS3 (Yarrowia lipolytica),AgCHS3 (Ahsbya gosypii), SeCHS3 (8. cerevisiae), K1CHS3 (Kluyveromyces lactis), HpCHS3 (Hansenulla
polymorpha), ThCHS3 (Trichoderma harzianum), McCHS3 (Mucor circinelloides) and PbCHS31 (Phycomyces blakesleeanus) are from T. Cos and C. Roncero, (unpublished data), CaCHS3 (C
albicans) [57], MgCHS3 (Magnaesfera griseus) (Specth, c., EMBL Acc.AF020528), NcCHS4
(Neurospora crassa) [58], AnCHSD (Aspergillus nidulas) [61], CnCHS3 (Criptococcus neoformans) (Specth, c., EMBL Acc. AF021318), AjCHSE (AspergillusjUmigatus) [46], and PbCHS32 (Phycomyces blakesleeanus) (A. Perez-Eslava, unpublished data).
most (90-95%) ofthe cellular chitin. However, the biological relevance of

this chitin synthesis is not yet clear since it has been shown that SeCHS3 is
only important for proper maturation of the spore wall [27] and that
CaCHS3 could play some role in virulence [34, 60].
In fungi, NcCHS4 [58] and AnCHSE [40, 61] have been characterised.
Mutants in these genes displayed a minor decrease in chitin content and no
obvious phenotypes during vegetative growth. However, some evidence in
both fungi indicates that class IV es in fungi serves as auxiliary or redundant
enzymes. It is conceivable that the new fungal class V es could assume the
role of yeast class IV in chitin synthesis. However, some work with A. nidulans chsD mutants indicate that this gene, although involved in chitin synthesis, cannot account for the majority ofchitin synthesis in this fungi [40, 61].
Obviously, much more work is required in the characterisation of class
IV and V chitin synthases in fungi, and the emergence in the near future of
new classes of chitin synthases is quite possible.
67
Acknowledgement
We thank all members of A. Duran 's lab for communicating unpublished results and for their
support during the course ofthis work. Special thanks are due to E. Mellado for helpful COffiments on Aspergillus chitin synthases, to A. Perez-Eslava for the Phyeomyees sequence and to
N. Skinner for correcting the English version. This work was supported by grants CICYT
BI095-0500, Junta de Castilla y Le6n SA13/97 and a EUROFAN 11 project.
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ed. by ~ Jolles and R.AA Muzzarelli
Function of chitin oligosaccharides in plant

and animal development
Jeroen Bakkers, Jan W. Kijne, Herman P. Spaink
Leiden University, Institute ofMolecular Plant Sciences, Wassenaarseweg 64,
NL-2333 AL Leiden, The Netherlands
Summary. In plant development chitin oligosaccharides have been studied intensively as part
of the communication between leguminous plants and Rhizobium bacteria. The Rhizobium
bacteria synthesize and secrete lipochitin oligosaccharides (LCOs) to induce the development
of a root nodule, in which the bacteria will infiltrate to start a symbiotic relation with the plant.
Here we will give an overview ofthe biosynthetic route used by the bacteria to synthesize these
LCOs. Perception by the plant will also be discussed as weil as early responses to the LCOs. By
working with the genes from the biosynthetic route, other genes were identified that share
homology with the ehitin synthase genes from Rhizobium. These genes are now isolated from
human, mouse, chick, Xenopus and zebrafish and can be divided into three classes. They are
mainly expressed during early development at the same stage as chitin oligosaccharide synthase
activity can be detected. A controversy has been risen about their biochemical activity and will
be further discussed here.
Introduction
Lipoehitin oligosaeeharides (LCOs) are signal moleeules that were diseovered during the study of the root nodulation proeess in leguminous
plants. Nitrogen-fixing root nodules are the result of an association of
plants with baeteria belonging to the family of Rhizobiaeeae, eommonly
ealled rhizobia. LCOs produeed by rhizobia are key factors in the speeific
reeognition proeess that underlies the formation of infection threads and
root nodules [1-3]. In the absence ofbacteria these LCOs in nanomolar
eoneentrations are able to elicit all the early events normally oeeurring in
the nodulation process such as root hair deformation, preinfection thread
formation, induction of eell divisions and expression ofnodulation-related
plant genes. Here we will give an overview ofthe present knowledge ofthe
biosynthesis and biologie al role of LCOs and related compounds in plant
and also animal development.
Structure and biosynthesis of lipochitin oligosaccharides
The biosynthesis of the LCOs by rhizobia has been studied in great extent
and reviewed in detail by many authors [4-6]. In response to flavonoids
present in the exudate of host plants, rhizobia secrete metabolites, which
72
J. Bakkers et al.
were initially detected due to their ability to elicit root hair deformation,
one of the earliest events in the nodulation process. These metabolites,
named nodulation (Nod) factors, have been purified from a large number
of rhizobial strains. Their chemical structure has been elucidated using
nuclear magnetic resonance and mass spectrometry [2, 7]. Most Nod
factors characterized so far share a common structure, which consists of an
oligosaccharide backbone of -l,4-linked N-acetylglucosamine (GlcNAc)
residues, with a fatty acid group attached to the nitrogen ofthe nonreducing
terminus (Fig. I). For this reason these compounds are named lipochitin
oligosaccharides. All rhizobial strains examined produce a mixture of different LCOs. They vary in (i) the presence of additional groups on the reducing or nonreducing terminus of the chitin oligosaccharide backbone,
(ii) the type of acyl chain present on the nonreducing terminus and (iii) the
length of the oligosaccharide backbone. These variations are major determinants of host specificity (for reviews see [5, 8, 9]). examples of host
range-determining modifications are a sulfate group in LCOs of Sinorhizobium meliloti and the O-acetyl group in LCOs of R. leguminosarum
biovar viciae. Mutant strains that lack the sulfate group on their LCOs no
longer nodulate alfalfa roots, the natural host for S. meliloti [1, 10]. LCOs
of R. leguminosarum biovar viciae that lack the O-acetyl group on the nonreducing terminus lost their mitogenic activity in vetch [2]. Also, the presence of special polyunsaturated fatty acids in the LCOs of R. leguminosarum and S. meliloti was shown to be important for these strains to
m
~
~o~o"'o~o\.
~o~o~
ID
/
/
o=c
/,
/,
LI:.LIo:iI
OH
~_~o
o=c,
CHs
Figure 1. Structure of an LCO molecule with known modifications. The LCO shown here
contains a C 18: 4 fatty acyl group. Different fatty acyl groups have been reported to be present
on LCOs, varying from C16:0 to C20:4. Ac, acetyl; Ara, arabinosyl; Cb, carbamoyl; Fuc,
fucosyl; Me, methyl; S, sulfate.
73
nodulate their natural host plants [2, 11-13]. The bacterial nodulation
(nod) genes are responsible for the synthesis and secretion of the LCOs.
Information on the biochemical function of nodulation proteins has mainly come from structural analysis of LCOs produced by strains carrying
mutations in nodulation genes and by analysis of LCOs after introduction
of cloned nodulation genes of one rhizobial strain into another. Insight in
the biochemical function of nodulation proteins has also been obtained by
expressing nodulation genes in Escherichia coli, followed by the characterization of nod gene-dependent metabolites produced by such strains. In
this way the functions ofthe nodA, Band C genes have been characterized.
When expressed in E. coli, the NodC protein can convert uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) into metabolites that migrate as
chitin oligosaccharides in thin layer chromatography (TLC) and high
pressure liquid chromatography (HPLC) systems and are degradable by
chitinases. It was shown by mass spectrometry that these metabolites are
indeed linear oligosaccharides of -l,4-linked GlcNAc residues with a
degree of polymerization of 2 to 5 [14, 15]. It has been shown that the
NodC protein synthesizes chitin oligosaccharides by a processive mechanism, since short chitin oligosaccharides are not used as substrates for
elongation. Several compounds that inhibit the synthesis of prenyl-linked
oligosaccharides or recycling of prenyl carriers do not affect the synthesis
of chitin oligosaccharides by NodC [4, 15]. It is therefore most likely that
NodC does not require lipid-linked intermediates in the synthesis of chitin
oligosaccharides. Since GlcNAc can be incorporated by NodC only at the
reducing terminus, and higher concentrations of GlcNAc do not affect
chain length ofthe chitin oligosaccharides, it was concluded that synthesis
of chitin oligosaccharides starts at the reducing terminus. This was confirmed by studies on the incorporation ofp-nitrophenyl--N-acetylglucosamine (pnp-GlcNAc) in chitin oligosaccharides by NodC, showing that this
component can be used by NodC as a starter for chitin oligosaccharide synthesis [4, 16]. Since GlcNAc and pnp-GlcNAc also stimulate fungal chitin
synthases, it was suggested that a similar mechanism is involved in the synthesis of fungal chitin.
The NodB protein has been characterized as a chitin deacetylase. This
activity is needed for the deacetylation of the nonreducing terminal GlcNAc
residue in order to allow a fatty acid to be coupled to the resulting free amino
group. The combined expression of nodC together with nodB in E. coli leads
to the synthesis of chitin oligosaccharides which are N-deacetylated at the
non-reducing-end residue [17]. It was also shown that purified recombinant
NodB protein can deacetylate chitin oligosaccharides [18]. The addition of
the acyl chain to the terminally N-deacetylated chain oligosaccharides produced by NodC and NodB is mediated by NodA. Expression of nodABC in
Rhizobium is sufficient for the synthesis of the core of the LCO, containing
a common acyl chain and lacking other modifications of the oligosaccharide
backbone [2]. NodA-dependent acylation of chitin oligosaccharide back-
74
J. Bakkers et al.
bones has been demonstrated in vitro with extracts of E. coli strains expressing S. meliloti nodA and nodB and extracts of S. meliloti cells [19-21].
Many other modifications of LCOs have been characterized, mainly by
mass spectrometry. These modifications are normally divided in two main
groups depending on whether they are at the reducing or nonreducing
terminus of the chitin oligosaccharide. An example of a modification at the
reducing residue is the presence of a fucosyl group on the LCOs of Bradyrhizobium japonicum. The presence of an O-fucosyl residue at C6 of the
reducing GlcNAc residue is due to the activity of the NodZ protein. The
NodZ protein, after overexpression in E. coli, has been shown to have
fucosyl transferase activity using guanosine 5'-diphosphate-a-L-fucose
(GDP-fucose) as fucosyl donor. Of all tested substrates, chitin tetra-,
penta- and hexamers were shown to be the preferred substrates by the NodZ
protein [22]. An example of a modification at the nonreducing terminus is
the addition of an O-acetyl group at C6 of the nonreducing GlcNAc residue
by the NodL protein. The NodL protein, after overexpression in E. coli, has
been shown to have acetyltransferase activity, using acetyl-coenzyme A
(acetyl-CoA) as acetyl donor [23]. Acceptors for acetylation by NodL are
GlcNAc, chitin oligosaccharides, chitosan oligosaccharides and LCOs. The
lowest Km values were observed with chitin oligosaccharides, which are
N-deacetylated at the nonreducing terminal position.
Perception of lipochitin oligosaccharides by leguminous plants

Since the identification ofLCOs, many groups have studied their effect on
plant development. Several factors involved in the signaling cascade induced by LCOs have been identified, and probably many more are still to
be discovered (reviewed in [9, 13, 24]). When LCOs in nanomolar concentrations are applied externallyon the plant root, they can elicit in the
root cortex the formation ofnodule primordia [1, 2, 25]. Furthermore, as in
plants that are infected by rhizobia, the primordia are induced only at
certain positions in the plant roots, namely the position were young root
hairs emerge, opposite to the protoxylem poles ofthe vascular bundle [1,
2, 25]. Besides their role in the formation of the root nodule primordia,
LCOs are also involved in the bacterial infection process, as suggested by
the inductions of preinfection thread structures in the outer cortex of vetch
roots by mitogenic LCOs in the absence ofbacteria [3]. These preinfection
thread structures are composed of so-called cytoplasmic bridges in cells of
the outer cortex, which are radially aligned, giving the impression of cytoplasmic treads that cross the outer cortex. The formation of cytoplasmic
bridges is preceded by polarization of the cell in which the nucleus moves
to the center of the cell just as in cells that are about to divide. Indeed, it has
been shown by expression patterns of cell cycle genes that these outer
cortex cells reenter the cell cycle but remain in the G2 phase. The inner
cortex cells that form the nodule primordium continue the cell cycle, re-
75
sulting in mitosis [26]. The final result apparently is determined by the

position of the cells in the cortex. Most likely this is achieved by a gradient
of a plant-derived factor, which makes plant cells respond according to
their position within this gradient. A good candidate for such a factor,
isolated from the central root cylinder (the stele), stimulates cell division in
pea root explants [27]. The factor was identified as uridine and may act as
a morphogen in plant roots at picomolar concentrations [28].
The first morphological event to be observed, after application of LCOs
on the plant roots, is the deformation of root hairs. Already I h after the
addition of nanomolar concentrations of the LCOs isolated from R.
leguminosarum bv viciae to the roots of vetch, the root hairs start to swell
[29]. Two hours later, in certain root hairs, a new tip will grow out of the
swelling. This effect is not observed when LCOs ofheterologous strains are
added. This root hair deformation is preceded by a cascade of responses
starting with the depolarization of the plasma membrane already after 15 s
of LCO addition [30, 31]. At the same time an increase in the cytoplasmic
pR in the root hair can be observed [32]. With a delay of 10 min, a regular
oscillation of spikes of intracellular calcium of root hairs is detected [33].
Since some ofthese early events can be induced by other factors, which do
not induce the formation of a nodule primordium, it is not clear what the
role of these events is for LCO signaling. For example, the increase in the
intercellular pR in root hairs of alfalfa can also be induced by LCOs that
lack the sulfate group, which is important for the induction of a nodule
primordium.
Current research interest has been focusing on how LCO signal perception and transduction are achieved. Though binding sites for LCOs are
found [34], no LCO receptor with specificity and high binding efficiency
towards LCOs has been characterized. Each plant may have its own
specific LCO receptor, which can recognize a small range of different
LCOs as produced by its symbiotic Rhizobium strain, or a host plant may
have a number of different LCO receptors each with a different specificity
towards LCOs and chitin oligosaccharides. It is also possible that these
receptors are present at different places of the plant root. Interestingly, it
was found that the O-acetyl group at the nonreducing terminus of the S.
meliloti LCO is not necessary for plasma membrane depolarization but
only for inducing cortical cell divisions [31]. It has been suggested that
O-acetylation is not essential for recognition of LCOs in root epidermal
cells but is required for a response by cells ofthe inner root cortex [35].
Chitin oligosaccharides and plant development
Schlaman et al. [36] have shown that O-acetylated chitin oligosaccharides

in combination with uridine can induce cell divisions when delivered inside
the root cortex of vetch by microballistic targeting. For this effect the
O-acetylation on the nonreducing terminus of the chitin oligosaccharide
76
1. Bakkers et al.
was found to be necessary. These results implicate that (i) a receptor is

present in/on the cells of the root cortex of vetch that recognizes an
O-acetylated chitin oligosaccharide and upon activation induces cell
divisions, and (ii) the fatty acyl moiety ofthe LCO is not necessary for the
induction of cell divisions in the inner root cortex. This could mean that the
fatty acyl group has a ftmction in the delivery ofthe chitin oligosaccharide
backbone inside the plant root. This is in agreement with the finding that
special unsaturated fatty acids are present in the LCOs of rhizobia species
that induce cell divisions in the inner cortex. In contrast, LCOs of rhizobia
species that induce cell divisions in the outer cortex elose to the epidermis
only contain common fatty acids [37].
There are several indications that chitin oligosaccharides are involved in
plant developmental processes other than nodulation. When LCOs or chitin
oligosaccharides are added to suspension-cultured tomato cells, a nonhost
of rhizobia, a rapid and transient alkalinization of the culture medium
occurs [38], suggesting that tomato cells recognize the chitin oligosaccharide by a receptor and release a signal upon stimulation. Also, a carrot
temperature-sensitive somatic embryogenesis mutant can be rescued by the
addition of achitinase purified from wild-type cultures [39]. The same
mutant could also be complemented by the addition of purified LCOs in
nanomolar concentrations, suggesting that the chitinase releases a signal
with structural homology with LCOs [40]. In carrot plants this chitinase
(ep3) is expressed in the tissue surrounding the developing carrot embryo,
suggesting a role in early embryogenesis [41]. In tobacco plants expression
of the Rhizobium nodA and nodB genes, either separately or in combination, has a strong effect on the plant growth and also leaf shape [42]. This
indicates that NodA and NodB interfere with the synthesis of plant
moleeules important in plant morphogenesis, which share a structural
homology with chitin oligosaccharides. In agreement with this is the
finding that chitinase-degradable compounds could be extracted from
flowers of Lathyrus plants [43]. These compounds labeled by 14C acetate or
35S sulfate were very hydrophobic and migrated on TLC like LCOs.
Chitin oligosaccharides in vertebrate development

Chitin oligosaccharides playa role not only in plant development but also in
vertebrate development. Injection of zebrafish embryos in the one-cell stage
with the rhizobial NodZ fucosyl transferase protein caused severe developmental defects in those embryos [44]. The NodZ protein used was shown to
modifY chitin oligosaccharides very efficiently by transferring a fucosyl
group on the reducing terminus [22]. Microinjection inside the developing
embryo of 0.5 ng of the NodZ protein resulted in the development of
embryos without normal tail structures. Microinjection of 10 times less
NodZ protein gave rise to less severe aberrant tail morphologies. These effects
77
were not only observed when the protein was injected but also when a plasmid, containing the nodZ gene under control of promoter active during early
embryo development, was injected [45]. Microinjection of athermo inactivated NodZ protein had no effect. Additionally, when protein extracts of zebrafish and carp embryos of gastrulation and neurulation stages are incubated
with UDP_[14C]GlcNAc, chitin oligosaccharides are synthesized [44, 46].
This chitin oligosaccharide synthase activity could also be detected
using other assays. (i) A Catharanthus roseus plant cell suspension culture
gives a very rapid alkalinization response when chitin oligosaccharides are
applied [44] as described for other plant cell suspension cultures [38].
(ii) The NodZ protein has been used as an efficient tool in the detection and
identification of chitin oligosaccharides. By incubating chitin oligosaccharides and NodZ protein in the presence ofradiolabeled GDP-fucose, the
radio label is transferred to the chitin oligosaccharide and can be easily
detected by HPLC or TLC [22, 44]. In all assays used, immunoprecipitation of the protein extract with antiserum raised against a protein named
DG42 of Xenopus leavis can reduce the chitin oligosaccharide synthase
activity. The DG42 protein is very homologous to the rhizobial NodC
protein and to proteins from the c1ass III of fungal chitin synthases [47].
Microinjection of the DG42 antiserum into zebrafish embryos of a singlecell stage had a similar effect on the development as the injection of the
NodZ protein. Microinjection of the preimmune serum had no effect on
zebrafish development [44]. This would implicate that in zebrafish
embryos a DG42-like protein is involved in the synthesis of chitin oligosaccharides whose function is very important for normal embryonic development. A sequence from zebrafish has been identified that shares high
homology with the Xenopus DG42 gene and also with the Rhizobium nodC
gene, fungal chitin synthase genes and a bacterial hasA [46].
The Xenopus DG42 gene and also the zebrafish sequence fall into a large
vertebrate gene family (Fig. 2) [48]. Up to now three members of this
family have been identified from human as well from mouse, two members
from chicken, four from Xenopus and one from zebrafish. For a long time
now there has been a controversy about the true biochemical function ofthe
members of this gene family [49]. The question is whether they are involved in synthesizing chitin oligosaccharides or whether they are involved
in synthesizing hyaluronan, a polymer consisting of altemating units of
-I,4-linked GlcNAc and -l,3-linked glucuronic acid (GlcA). In vitro
experiments with the Xenopus DG42 gene by different groups gave different results. Expression of the gene in an in vitro transcription/translation
system resulted in chitin oligosaccharide synthase activity that could be
reduced by immunoprecipitation by the DG42 antiserum. In this system no
hyaluronan synthase activity could be detected [50]. The same group
showed that overexpression ofthe Xenopus DG42 gene in mouse 3T3 cells
resulted in chitin oligosaccharide synthase activity in these cell extracts
and no increased hyaluronan synthase activity compared to control trans-
78
1. Bakkers et al.
100 90 80 70 60 50 40 30%
I
m[
chas3
hhas3
mhas3
xhas3
hhas2
mhas2
TI
rhas2
chas2
xhas2
I[
zDG42
hhas1
mhas1
xDG42
xhas-rs
nodC
fbfa
chs3
Figure 2. Homology tree ofthe genes homologous to the rhizobial nodC gene. In the figure is
shown which genes belong to the vertebrate class I, 11 or III as described in the text. The jbfa
gene of Stigmatella aurantiaca is included for its high homology towards nodC. The gene is
involved in fruiting body formation, but it is not known whether it is involved in synthesizing
chitin oligosaccharides [58]. For the alignment, a progressive sequence is used as described by
[59]. The scale bar shows the percentage of homology at the branch points. The accession
numbers for the genes listed above are: S. aurantiaca fbfa (ZI1601), R. loti nodc (X52958),
Xenopus leavis xDG42 (X52958), X leavis xhas-rs (AFOI5780), Danio rerio (zebrafish)
zDG42 (DRU53223), Gallus gallus (chick) chas3 (AFOI5777), Homo sapiens (human) hhas3
(HSU86409), Mus musculus (mouse) mhas3 (MMU86408), X leavis xhas3 (AFOI5778), H.
sapiens hhas2 (HSU54804), M. musculus mhas2 (MMU52524), Rattus norvegicus (rat) rhas2
(AF008201), G. gallus chas2 (AFOI5776), X leavis xhas2 (AFOI5779), H. sapiens hhasl
(HSU59269), M. musculus mhasl (D82964), S. cerevisiae chs3 (M73697).
formed cells [46]. Other groups reported that overexpression of the Xenopus DG42 gene resulted in induction of the hyaluronan synthase activity.
These results were obtained using Saccharomyces cerevisiae [51, 52] as
weIl as rabbit kidney and human osteosarcoma cells [53]. Unfortunately,
these authors never tested the presence of chitin oligosaccharides in their
system, which makes it even more difficult to explain the controversy. It is
not known whether the hyaluronan synthase activity can be inhibited by the
DG42 antiserum as reported for the chitin synthase activity.
79
In spite of this controversy several authors have designated all the members of this gene family, induding DG42, as hyaluronan synthase (has)
genes [48]. The gene family can be divided into three dasses based on
sequence comparison. Mouse, human and Xenopus have one gene from
each dass except for Xenopus dass I, which has two genes (DG42 and
xhas-rs) [48]. Members of the dass 11 and III from human and mouse,
when expressed in mammalian cells, induce the synthesis ofhyaluronan in
vitro and induce the formation ofhyaluronidase-sensitive pericellular coats
in vivo [48,54]. However, members of dass I, induding DG42, only show
induction ofhyaluronan synthesis depending on the cell type in which they
are expressed. They do not induce the formation of pericellular coats in any
cell type tested. This is also the case for the extra gene identified in
Xenopus (xhas-rs), which does not show any in vitro hyaluronan synthase
activity in any cell type tested [48]. All this indicates that there is a difference in biological function between members of the different dasses. In
agreement with this is the difference in expression pattern. Expression of
the dass I and 11 members seems to be restricted to early embryo development, whereas expression of dass 111 members can be detected only in
adult tissue. A possible explanation would be that during vertebrate development different forms of hyaluronan or chitin are needed for anormal
development. It has already been suggested that chitin oligosaccharides
might act as primers for hyaluronan synthesis [46], as has been found for
the synthesis of glycogen and starch. In this model chitin oligosaccharides
could regulate hyaluronan synthesis. It would be too simple to state that the
chitin oligosaccharide synthase activity observed would be due to an
artifact introduced by the in vitro experiments, since the experiments in
zebrafish show a strong correlation between the presence of a DG42-like
protein and that of chitin oligosaccharides. The NodZ enzyme can only use
oligosaccharides containing at least two GlcNAc residues but preferentially more at the reducing end [22]. Results similar to those with NodZ protein
have been obtained by injection ofa chitinase in zebrafish embryos [55].
Only for Xenopus DG42 has the expression pattern in the developing
Xenopus embryo been shown by in situ hybridization and by immunohistochemical localization [56]. Xenopus DG42 expression moves as a
wave or gradient through the Xenopus embryo. The protein is present for a
longer time in posterior regions where the tail is formed. This corresponds
very weH with the effects on tail development found in microinjection
experiments done in zebrafish embryos.
Conclusion
All the data reviewed above indicate that chitin oligosaccharides or chitin
oligosaccharide derivatives play an important role during plant and animal
development. What might be a common theme in plant nodule formation
80
J. Bakkers et al.
and development of the vertebrate embryo? To begin with the latter, the
posterior region where transcripts of the Xenopus DG42 gene can be detected last, the tailbud, can be regarded as a center for multipotent cells. From
these cells, all the different cell types present in the tail in a later stage can
be formed. These cells must remain in an undifferentiated state and are
characterized by their high migration rate. Cells of the root cortex that are
induced to form a preinfection thread or a nodule primordium must go from
a differentiated state back into an undifferentiated state and reenter the cell
cycle. It has been suggested that this G 1 ~ G 2 transition is related to animal
cell migration, since this is the only state in which the plant nucleus can
move around freely and cytoplasmic rearrangements occur [57]. This
would suggest that chitin oligosaccharides are important in giving cells the
potential to migrate and inducing cells to be in an undifferentiated state.
More effort has to be put in clearing up the controversy between the
chitin and hyaluronan synthase activity observed for the DG42 class of
genes. This must be done by comparing both activities for the different
genes in one and the same system, preferably without background activities
for hyaluronan or chitin synthase. A suitable test system would be E. eoli
or an in vitra transcriptionltranslation system. The strong genetics of the
zebrafish system could help in solving the regulation of and biological role
for the different classes, since most genes are expressed during embryogenesIs.
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1999 Birkhuser Verlag BaseVSwitzerland
Molecular and biochemical aspects of chitin

synthesis inhibition
Subba Reddy Palli 1 and Arthur Retnakaran 2
1
2
Rahm and Haas Research Laboratories, 727 Norristown road, Po. Box 904, Spring House,
PA 19477, USA
Natural Resources Canada, Canadian Forest Service, Great Lakes Forestry Centre,
1219 Queen Street East, po. Box 490, Sault Ste. Marie, Ontario P6A 5M7, Canada
Summary. Chitin, is a -I ,4-linked aminopolysacharide homopolymer of GlcNAc that occurs

as a glycoprotein in the exoskeleton of arthropods, the cell wall of fungi and in various components of diverse invertebrates. It is synthesized in two different ways: in fungi the chitin
synthase enzyme occurs as an inactive zymogen in vesicles called chitosomes and requires
proteolytic activation; in arthropods this enzyme is membrane-bound and catalyzes the addition
of G1cNAc units to a dolichol carrier. Chitin is degraded by three different chitinases, the
endochitinase that degrades chitin into oligosaccharides of differing chain lengths, the exochitinase that degrades oligosaccharides into diacety1chitobiose and chitobiase, which degrades
diacetylchitobiose into G1cNAc monomers. Inhibition of chitin synthesis as well as degradation
can both result in deleterious effects that are often similar. Chitin synthesis can be blocked
during the various steps by a variety of antibiotics, metabolie inhibitors, insect growth regulators, alkaloids and hormone analogs. During the molting process in arthropods, genes are
sequentially expressed and repressed by developmental hormones. When these hormones or
their analogs are administered temporally out of sequence, it can result in the blocking of cutic1e
formation, inc1uding chitin synthesis. With the advent of biotechnology and the availability of
both complementary DNA and antibody probes, it is possible to develop high throughput assays
for discovering new chemieals that can block chitin formation. Chitin synthesis inhibitors as
well as inhibitors of chitin degradation that produce similar effects are promising agents for
controlling insect pests, fungal pathogens and helminthic parasites.
Introduction
Chitin synthesis is c10sely associated with specific glycoproteins, and the

covalent binding of chitin to proteins has been well documented in insects
as well as in fungi [1-5]. The enzyme chitin synthase (chitin-UDP-acetylglucosaminyltransferase, E. C. 2.4.1.16), which has been well characterized
in fungi [6], has probably undergone sequential gene duplication and divergence during evolution and at present is manifested in different forms in
diverse species, one of which is the human hyaluronan synthase. This
enzyme has the conserved amino acid sequence essential for chitin biosynthesis in yeasts [7, 8]. Chitin synthase is thought to have evolved into
two types, the fungal form, which occurs as an inactive zymogen requiring
proteolysis for activation [9], and the arthropod form, which is membranebound [10].
86
S. R. Palli and A. Retnakaran
Inhibition of chitin synthesis: biochemical aspects
Chitin biosynthesis is a complex process consisting of aseries of

enzymatic steps beginning with a glucose unit which is converted to
N-acetyl glucosamine (GlcNAc), linked with uridine triphosphate (UTP),
transported within the cell in combination with dolichol phosphate, polymerized into chitin and covalently linked to proteins to form chitin microfibrils in the cuticle. The entire sequence of events is schematically represented in Figure 1 [1-15]. It is then sclerotized [17 -19], and in crustaceans
it is mineralized with CaC0 3 deposition in the cuticular canals [20]. The
process of chitin formation can be blocked at any one of these biosynthetic
steps by various inhibitors [21-23].
A wide variety of chitin synthesis inhibitors belonging to different
chemical groups have been reported in the literature, and some of the more
common ones are listed in Table 1. Several types of bacterial and fungal
antibiotics have been shown to block chitin synthesis by inhibiting specific
enzymes or interfering with intracellular functions of the Golgi apparatus
and the endoplasmic reticulum. Certain metabolic inhibitors that block
dihydrofolate reductase have been shown to inhibit chitin formation by
interfering with nucleic acid synthesis. It appears therefore that since the
synthesis of chitin occurs in the epidermal cell, it serves as the site of action
for most of these inhibitors.
There are a variety of compounds commonly referred to as insect growth
regulators that adversely interfere with the normal growth process, including chitin synthesis. One such compound is buprofezin, which has
been shown to interfere with the formaton of the mitotic apparatus but
manifests its effects by inhibiting chitin formation.
One of the largest groups of insect growth regulators that deserve special
attention is the benzoylphenylureas. The first compound in this series is diflubenzuron (Dimlin), which has been extensively studied ever since it was
serendipitously discovered by scientists at the Philips-Duphar company in
1970, and since its discovery it has spawned the development of a whole array
ofnew analogs [31, 35]. Recently, some ofthe newer analogs such as chlorfluazuron, hexaflumuron, lufenuron and novaluron have been found to be
effective against a variety ofinsects [36, 37]. Benzoylphenylureas have long
since been considered to be inhibitors ofthe enzyme chitin synthase in arthropods, although the evidence is not unequivocal. However, this class of compounds does not inhibit fungal chitin synthase, which is difficult to explain. In
addition to chitin synthase inhibition, various other modes of action have
been suggested for benzoylureas such as (i) inhibition of transport of
UDP-GlcNAc across biomembranes, (ii) inhibition ofthe protease that activates chitin synthase, (iii) blocking ecdysone metabolism resulting in ecdysone accumulation, which stimulates chitinase, which in turn digests nascent
chitin, (iv) blocking ofthe conversion of glucose to fructose-6-phosphate, (v)
blocking chitin synthase activity by an active metabolite ofthe parent benzoyl-
87
Microvilli with
Plasma Membrane Plaques
, . Dolichol-p
,
Protein-asn
Oligosacch-protein-transferase
t
t
ER
Dolichol-Oligosaccharide
Dolichol-p-p-NAGA-NAGA-NAGA
Nucleus
Cytosol
NAGA-1-p
t .
N-Acetylglucosam,ne-6-p
Glucosamine-6-p
Fructose-6-p~
Glucose-6-p
t
==-========-4-4-=-=-====-=--D-Glucose
~
Hemolymph
Trehalose
Figure 1. A schematic representation of an epidermal cell showing the generalized pathway of

chitin biosynthesis in insects_ The entry of trehalose from the hemolyrnph and the sequential
changes in the cytosol, endoplasmic reticulum (ER), processing in the Golgi apparatus and
transport through vesicles culminating in exocytosis into the assembly zone are indicated.
88
S.R. Palli andA. Retnakaran
Iable 1. Inhibitors of chitin biosynthesis

Inhibitor
Mode of action
Effect
A. Antibioties
1. Puromycin
A nuc1eoside antibiotic that interferes Inhibits chitinlprotein synthesis in

with transfer RNA (tRNA) function the blue crab [16]. Indicates that
and inhibits protein synthesis.
concurrent protein synthesis is
essential for chitin formation.
2. Cyc10heximide Binds to subunit ofribosomes and

prevents protein synthesis.
Inhibits G1cNAc uptake in Plodia

cells, indicating protein synthesis
is essential [24].
3. Iunicamycin
Prevents transfer of G1cNAc to

dolichol phosphate and prevents
glycosylation.
Blocks chitin synthesis in

Triatoma [11]. Dolichol pathway
is blocked.
4. Polyoxin-D
Structural analog ofUDP-GlcNAc

and competitive1y inhibits chitin
synthase.
Inhibits chitin biosynthesis in

Chilo [25].
5. Nikkomycin
Structural analog ofUDP-GlcNAc

and is a more powernd chitin
synthase inhibitor than polyoxin-D.
Inhibits G1cNAc incorporation

into chitin in Tribolium [26].
6. Brefeldin
Disrupts the glycosylation function

ofGolgi.
Chitin-protein complex not

formed in the blue crab [14].
7. Monensin
Inhibits glycosylation in the

endoplasmic reticulum during the
process of extracellular secretion
(Palade pathway is blocked).
Antiparasitic drug that prevents chitin
synthesis; effective on helminth
parasites.
Prevents chitin complexing with

protein for vesic1e formation
enroute to cutic1e in the blue crab
[14].
8. Avermectin
Inhibits chitin formation in

Artemia [27].
B. Metabolie
Inhibitors
9. Aminopterin
10. Cyromazine
Inhibits dihydrofolate reductase and lndirectly inhibits normal chitin

interferes with nuc1eic acid synthesis deposition in Musea [28].
in the epidermis.
Abnormal chitin formation in
An S-triazine inhibitor of dihydroLucilia [29].
folate reductase.
C. Insect Growth
Regulators
11. Buprofezin
Interfercs with the mitotic apparatus. Inhibits cutic1e formation and

chitin biosynthesis in Nilaparvata
[30].
12. Diflubenzuron
(and analogs)
It is the harbinger of all benzoylphenylureas; widely considered to

be an inhibitor ofthe arthropod
chitin synthase.
Inhibits chitin synthesis in

numerous insects [31].
89
Table 1 (continued)
Inhibitor
D. Alkaloids
13. Vinblastine
14. Colcemid
E. Hormones!
Analogs
15. 20-Hydroxyecdysone
16. Tebufenozide
(RH-5992)
andanalogs
Mode of action
Effect
Combines with tubulin and inhibits

the assembly of microtubules
resulting in blocking mitosis.
Binds to tubulin and prevents
tubulin assembly into microtubules
and blocks mitosis.
Inhibits cutic1e formation in

Plodia wing discs [32].
Molting hormone expresses and

represses molting cyc1e genes and is
released at a precise time frame.
Chitin is synthesized in its

absence during the intermolt stage
in Manduca [33].
Dibenzoyl hydrazines that are

agonists of 20-hydroxyecdysone;
stable and persist in the epidermis.
Persistence during intermolt stage

prevents chitin synthesis [34].
Cutic1e is not formed in Plodia

wing discs [32].
phenylurea and (vi) blocking the binding of chitin to cuticular proteins

[38,39].
Chitin has been shown to be present in the eggshells of nematodes, and
many benzoylphenylureas adversely interfere with egg development [40]. In
addition to the eggshell, the microfilarial sheath appears to con-tain chitin
[41], and benzoylphenylureas such as hexaflumuron have been found to
be effective in controlling the filaria! parasite [42]. Absence of chitin in
vertebrates makes it an attractive target for control of invertebrate pests,
inc1uding parasitic nematodes. With the advent of recombinant DNA
technology, we are now embarking on the development of designer methods
of targeting specific synthetic pathways as weIl as regulatory steps in chitin
biosynthesis as control methods of the future.
Inhibition of chitin synthesis: molecular aspects
It is becoming increasingly evident that many of the metamorphic events
especially in insects are regulated by developmental hormones that act at

the molecular level very often by inducing the expression of transcription
factors that down- or upregulate other functions. Hormone analogs that
have been shown to inhibit chitin synthesis probably do so by acting in such
a fashion. To understand the mode of action of some of these analogs, we
need to examine the hormonal control of insect development, especially the
molting process.
Insect development and metamorphosis are orchestrated by aseries of
temporally regulated endocrine secretions. Several key hormones such as
the prothoracicotrophic hormone (PTTH), 20-hydroxyecdysone (20E) and
90
s. R. Palli and A. Retnakaran
juvenile hormone (JH) play important regulatory roles. Because of the

rigid exoskeleton, arthropods in general and insects in particular have to
periodically replace the exoskeleton by molting to accommodate for
growth. The molting cycle consists of an intermolt period when the larva
feeds and grows and a molt period when the old cuticle is degraded and in
its place a new one is synthesized. During the intermolt period the insect
actively synthesizes components for the new cuticle, including chitin.
When the larva reaches a critical weight, proprioreceptors send signals to
the neurosecretory cells of the brain to release PTTH. The PTTH in turn
stimulates the prothoracic glands to secrete ecdysteroids into the hemolymph. In most insects, including the spruce budworm, Choristoneura
fumiforana, an ecdysteroid peak is observed in the hemolymph at the
middle of each of the embryonic, larval, pupal and adult stages [43]. Ecdysteroids then downregulate the intermolt synthetic activities such as chitin
synthesis and initiate the molting process.
20E, like other steroid hormones, upon reaching the target tissue binds
to its receptor and initiates a sequence of events. Aseries of pioneering
experiments by Clever and Karlson [44] and Ashburner et al. [45] showed
the induction of a sequence of specific puffs in the polytene chromosomes
of the salivary glands in Chironomus tentans and Drosophila melanogaster
in response to 20E that reflected the molting events. Ashburner et al. [45]
hypothesized that ecdysone binds to its receptor, and the ecdysone-receptor
complex induces the transcription of"early" genes, whose protein products
in turn induce the transcription of "late" genes and suppress the transcription of"early" genes. During the last 2 decades this hypothesis has become
the central paradigm for explaining the mode of action of ecdysone.
The two components ofthe ecdysone-receptor complex, that is ecdysone
receptor or EcR [46] and ultraspiracle or USP (47) were cloned from C.
fumiferana and found to be members of the steroid hormone receptor
superfamily. The complementary DNAs (cDNAs) for the two isoforms of
ecdysone-induced early Choristoneura hormone receptor 75 (CHR75), the
two isoforms ofearly-Iate gene Choristoneura hormone receptor 3 (CHR3)
and the late genes such as dopadecarboxylase have been cloned, characterized or weB studied [48-51]. Developmental Northem hybridization
analysis showed that EcR messenger RNAs (mRNAs) are present throughout C. fumiferana development, and the mRNA levels increase during the
20E peaks. USP mRNAs are maintained at constant levels throughout
development in all tissues except in the larval midgut of the sixth instar,
where the mRNA levels increase during the prepual peak of 20E. The
mRNAs for ecdysone-induced transcription factors CHR75 and CHR3 are
undetectable during the intermolt periods. However, the mRNA levels
increase during 20E peaks (Fig. 2).
As outlined in Asburner's model [45], the products of ecdysone induced
early genes can repress the transcription of late genes such as chitin
synthase, resulting in the inhibition of chitin synthesis. CHR3 and
91
20E
:'.
Di!'P'!Ulie
o--:E::-m':"bryo--l"';":'s:~I~star:-:-lr~' ~ JDS:.:
::
::
:.... :
; ........;
, ;';"1_ r4th Insw I5th Insw I
6th Instar
I Pupe
EcR
USP
CHR75
CHR3
~~::~ns7o"!=~~nl A_L-_. .A
__. .A_'
. --____.A_L-"__. .,_'--.
. .,_____
..
A ____
A_ _
AL-..-._______...A
___.
A _____
A_
Chilinase
....
(Chilindegradalionl _ _ _ _
..
....
..
Figure 2. Schematic diagram of ecdysteroid titers, expression of ecdysone receptor complex

(ECR and USP), ecdysoneinduced transcription factors (CHR75 and CHR3) mRNA, chitin
formation and chitin degradation during deve10pment of the spruce budworm Choristoneura
fumiferana. The ecdysteroid titers for 5 th instar and 6 th instar were measured as described in
Palli et al. [43]. The ecdysteroid titers for embryo and early larval instars are extrapolated from
the ecdysteroid titers of other lepidopteran insects. The mRNA profiles ofEcR [46], USP [47],
CHR75 [50] and CHR3 [48] are based on published results. Chitin synthesis and degradation
were measured during three to six larval instars [34]. The chitin synthesis and degradation for
embryo and early instars are extrapolated from the chitin synthesis and degradation patterns
during the last three larval instars. EcR, ecdysone receptor; USP, ultraspiracle; CHR75, Choristoneura hormone receptor 75; CHR3, Choristoneura hormone receptor 3; 20E, 20 hydroxyecdysone.
CHR75B gene products are prime candidates that can act as repressors of
late gene activity. As shown in Figure 2, chitin synthesis decreases as
CHR3 and CHR75 mRNA levels increase, concomitant with the ascent of
the 20E peak at each molt. In the spruce budworm chitin synthesis occurs
during the first 48-h period of each larval stadium as shown by incorporation of 14C-GlcNAc by isolated abdominal epidermis in vitro [34, 38].
Electron microscopic studies of cuticle development showed that newly
molted sixth instar spruce budworm larvae had a thin cuticulin layer overlaid on a dense epicuticle layer. The rest of the cuticle is elaborated during
the early part of the stadium. In order for this to happen, chitin has to be
synthesized, transported and cross-linked. The entire cuticle, including
chitin deposition and sclerotization, is fully formed by 48 h after ecdysis
s. R. Palli and A. Retnakaran
92
[51]. We have observed in the spruce budworm that chitin synthesis occurs
at the beginning of each stadium before the appearance of the ecdysteroid
peak, and this is probably true of all insects. It follows therefore that if 20E
or any compound that mimics 20E is applied early in the stadium when 20E
is absent, we should be able to inhibit chitin synthesis. This is precisely what
happens to larval epidermis isolated from newly ecdysed larvae (before
48 h) and cultured in vitro in the presence of 20E or its stable analog, RH5992. It is conceivable that 20E or RH-5992 elicit ecdysone-induced transcription factors such as CHR3 and CHR75, whose gene products in turn
repress the transcription of the enzyme chitin synthase, resulting in the
inhibition of chitin synthesis. It is also possible that 20E or its analog
RH-5992 evokes ecdysone-inducible transcription factors which in turn upregulate the transcription of chitinases, which can degrade nascent chitin,
giving the same end result as inhibition of chitin synthesis. Other chitin
synthesis inhibitors whose mode of action is not understood at the present
mayaIso act similarly to RH-5992 by interfering with the normal developmental gene expression cascade. Since cDNA and antibody probes are
currently available for several genes involved in the molting cascade, it is
possible to design high-throughput screening assays for discovering new
compounds with different chemistries that can block chitin synthesis by
interfering with the cascade of gene expression during the molting process.
Application of chitin synthesis inhibitors: commercial use
Chitin synthesis inhibitors, primarily the benzoylphenylureas, have been

considered to be "soft insecticides" belonging to the group of biorational
control agents often referred to as insect growth regulators (IGRs). For the
most part they are effective as larvicides, and upon ingestion they block
chitin formation, resulting in the mortality of the insect at moult. They also
act as ovicides, blocking embryonic development. Some of the commercial
applications ofthese inhibitors are summarized in Table 2.
Table 2. Chitin synthesis inhibitors used for controlling insect pests of agriculture, forestry and
public health
Pest species
Inhibitor used
Activity
Anthonomus grandis (cotton boll

weevil) - a severe pest of cotton
Diflubenzuron, Penfluron
Treated females lay

sterile eggs [52]
Spodoptera littoralis (Egyptian cotton Diflubenzuron, Alsystin,

leafworrn) - pest on cotton, beet,
Penfluron, Novaluron
tomato, alfalfa
Contact and feeding

toxicity to different
stages [36, 53-56]
Laspeyresia pomonella (coddling

moth) - apple, pear
Provides excellent
larvicidal control
[57,58]
Diflubenzuron, Alsystin,
Penfluron
93
Table 2 (continued)
Pest species
Inhibitor used
Anticarsia gemmatalis (velvet bean

caterpillar) - soybean
Diflubenzuron
Activity
Excellent control
[59,60]
Leptinotarsa decemlineata (Colorado Diflubenzuron, Alsystin
Ovicidal and larvicidal
potato beetle) - potato
control [61, 62]
Excellent control
Lycoriella maU (mushroom fly)Diflubenzuron, Alsystin
[63,64]
mushroom
Tribolium, Acanthoscelides,
Diflubenzuron, Triflumuron, Ovicidal and larvicidal
Oryzaephilus, Plodia, Sitophilus and Teflubenzuron,
control [65]
Rhyzopertha species (stored product Chlorflubenzuron
insects) - various stored grains
Ingestion and contact
Bemisia tabaci (cotton whitefly) Novaluron> Chloreffects on larvae [36]
cotton
fluazuronITeflubenzuron
Aubeonymus mariaefranciscae (sugar Hexaflumuron
Adult and embryonic
beet weevil) - sugar beet
effects [66]
Aonidiella aurantii (California red
Buprofezin
Suppresses embryogenescale) - lemon, orange
sis [67]
Thaumetapoea pityocampa (pine
Diflubenzuron
Excellent larvicidal
processionary caterpillar) - pine and
control [68]
other conifers
Lymantria dispar (gypsy moth) Diflubenzuron, Alsystin
Excellent control at very
hardwoods, especially oak
low dosages [69,70]
Malacosoma disstria (forest tent
Diflubenzuron
Excellent control at very
caterpillar) - hardwoods
low dosages [71, 72]
Croesia semipurpurana (oak leaf
Diflubenzuron, Alsystin
Very good control
shredder) - oak
[73,74]
Haematobia irritans (horn fly) Diflubenzuron
Ovicidal control by feedrange cattle-biting fly
through method [75]
Stomoxys calcitrans (stable fly) -live Diflubenzuron
stock biting fly
Diflubenzuron
Ovicidal by surface
treatment and feed
through [76]
Ovicidal and larvicidal
at dosages as low as
51 ppb [77]
Excellent control [78]
Diflubenzuron
Aedes aegypti (yellow fever mosquito) Diflubenzuron, Alystin,

- vector for yellow fever virus
Penfluron
Aedes triseriatus (encephalitis
mosquito) - vector for California
encelphalitis virus
Anopheles albimanus - malarial
mosquito - vector for malaria in
Central America
Culex pipiens fatigans (house

Diflubenzuron, Penfluron
mosquito) - vector for filarial parasite
Reticulitermes jlavipes (termite) wooden structures
Bait control [81, 82]
Hexaflumuron, Lufenuron
94
S.R. Palli andA. Retnakaran
Table 3. Ecological consequences of some chitin synthesis inhibitors [84]

Inhibitor
Crustacean
Fish
Bees
Parasitoids
Chlorfluazuron Daphnia (48 h)

EC so 0.9 1lg!L
Flucycloxuron Daphnia (48 h)
EC so 4.4Ilg/L
carp (48 h)
> 100 Ilglbee
LC so > 300 mg/I
rainbow trout
> 100 Ilglbee
(96 h) LC so > 100
mg/I
very little effect
Flufenoxuron
rainbow trout
LC so > 100 mg/L
very little effect
Hexaflumuron
Daphnia (96 h)
EC so O.IIlg/L
Tilapia
LC so > 3 mg/I
Lufenuron
Teflubenzuron
carp (96 h)
> 500 mg/I
Triflumuron
Buprofezin
Daphnia (48 h)
EC so 225 1lg!L
Daphnia (3 h) EC so
50.6 mg/L
rainbow trout
(96 h) > 320 mg/I
very little effect
> 100 Ilglbee
extoparasites
affected
> 381lglbee
moderately
harmful
> 1000 Ilglbee
very little effect
toxic
moderately
harmful
carp (48 h)
> 2.7 mg/I
Application of chitin synthesis inhibitors: ecological aspects
The benzoylphenylureas adversely interfere with chitin formation in all

arthropods and therefore are detrimental to many nontarget arthropods,
including crustaceans. The fears that spray runoff into streams might cause
widespread mortality of nontarget species have not been realized. The
environmental fate of diflubenzuron, the harbinger of all benzoylphenylureas, has been extensively investigated, and it has been shown that the
material is broken down by various microbial agents and does not accumulate in the soil [83]. By virtue of its arthropod specificity, benzoylureas have varying degrees of adverse effects on crustaceans as weH as
beneficial insects and must be used with care. Since most of the formulations have to be ingested to be effective, topical effects on parasites,
predators and pollinators are minimal. A representative list of IGRs that
inhibit chitin formation and their salient environmental effects [84] are
listed in Table 3.
Acknowledgments
The authors thank Karen Jamieson for editorial help, Marilyn Scott for help with the typing and
Bill Tomkins for the illustration.
95
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ed. by P. Jolles and RA A. Muzzarelli
1999 Birkhuser Verlag BaselfSwitzerland
Characteristics of chitin-bin ding proteins from

Streptomycetes
Hildgund Schrempf
FB Biologie/Chemie, Universitt Osnabrck, Barbarastrae 11,
D-490690snabrck, Germany
Summary. During growth in the presence of chitin-containing substrates, many Streptomyces
strains have been shown to secrete formerly unknown, small chitin-binding proteins (CHBs)
which lack enzymatic activity, specifically target and invade, like a glue, a-chitin, but not chitin or other polysaccharides. CHBs were purified, and their N-terminal amino acids were
determined. Deduced oligonucleotides were used to identifY the corresponding genes, which
were then sequenced. The deduced CHB land CHB2 proteins contain 20 land 200 amino acids,
respectively, 77.7% ofwhich are identical. Several motifs, including the relative location and
spacing of four tryptophan residues, are conserved in CHB 1 and CHB2. The affinity of CHB 1
to crab shell chitin is two times higher than that of CHB2. Comparative studies of various generated mutant CHB 1 proteins led to the conclusion that mainly onc of the exposcd tryptophan
residues directly contributed to the interaction with chitin. Using CHB doupled with FITC
(fluoresceine isothiocyanate), a highly specific and rapid assay was developed to visualize the
location of crystalline a-chitin within native sampies by fluorescence or confocallaser microscopy. In contrast, the N-terminal domain (12 kDa) ofthe S. olivaceoviridis exochitinase can be
used to detect a- and -chitin. The structural parameters inducing the recognition and possible
loosening of a-chitin or of a- and -chitin are at present being investigated.
Introduction
Chitin can be hydrolysed by chitinolytic enzymes produced by different
bacteria, fungi and plants. In addition to a few species of the bacterial
genera Aeromonas [1], Bacillus [2], Cellvibrio [3], Serratia [4], Vibrio [5],
nearly every Streptomyces species is chitinolytic.
Streptomycetes are mycelia- and spore-forming Gram-positive bacteria
abundant in soil. They produce a wide range of antibacterial and antifungal
compounds. In addition, they hydrolyse a number of macromolecules,
induding chitin. Some chitinases were enriched from Streptomyces antibioticus [6], S. griseus [7], S. plicatus [8], S. erythraeus (now Saccharopolyspora erythraea) [9] and S. lividans [10]. S. olivaceoviridis was shown
to degrade chitin very efficiently [11], and several of its chitinases were
purified [12].
Our recent studies have revealed that the chitinolytic systems of S. olivaceoviridis, S. reticuli and several other investigated streptomycetes comprise the synthesis of a formerly unknown type of extracellular small proteins (18-19 kDa) targeting specifically a-chitin. The characteristics of
these chitin-binding proteins (CHB s) have been investigated in more detail.
100
H. Schrempf
Identification of the chitin-bin ding proteins
During growth with chitin, S. olivaeeoviridis secretes on 18.7-kDa protein

which was shown to adhere strongly to the insoluble substrate and was
hence named CRB I. The protein could neither be released by detergents nor
by increasing concentrations of NaCI (up to 1 M), but by high concentrations of guanidine hydrochloride [13]. The protein was purified to homogeneity by consecutive chromatography. Using antibodies raised against
CRB 1, we found that, when cultivated with chitin, S. retieuli also secretes a
protein (18-19 kDa) that cross-reacts with CRBl. The cross-reacting protein is predominantly associated with the insoluble substrate and, like
CRB I, can on1y be re1eased by guanidine hydrochloride. The chitin-binding
protein produced by S. retieuli, which was named CRB2, was purified to
homogeneity. The purified CRBs were subjected to Edman degradation.
The NHrterminal amino acids were used to deduce the respective oligonucleotides. Rybridisations of genomic libraries and sublibraries, as well
as further cloning, resulted in the identification of the corresponding genes,
ehbl [13] and ehb2 [14], which were subjected to DNA sequence analysis.
The CRB1 protein, which was deduced from the S. olivaeeoviridis ehbl
gene, contains 201 amino acid residues as wen as an N-termina1 signal
peptide of 30 amino acids. The deduced S. retieuli CRB2 protein has a
length of200 amino acids and comprises a 30-amino acid signal peptide. It
shares 77.7% amino acids with CRB1 (Fig. 1).
Properties of chitin-binding proteins
Neither CRB displays any cata1ytic or antifungal activity. The proteins

interact strongly with various types of a-chitins, including crab shell
powder. Weaker adsorption of CRBs has been found with colloidal chitin;
this is derived from native crab shell a-chitin after treatment with acids,
and its structure is probably predominantly amorphous. The proteins
adhere neither to -chitin from various sources (i. e. diatom spike, squid
pen, ciliates) nor to carboxychitin or chitosan. CRBs do not interact with
crystalline bacterial cellulose [chains arranged in parallel orientation (cellulose I)], plant-derived cellulose [Avicel, consisting of crystalline (type I)
and amorphous regions], or mureine from Eseheriehia eoli, either (Fig. 2).
From these results we concluded that both proteins recognise and interact highly specifically with a-chitin. More detailed binding studies
revealed that CRB I has a binding capacity ofO.063 pM (i. e. 1.2 mg protein/
mg purified crab shell powder). CHB2 has about half (i.e. 0.037 pM) of
this capacity. The dissociation constants (K d ) for CRBI and CRB2 have
been determined to be 0.11 pM and 0.27 pM, respectively [14, 15].
1.32
----------------------------------------- - - - -- -- ------ --------------
Position of aminoacids
A
CH8I
C R :B2
eRBl
CR\!2
99
PS~GPADGRICSAGNTSPAO~DS
134
a'psGG~.P"~~,GG
PAaGPA~ROLCNAGLGQ'sO~sAPa'P8GAA"KY'GG
CH81
C882
Figure 1. (A) Hydrophobicity plot of CHBL (B) The deduced CRBI was aligned with the
dedueed CHB2 protein_Aromatie amino aeid residues are marked by a dot; the positions of the
tryptophan residues are numbered.
Figure 2 . Crab shell powder (B, left and C, left) or a eonidiophore from Aspergillus proliferans
(B, middle and C, middle) and -chitin from squid (C, right) were treated with FITC-labelled
CRBl, and inspected by eonfocal microseopy (A, B) under ultraviolet (UV) light using a light
microscope (C). Controls were performed with erab shell powder using FITC-labelled WGA
(A, left) binding only to the surface of the substrate, or with the nonbinding FITC-labelled
-lactoglobulin (A, right), and inspeeted by eonfoeal mieroscopy.
102
H. Schrempf
Targeting a-chitin: the role of individual tryptophan residues
Previous studies indicated that the binding of proteins to carbohydrates is

govemed by hydrogen bonding and hydrophobic interactions. The latter are
due to aromatic residues (primarily tryptophan and tyrosine) stacked along
the face of pyranose rings [16, 17].
Our spectroscopical investigations suggested an involvement of tryptophan residues in the interaction of CRBI with a-chitin [13]. In order to
explore the role of tryptophan (W) residues within CRBI in more detail
(Fig. I), they were individuaIly changed to leucine, in some cases to tyrosine residues. Based on the fluorescence spectra, the presence of three
buried W-residues can be calculated for CRB 1 and the mutant protein W57.
Contrary to CRB I, the protein W57 lacks one exposed W-residue. The CD
spectra and the affinity studies with antibodies suggest that there is comparatively little conformational change of the mutant protein W57L,
compared with CRB I. The significant reduction of the binding efficiency
ofW57L to a-chitin can be primarily attributed to the lack of one exposed
W-residue. As the replacement ofW57 by a tyrosine (Y) residue also leads
to the formation ofthe mutant protein W57Y, whose affinity to a-chitin is
even slightly lower than that ofW57L, it can be concluded that the W57residue is essential and cannot be replaced by the other aromatic Y-residue.
The CD spectrum suggests that the secondary structure of the mutant
protein W134L diverges from that of CRBl. The data obtained by
fluorimetry indicate that W134L has two buried and two exposed Wresidues. Rowever, according to the hydrophobicity plot of the deduced
protein, the mutant protein retains one exposed and three buried Wresidues. Thus the secondary structure ofthe mutant W134L protein must
significantly differ from that of CRB 1; this is also indicated by its considerably reduced affinity to anti-CRB I antibodies. Consequently, no
conclusion can be drawn about the contribution ofthe W134-residue to the
binding affinity of CRB 1 to a-chitin. The CD and fluorescence spectra, as
weIl as the slightly lower affinity to anti-CRB 1 antibodies suggest that the
conformational change in Wl84 is less pronounced than that in W134L.
Therefore it can be assumed that the Wl84-residue also plays a part in the
specific interaction of CRB 1 with a-chitin [15].
The number and relative position of the tryptophan residues within CRB2
correspond to those of CRB 1 [14]. Therefore their role may correspond to that
ofthe CRBI residues, too. No common amino acid motifs were identified
among the deduced CHBs and chitin binding domains from several chitinases,
including a chitin binding domain from the S. olivaceoviridis exochitinase
(see below) [18]. A clustering of cysteine and glycine residues typical of
WGA (wheat germ agglutinin) is also missing in the deduced CRBs. The
novel CRBs do not share amino acid identities with a new Anopheles gambiae
type I peritrophic, glycosylated matrix protein (30 kDa) binding to chitin, or
with a chitin-binding lectin (39 kDa) secreted by human macrophages [19].
Characteristics of chitin-binding proteins from streptomycetes
103
Occurrence of chitin-binding protein! homologues
Several other Streptomyces species, ineluding strains of S. albus, S. canescens, S. citrojluorescens, S. coelicolor A3(2), S. coelicolor Mller, S. lividans, S. parvulus, S. vinaceus, S. rimosus and S. tendae secrete homologues of CHB 1 and CHB2 during cultivation with insoluble chitin. We
also detected proteins cross-reacting with anti-CHBI from several other
chitinolytic bacteria.
Recently a 21-kDa protein (CBP21) was found to be secreted by Serratia
marcescens 2170. It binds most strongly to -chitin (from squid), followed
by colloidal and regenerated chitin [20]. It is interesting that the CBP21
protein deduced from the corresponding gene shares 45.3 % amino acid
identity with the Streptomyces CHB 1.
The biological role of chitin-binding proteins
Detailed investigations revealed that Streptomyces strains secrete the CHBs

only when grown with ground crab shells, or with living or autoelaved
mycelia from chitin-containing fungi (i. e. Aspergillus proliferans, Neurospora crassa), but not in the presence of chitobiose or glucose. The
Streptomyces hyphae adhere closely to the chitin-containing substrates, and
the secreted CHBs seem to act like a glue. We showed by confocal microscopy (using CHBI labelled with fluorescence dye, see be1ow) that, in the
course of the elose interactions, CHBs invade (in contrast to wheat germ
agglutinin [WGA], see below) deeper layers of crab chitin or of the fungal
weIl wall containing a-chitin (Fig. 3).
In their natural habitat, streptomycetes encounter different a-chitin
types, which vary as to the length of their N-acetylglucosamine chains
and the degree of crystallinity, and in dependence on the presence of accessory inorganic compounds or proteins. As a consequence, the consistency of the chitinous layers in organisms like arthropods (comprising
crustaceans and insects), molluscs, nematodes, worms, and fungi differs
considerably [21]. Within fungal cell walls chitin is embedded in several
types of other polysaccharides, including glucans. It is thus assumed that
binding constants ofCHB 1 and CHB2 and of the different chitin types vary
and consequently affect the contact to CHB-producing Streptomyces
strains.
The marine bacterium Vibrio parahemolyticus secretes a large lectin
(134 kDa) called chitovibrin that shows a high atImity to swollen a-chitin
prepared from crab chitin, as weIl as longer chitooligomers (> dp 9). It has
been speculated that chitovibrin plays a part in the adhesion to chitin [22].
Within Vibrio harveyi, cell-associated proteins (55 and 150 kDa) lacking
catalytic activities were discovered, which perhaps mediate the specific
attachment of V. harveyi to chitin [23].
104
R. Schrempf
Figure 3. Streptomyces reticuli growing with chitin (crab shell powder) (A) or mycelia from
Aspergillus proliferans (B) were inspected by light (A, left) or electron (B, left) microscopy,
treated with anti-eRBl antibodies and analyzed under UV light (A, B, right).
The use of chitin-binding proteins for the in situ localisation of a-chitin
Fluorescent dyes, such as calcofluor [24], Congo red [25] and primulin [26]
interact with chitin and other polysaccharides. Calcofluor, the most commonly used, was found to bind to many -linked polysaccharides [24],
including cellulose and exopolysaccharides from Rhizobium strains [27].
Thus the detection of Calcofluor-binding material does not prove the
presence of chitin.
WGA (wheat germ agglutinin) is a plant lectin consisting of 171 amino
acids. It recognises and binds to N-acetylglucosamine (GlcNAc) and exhibits an expecially high affinity for (1 ~ 4)-linked GlcNAc oligomers
which are composed ofthree or more residues [28]. Sectioned cells can be
treated with gold-conjugated WGA, and the location ofthe bound electrondense particles be determined by transmission electron microscopy [29].
Although this method provides the highest resolution currently obtainable,
it has several drawbacks. First, it requires considerable technical skill and
time. Second, WGA is not absolutely specific for GlcNAc; it also interacts
weakly with N-acetylneuraminic acid, N-acetylgalactosamine, and other
compounds containing GleNAc. To achieve a more rapid assay, fluorescein
isothiocyanate (FITC) or rhodamine can be coupled with WGA and used
for binding assays [30].
105
In order to quantify chitin, it can be treated with a chitinase, and the

released chitobiose can be enzymatically cleaved to GlcNAc. Although this
is a highly reliable method to definitely identify chitin, its sensitivity is
relatively low, and it cannot be applied to ascertain the location of chitin
within biological material [31].
None of the above described methods can discriminate between polymer
chains arranged in a parallel () and in an antiparallel (a) fashion; this is
only possible by X -ray diffraction spectra of purified chitin [21]. However,
FITC-Iabelled CHB 1 is most suitable to detect the relative position of
a-chitin in native sampies (including native a-chitin-containing fungi,
shells or cuticles of arthropods, insects and crustaceans) by fluorescence
microscopy [l3, 32]. Thus the novel chitin-binding protein has proven to be
superior to any of the other known methods to rapidly detect the relative
location of crystalline a-chitin. It is also expected that a gold-conjugated
CHB 1 could be utilised for an analysis of sections through cells. With confocal laser microscopy we found that FITC-Iabelled WGA binds more
strongly to the surface of chitin, whereas FITC-marked CHB I invades its
deeper layers [32]. The CHB l-labelled protein is therefore weH suited, too,
to estimate the width of chitin layers. Since CHB 1 binds neither to
chitotriose or chitoligomers, nor to chitin of low crystallinity (situated at
the tips of fungi), it can in future be used to study the crystallisation process
of chitin within various organisms.
A protein domain recognizing
lr-
and -chitin
S. olivaceoviridis secretes an exochitinase (exo-chiOl) of 59 kDa. The

amino acids deduced from the exo-chiO 1 gene consist of a C-terminally
located catalytic domain, a central region containing an FnIII module [33]
and an N-terminal region [34]. The predicted catalytic domain belongs to
family 18 of glycosyl hydrolases [35] and shares high amino acid identity
with the deduced Bacillus circulans chitinase D [2].
The 59-kDa enzyme was shown to adhere very strongly to crystalline
chitin and to efficiently hydrolyse native crab and fungal a-chitin. Neither
standard physiologica conditions nor elevated NaCI and detergent concentrations allowed the removal of the bound Streptomyces exochitinase (59
kDa); only high concentrations ofurea and guanidine hydrochloride led to
its release. In the course of cultivation, the 59-kDa chitinase is specifically
proteolytically processed to a 47-kDa truncated form which retains the
catalytic and the FnIII domain, but lacks the N-terminal part. By immunological studies, we could demonstrate that the 59-kDa chitinase, unlike the 47 -kDa truncated form, mediates a very specific and strong
binding to crystalline a- or -chitins from various sources, but does not
adhere to colloidal chitin. Moreover, the 59-kDa enzyme hydrolyses crab
chitin and even better the chitin within the fungal cell wall. These data reve-
106
H. Schrempf
Figure 4. a-Chitin (crab shell, left and -chitin, squid, right) was incubated with the 59 kDa
exochitinase containing the l2-kDa binding domain. As control, a-chitin from crab (middle)
was treated with the truncated exochitinase (47 kdA) lacking the binding domain. All sampies
were treated with antibodies raised against the exochitinase and inspected microscopically
under UV (A) or visual (B) light.
al that a strong adhesion of the large form of the enzyme is aprerequisite

for effective hydro lysis of the crystalline structure of chitin. In addition, it
can be concluded that the NHrterminal domain (12 kDa) ofthe 59-kDa
enzyme is a chitin-binding domain (Fig. 4).
The chitin-binding domain of the S. olivaceoviridis exochitinase shares
no significant similarity with the above-described CHBs or with chitin
binding domains from other chitinases identified in various organisms. An
exchange of individual tryptophan codons within part of the S. olivaceoviridis exo-chi gene encoding the binding domain led to the generation of
mutant genes, and subsequently to mutated proteins with varying degrees
ofbinding capacity. As with CHBI, tryptophan residues playa key part in
the interaction with insoluble chitin.
Acknowledgements
I am grateful to M. Lemme for supporting the writing of the manuscript and to D. Mller for
taking the photographs. The work was financed by the Deutsche Forschungsgemeinschaft
(DFG).
107
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4 Fuchs RL, McPherson SA, Drahos DJ (1986) Cloning of a Serratia marceseens gene
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6 Jeuniaux C (1966) Chitinases. Methods Enzymol8: 644-650
7 Tarentino AL, Maley F (1974) Purification and properties of an endo--N-acetylglucosaminidase from Streptomyees griseus. J Biol Chem 249: 811-817
8 Robbins pw, Albright C, Benfield B (1988) Cloning and expression of a Streptomyees
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11 Beyer M, Diekmann H (1985) The chitinase system of Streptomyees sp. ATCC 11238 and
its significance for funga1 cell wall degradation. Appl Mierobiol Bioteehnol23: 140-146
12 RomagueraA, Menge U, Breves R, Diekmann H (1992) Chitinaes of Streptomyees olivaeeoviridis and significance ofprocessing for multiplicity. J Baeteriol174: 3450-3454
13 Schnellmann J, Zeltins A, Blaak H, SchrempfH (1994) The novellectin-like protein CHB I
is encoded by a chitin-inducible Streptomyees olivaeeoviridis gene and binds specifically to
a-chitin offungi and other organisms. Mol Mierobiol13: 807-819
14 Kolbe S, Fischer S, Becirevic A, Hinz p, Schrempf H (1998) The Streptomyees retieuli
a-chitin-binding protein CHB2 and its gene. Mierobiology 144: 1291-1297
15 Zeltins A, Schrempf H (1997) Specific interaction of the Streptomyees chitin-bindin protein
CHB 1 with a-chitin: the ro1e of individual tryptophan residues. Eur J Bioehem 246: 557 - 564
16 Spurlino JC, Rodseth LE, Quiocho FA (1992) Atomic interactions in protein-carbohydrate
complexes. Tryptophan residues in the periplasmic ma1todextrin receptor for active
transport and chemotaxis. J Mol Bio1226: 15-22
17 Quiocho FA (1986) Carbohydrate-binding proteins: tertiary structures and protein-sugar
interactions. Ann Rev Bioehem 55: 287 - 315
18 B1aak H, SchrempfH (1995) Binding and substrate specificities ofa Streptomyees olivaeeoviridis chitinase in comparison with its proteolytically processed form. Eur J Bioehem
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19 Renkerna GH, Boot RG, Au FL, Donker-Koopman WE, StriklandA, Muijsers AO, Hrebicek
M, Aerts JMFG (1998) Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted
by human macrophages. Eur J Biochem 251: 504-509
20 Suzuki K. Suzuki M, Taiyoji M, Nikaidou N, Watanabe T (1998) Chitin binding protein
(CBP21) in the culture supematant of Serratia marceseens 2170. Biosci Bioteehnol
Bioehem 62: 128-135
21 Muzzarelli RAA (1977) Chitin. Pergamon Press, Oxford
22 Gildemeister OS, Zhu BCR, Laine RA (1994) Chitovibrin: a chitin-binding lectin from
Vibrio parahemolytieus. Glyeoeonj J 11: 526
23 Montgomery MT, Kirchman DL (1994) Induction of chitin-binding proteins during the
specific attachment ofthe marine bacterium Vibrio harveyi to chitin. Appl Environ Mierobiol60: 4284-4288
24 Pringle JR, Adams AEM, Drubin DG, Haarer BK (1991) Immunofluorescence methods for
yeast. In: C Guthrie, GR Fink (eds): Guide to yeast geneties and moleeular biology.
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25 Vannini GL, Poli F, Donini A, Pancaldi S (1983) Effects ofCongo Red on wall synthesis and
morphogenesis in Saccharomyces cerevisiae. Plant Sei Lett 31: 9-17
26 Schekman R, Brawley V (1979) Localized deposition of chitin on the yeast cell surface in
response to mating pheromone. Proc Natl Acad Sei USA 76: 645-649
27 Glazebrook J, Walker GC (1989) A novel exopolysaccharide can function in place ofthe
Calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti. Cell
56: 661-672
28 Raikhe1 NY; Lee H-I, Broekaert WF (1993) Structure and function of chitin-binding
proteins. Annu Rev Plant Physiol Plant Mol Biol 44: 591-615
29 Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A, Cabib E (1991) The
function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol
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30 Molano J, Bowers B, Cabib E (1980) Distribution of chitin in the yeast cell wall: An ultrastructural and chemical study. J Cell Bio185: 199-212
31 Bulawa CE, Slater M, Cabib E, Au-Young J, Sburlati A, Lee WA Jr, Robbins PW (1986) The
S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.
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32 Zeltins A, Schrempf H (1995) Visualization of a-chitin with a specific chitin-binding
protein (CHB 1) from Streptomyces olivaceoviridis. Anal Biochem 231: 287-294
33 Bork P, Doolittle RF (1992) Proposed acquisition of an animal protein domain by bacteria.
34 Blaak H, Schnellmann J, Walter S, Henrissat B, Schrempf H (1993) Characteristics of an
exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein
domains and relationship to other chitinases. Eur J Biochem 214: 659-669
35 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence
similarity. Biochem J 280: 309-316
Chitinases

ed. by r Jolles and R.A.A. Muzzarelli
Biochemistry of chitinases
Daizo Koga 1, Masaru Mitsutorni 2, Michiko Kono 3 , and
Masahiro Matsumiya 4
J Laboratory 0/ Biochemistry, Department 0/ Biological Science, Faculty 0/Agriculture,
Yamaguchi University, Yamaguchi 753-8515, Japan
2 Laboratory 0/Food Chemistry, Department 0/Applied Biological Sciences,

Faculty 0/Agriculture, Saga University, Saga 840-8502, Japan
3 Fisheries Research Laboratory, Faculty 0/Agriculture, The University o/Tokyo, Maisaka,
Shizuoka 431-0211, Japan
4 Laboratory 0/Marine Products Utilization, Department 0/Marine Science and Resources,

College o/Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Summary. Chitinases are found in many organisms, and their properties seem to be c10sely
related to their biological function. In this chapter, the physicochemical properties of chitinases
such as molecular size are compared among organisms, and the optimum and stability conditions for chitinase activity are described. Furthermore, considering their c1assification based
on amino acid sequence, kinetic behaviors are discussed together with their biological
functions. In particular, hydrolytic mechanisms such as inversion and retention of the substrate
are discussed in relation to allosamidin inhibition.
Introduction
In higher plants, chitinases are used for defense against plant pathogens
and pests [1]. The seaweed chitinases also playa role in defence sirnilar to
plant chitinases [2, 3]. In insects and crustaceans, chitinase acts by
degrading the exoskeletal chitin in the cuticle OT shell for ecdysis [4, 5].
Microorganisrns produce chitinase to digest the chitinous nutrient or to
partially hydrolyze the chitinous cell wall for cell proliferation [6]. Recently, chitinase was even found in rnarnrnals [7 -11]. Fish and rnammals also
use chitinase for defense [6, 8]. Furthermore, chitinases are found in other
organisrns [6]. Thus, these living organisrns produce and use chitinase for
their own specific and biological purposes.
Physicochemical properties
Molecular size
Chitinases found in higher plants and seaweeds (algae) have a rnolecular

weight of about 30 kDa. Chitinases of 40-90 kDa and sorne as high as
120 kDa have been obtained frorn rnollusks, arthropods and sorne vertebrates such as fishes, arnphibians and rnammals. A wide range of rnole-
112
D. Koga et al.
cular sizes from 30 to 120 kDa is observed in bacteria and fungi. Some of
these small chitinases may possibly be processed from a larger enzyme by
limited proteolysis [18-21]. Some chitinases from higher plants such as
carrot [22] and from insects such as the tobacco homworm [23] and silkworm [21] are glycoproteins. The enzymes from microorganisms such as
Aeromonas [24] and Rhizopus [25] were reported to be glycoproteins.
Isoelectric point
Chitinases have the following wide range ofpI values: 3.0-10.0 in higher
plants and algae; 4.7-9.3 in insects, crustaceans, mollusks and fishes; and
3.5-8.8 in microorganisms. Therefore, acidic and basic chitinases are present in these organisms.
Enzyme activity
OptimumpH
The optimum pR's ofchitinases are pR 4-9 for higherplants and algae, pR
4.8-7.5 for animals and pR 3.5-8.0 for microorganisms. The optimum pR
seems to be dependent on the substrate used. For analytical purposes, the
soluble substrates glycol chitin and N-acetyl chito-oligosaccharides were
used instead of chitin. The optimum pR of chitinase toward glycol chitin
was observed at a slightly alkaline pR or at two pRs compared with the
short substrates such as the N-acetyl chito-oligosaccharides and their
derivatives. For example, the chitinases from the silkworm [21] and plant
yam [26] showed two optimum pR values such as 4 and 8-10 toward glycol
chitin. Rowever, these chitinases show only one optimum pR in the acidic
pR range, such as pR 4-6 toward the N-acetyl chito-oligosaccharides [21,
27]. This fact may be due to the chitin-binding ability of chitinase or to the
existence of another chitin binding domain. The chitinase with a high
chitin-binding ability would show two optimum pRs in the reaction with
glycol chitin. Of course, a certain yam chitinase shows only one optimum
pR at 4 even during the re action with glycol chitin [27]. Therefore, the two
optimum pRs do not necessarily come from the substrate glycol chitin.
Stability
Regarding thermal stability, it is remarkable that class III plant chitinases

[27] and the chitinases from Bacillus licheniformis [28] found in a hot
spring show a resistance to high temperatures such as 80C. On the other
hand, the chitinases from insects such as the silkworm are not very stable
113
above 40C [21] because the insect grows at ca. 25C. Therefore, insect
chitinase does not require stability against such high temperatures. Insects
hydrolyze their own cutic1e chitin in vivo during ecdysis, whereas plants
must degrade other organisms (pathogens and pests). Considering that
insect chitinases are generally larger than plant chitinases, the small and
compact chitinase may be thermally more stable. Accordingly, the optimum temperature is weIl related to the thermal stability.
Inhibitor and activator

Allosamidin was first reported to be a specific inhibitor against insect
chitinase [29]. Its K i value is about 0.1 pM. Allosamidin inhibits chitinase
competitively. The inhibitor has a structure similar to the intermediate of
the substrate such as an oxazoline ring that may be formed between the
carbonyl oxygen of the N-acetyl group and the C-l of N-acetylglucosamine
during hydrolysis. So far allosamidin and its derivatives have been found
only to inhibit certain chitinases from the silkworm [29], prawn [30] and
microorganisms such as Piromyces communis [31], Streptomyces sp. [32]
and S. olivaceoviridis [33]. Recently, allosamidin was found to bind to
plant chitinase such as hevamine [34] and inhibit plant chitinase such as
yam chitinase H with ID 50 (dose for 50% inhibition) of 44.7 pM, which
corresponds to the K 1 value [27]. These plant chitinases belong to family
18 of the glycosyl hydrolases. Allosamidin is produced by Streptomyces sp.
[35]. Interestingly, this microorganism produces two types of chitinases:
one is allosamidin-sensitive and the other is resistant [32]. Therefore,
allosamidin and its derivatives inhibit only chitinases belonging to family
18 ofthe glycosyl hydrolases but not to family 19.
Regarding metal ions, chitinase is commonly inhibited by Hg2+ and
Ag+. In the case ofCu 2 +, there are two types ofchitinases: one is inhibited
and the other enhanced. The chitinase enhanced by Cu 2 +was found in some
fishes [36-38] and microorganisms such as Pseudomonas aeruginosa
[39].
Reaction mechanism of chitinase
Comparison with similar enzymes

Lysozyme (EC 3.2.1.17) is weIl known as a chitinolytic enzyme. Hen egg
white lysozyme hydrolyzes the -l,4 linkage between C-l of N-acetylmuramic acid and C-4 of N-acetylglucosamine in the cell wall of grampositive bacteria such as Micrococcus lysodeikticus (luteus). Certain plant
chitinases such as hevamine also have lysozyme activity in addition to
chitinase activity. However, this chitinase hydrolyzes the -l,4 linkage
114
D. Koga et al.
between the C-I of N-acetylglucosamine and C-4 ofN-acetylmuramic acid,

which is different from the lysozyme at the hydrolytic site of the substrate
[40]. Such chitinases with lysozyme activity were also found in microorganisms such as Pseudomonas aeruginosa [39], Choanephora cucurbitanum [41] and Phascolomyces articulosus [41] and in plants such as bean
[42], pea [43], sweet orange [44] and Wasabi [45]. It is also difficult to
distinguish between chitosanase (EC 3.2.1.132) and chitinase because the
chitin used as the substrate for chitinase is usually partially deacetylated.
However, we can now distinguish these enzymes by investigating the products as to whether the reducing end and nonreducing end are N-acetylglucosamine or glucosamine [46]. As show in Table I, there are two types
of chitinases [47]. One produces the N-acetyl chito-oligosaccharides containing N-acetylglucosamine at the reducing end and is from family 18,
whereas the other is from family 19. On the other hand, chitosanases show
that glucosamine is necessarily observed at the reducing end or at the nonreducing end [46]. Furthermore, N-acetylglucosaminidase or N-acetylhexosaminidase is an exo-type chitinolytic enzyme and hydrolyzes the
chitin oligosaccharide from the nonreducing end to release a monomeric Nacetylglucosamine [5, 48]. These chitinolytic enzymes are different from
chitinase in antigenicity, amino acid sequence and three-dimensional
structure. These exo-type enzymes act synergistically with an endo- or
random-type chitinase for degradation of the exoskeletal chitin in insects
[5] and crustaceans [49].
Anomer formation
There are two methods ofwater ingress to perform hydrolysis. As an intermediate, the oxazoline ring would be formed in the substrate between the
carbonyl oxygen of the N-acetyl group and C-I of the N-acetylglucosamine. If this oxazoline ring is formed, the water should enter from the
other side to form the Panomer and retain the configuration at C-I. However, the formation of such an oxazoline ring may be dependent on the
three-dimensional structure. If steric hindrance occurs during the reaction,
the oxazoline ring cannot form. In such a case, the configuration may invert
to the a anomer. In fact, two anomeric forms at C-l such as a and p were
found in the hydrolytic reaction as shown in Table 2. This table suggests
that family 18 chitinases act in the retaining mechanism, whereas family 19
chitinases act in the inverting mechanism. This may be related to inhibition
by allosamidin, since it has a conformation similar to an oxazoline ring as
mentioned in the section on inhibition. As shown in Tables 3A and H,
allosamidin inhibited family 18 chitinases such as the silkworm chitinase
[21] and a cIass III yam chitinase [27]. In fact, these chitinases produce the
Panomers. However, hen egg white lysozyme (family 22 of glycosyl hydrolases) produces the p anomer [50] but is not inhibited by allosamidin [29].
os
00
e0
oe0
eo0
ee0
oee0
eeo0
0
eeeo0
0 0
eoo0
00
e0
e00
oe0
oeo0
ehitinase Al
[58]
o ,GleNAe; e, GleN; 12', _, reducing end residue.
00
e0
eo0
oe0
oeo0
ehitinase
[56]
Bacillus circulans
WL-12
00
eo0
oeo0
eeo0
eeeo0
ehitinase D
[58]
Bacillus circulans
WL-12
ehitinase
[57]
Streptomyces
griseus
eOO0
eeo0
eoe0
ee0
oeo0
e00
00
000
e0
oeeo0
oeo.
oa.
o
00
a.
ehitinase C-l
[59]
Streptomyces
griseus HUT 6037
nO.l0S-24
ehitinase II
[46]
Aeromonas
hydrophila
Aeromonas sp.
Family 19
Family 18
Table 1. Produets ofpartially N-aeetylated ehitosan hydrolyzed by ehitinases
()()()e()0
ooeoo0
oeoo0
00e00
00.
oeo0
a.
00
000
Pokeweed ehitinase
PLC-A
[47]
VI
--
CD
'"
e,
t.
c:;
116
D. Kogaetal.
Table 2. Anomeric configuration of N-acetylglucosamine moiety at reducing end

Organism
a-Anomer formation
(Inverting mechanism)
Plant chitinase
barley
bean
yam
Microorganism chitinase
Streptomyces griseus
HUT 6037
Other enzyme
papaya
-Anomer formation
(Retaining mechanism)
Plant chitinase
cucumber
rubber tree
Enzyme
(dass, family)
Ref.
chitinase (dass II, family 19)

[60]
chitinase (dass I, family 19)
[61]
chitinase E (dass IV,
not inhibited by allosamidin [62]
family 19)
chitinase C-I (family 19)
similar to dass IV plant

chitinase
lysozyme
chitinase (dass III)

hevamine (dass III,
family 18) chitinasel
lysozyme
Microorganism chitinase
Bacillus circulans WL-12 chitinase Al and D
(family 18)
chitinase (family 18)
Other enzyme
hen (egg-white)
human
Comments
lysozyme (family 22)

lysozyme
[63]
[50]
inhibited by allosamidin
[61]
[34]
[64]
[62]
not inhibited by allosamidin [50]
[50]
Splitting pattern
Not rnuch information is available on splitting patterns; however, sorne

details are surnrnarized in Tables 3A and B. To investigate the splitting pattern, N-acetyl chito-oligosaccharides were used as soluble substrates
instead of chitin. Chitinase hydrolyzes these oligosaccharides in several
patterns [51]. Such oligosaccharides are usually hydrolyzed in an endo or
randorn mann er. However, sorne chitinases are able to hydrolyze the trisaccharide, whereas other chitinases do not. There are also two types of
chitinases that hydrolyze the pentasaccharide [52]: one is an endo-type and
produces disaccharides and trisaccharides, and the other is an exo-type and
produces monosaccharides and tetrasaccharides. Therefore, it is generally
better to say that chitinase is a randorn-splitting hydro lase.
0.249-0.005 mM
1.68 mg/mI
not available
0.23 mg/mI
not available
0.15 mg/mI
not available
0.3 mg/mI
1.6 mg/mI,
5.2 mg/mI
[23]
endo splitting
8.7 mI/mg/sec
13.6-502/sec/mM
1.30/sec
3.38-2.72/sec
endo splitting
inhibited by
allosamidin
random splitting
[23]
endo splitting
13.4 mI/mg/sec
3.08/sec
[36]
[30]
[67]
[67]
[21]
[23]
2.60 mI/mg/sec
7.3 ml/mg/seg
0.059/sec
1.68/sec
[21]
[65]
[66]
[27,51]
Ref.
endo splitting
not inhibited by
allosamidin
endo splitting
[27]
except for G1cNAcs
inhibited by
allosamidin
endo splitting
[27]
not inhibited by
allosamidin
endo splitting
[2]
Comment
endo splitting
inhibited by
allosamidin
endo splitting
inhibited by
allosamidin
endo splitting
0.37 mI/mg/sec
0.116-2.84/sec/mM
0.984 mI/mg/sec
1.25 mI/mg/sec
0.05-0.743/sec/mM
2.80 mI/mg/sec
1.83 mI/mg/sec
0.30-41.9/sec/mM
kc./Km
0.63 mI/mg/sec
0.084/sec
0.829/sec
0.049-0.395/sec
3.7 mg GlcNAclhlml
0.629/sec
0.645/sec
0.044-0.99/sec
1.07/sec
0.591/sec
0.033-2.9/sec
kcat orVrnax
G1cNAc, N-acetylglucosamine; G1cNAcn , N-acetyl chito-oligosaccharide ofn-mer ofGlcNAc; CM-chitin, carboxymethyl chitin.
Japanese eel (stornach)
GlcNAc3_6 (pH 5.5)
GlcNAc4_6 (pH 6.7)
Prawn
(family 18)
Glycol chitin (pH 9)

G1cNAc3_6
G1cNAc3_6
GlcNAc3_6
CM-chitin (pH 7.7)
Chitin
Glycol chitin (pH 6.5) 0.023 mg/mI

not available
G1cNAc3_6
88kDa
(family 18)
Tobacco homworm 50 kDa

(family 18)
62kDa
(family 18)
75kDa
(family 18)
Krill Antarctic
Krill North America
Glycol chitin (pH 5.5) 0.134 mg/mI

not available
G1cNAc3_6
2.24 mg/mi
0.424-0.139 mM
Animals
Silkworm 65 kDa
(family 18)

G1cNAc4_6 (pH 4)
76}lM
0.408 mg/mI
0.639 mg/mI
0.518 mg/mI
0.88-0.017 mM
0.381 mg/mI
0.323 mg/mI
0.11-0.07 mM
Km
G1cNAcs_6 (pH 6.7)
(family 19)

Chitin (pH 5.5)
(pH 8)
G1cNAc3_6 (pH 4)
(pH 8)
G1cNAc4_6 (pH 4)
Substrate
Red algae (Gigartina mikamii)
chitinase GI
chitinase H class III

(family 18)
Plants
Cabbage (30 kDa)
Tomato
Yam chitinase E class CV
(family 19)
A. Chitinase
Table 3. Kinetic parameters

l:l:I
o'
g-
-.l
......
'"
'"<>
!l>
()
a
es
0
....,
'"
r:t
'<
e.
()
S
IS
Chi42
2.904 x 10 3
jlmol/min/jlmol enzyme
7.67 x 10 3
jlmol/min/jlmol enzyme
1.1 jlM/min
0.5 jlMlmin
endo splitting
hydrolyzes GlcNAc2
hydrolyzes GlcNAc2
inhibited by allosamidin
not inhibited by allosamidin
endo splitting
endo splitting
endo splitting
endo splitting
endo splitting
O.77mM
0.50mM
0.33 mM
6.3jlM
5jlM
0.5jlM
0.56 mg/mi
0.056 mg/mi
16jlM
18jlM
0.63 mg/mi
0.80 mg/mi
0.47 mg/mi
2.3 mg/mi
0.8 mg/mi
1.0 mg/mi
1.33 mg/mi
2.85 mg/mi
pNp-GlcNAc2 (pR 6.0)

4Mu-GlcNAcz (pR 7.0)
4MU-GlcNAc2 (pR 5.5)
4MU-GlcNAc3 (pR 5.5)
colloidal chitin (pR 5.0)
chitosan (pR 4.5)
CM-chitin (pR 4.5)
regenerated chitin (pR 5)
glycol chitin (pR 5.0)
regenerated chitin (pR 5)
CM-chitin (pR 5.0)
II
3.1 jlmol/min/mg
3.8 jlmol/min/mg
2.9 jlmol/min/mg
46 jlmol/min/mg
ende splitting
0.11 mM
111
IV
ChiB
Chi63
ende splitting
endo splitting
exo splitting
1.4 mg/mi
0.8 mg/mi
34.1 jlM
squid chitin (pR 7.0)

squid chitin (pR 7.0)
4MU-GlcNAc2
(pR 6.1)
pNp-GlcNAc2
endo splitting
Comment
CI
C3
B
3.8 jlmol/min/mg
k cat orVrnax
2.8 mg/mi
Km
Substrate
pNp, para-nitrophenyl; 4MU, 4-methylumbelliferyl; CM, carboxymethyl.
Myrothecium verrucaria
Phascolomyces articulosus
Choanephora cucurbitarum
Streptomyces kurssanovii
Streptomyces sp. AJ9463
Clostridium paraputrificum
Streptomyces plicatus
Serratia marcescens
BJL2000
Bacillus licheniformis
X-7u
Microorganisms
Aeromonas hydrophyla
subsp. anaerogenes
Vibrio alginolyticus R-8
B. Chitinase
Table 3. (continued)
[73]
[41]
[41]
[53]
[32]
[71]
[72]
[28]
[70]
[69]
[68]
Ref.
):J
00
119
Transglycosylation reaction
Like lysozymes, some chitinases can do the transglycosylation reaction,

which is the reverse ofhydrolysis. Such chitinases are found in microorganisms such as Bacillus licheniformis [28], Streptomyces kurssanovii [53],
S. thermoviolaceus [54] and Nocardia orientalis [55].
Kinetics
The Km value indicates the dissociation constant of an enzyme-substrate

complex; therefore, the smaller the Km value, the stronger the affinity
toward the substrate. The k cat value indicates the rate constant for the reaction from the enzyme-substrate complex to enzyme and product. The
parameter Vmax, which measures the maximum velocity, is almost the same
as kcat' but its value equals kcat x (total concentration ofthe enzyme). The
kinetic parameters are summarized in Tables 3 A and B. The kinetic parameter values for long substrates such as chitin, colloidal chitin, CM -chitin
(carboxymethyl chitin) and glycol chitin differ from those for short substrates such as the N-acetyl chito-oligosaccharides. Perhaps the long
substrates are the real substrates for chitinases, whereas the short substrates are the products. The Km value is especially interesting in relation to the
affinity for chitin. For the long substrates, Km values are 0.023-0.23 mg/mI
for insect chitinases, 0.3-1.6 mg/mI for crustacean chitinases, 0.47-2.85
mg/mI for microbial chitinases, 0.38-2.24 mg/mI for plant chitinases.
Such wide-ranging values may be due to the solubility of the substrate in
the reaction solution. Furthermore, there are different types of chitinase in
each organism. For example, plant chitinases from yam have different
values [27]. Yam chitinases E and H have small Km values, whereas yam
chitinase G has a large value. In fact, the former chitinases have a high
affinity to chitin compared with the latter enzyme. Furthermore, chitinases
such as yam chitinases E and H have high antipathogenic activity, whereas
yam chitinase G has low antipathogenic activity. Yam chitin ase E belongs
to dass IV and has another chitin binding domain in the N-terminal region.
Yam chitinase H belongs to dass III (family 18). This enzyme might have
such a chitin binding domain. Therefore, the chitinases with small Km
values can be expected to have high affinity for chitin, and may have strong
antipathogenic activity. On the other hand, for the short substrates such as
the N-acetyl chito-oligosaccharides, Km values could not be obtained from
the insect chitinases of the tobacco homworm [23] and silkworm [21]. It
may be postulated that such insects produce an exo-type chitinolytic
enzyme such as N-acetylglucosaminidase that hydrolyzes the N-acetyl
chito-oligosaccharides produced from the old cutide chitin by insect
chitinase [5]. Therefore, insect chitinase does not necessarily hydrolyze
small oligosaccharides. On the other hand, the coexistence of chitinase and
120
D. Koga et al.
the exo-type chitinolytic enzyme has not been reported in plants and algae
[2]. Therefore, the chitinases from plants and algae may even hydrolyze
these small substrates. There is also another reason to consider: insects
need only hydrolyze their own cuticle chitin, and not exogenous chitin.
That is, the substrate for insects is only the cuticle chitin, whereas the substrate for plants is the chitin of pathogens and pests. Aplant needs to produce the chitinase that is able to degrade the exoskeletal chitin of the invaders, thus requiring wide substrate specificity far the chitins of various
orgamsms.
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1999 Birkhuser Verlag BaseVSwitzerland
The structure and action of chitinases

Jon D. Robertus and Arthur F. Monzingo
Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University ofTexas, Austin, TX 78712, USA
Summary. Chitin is second only to cellulose in biomass and it is an important component of
many cell wall structures. Several families of enzymes, of distincdy different structure, have
evolved to hydrolyze this important polysaccaride. Glycohydrolase family 18 enzymes, chitinases,
are characterized by an eight-fold al barrel structure; it has representatives among bacteria,
fungi, and higher plants. In general these chitinases act through a retaining mechanism in which
linked polymer is c1eaved to release a anomer product. Family 19 chitinases are found
primarily in plants but some are found in bacteria. Members of this family are related to one
another by amino acid sequence, but are unrelated to family 18 proteins. They have a bilobal
structure with a high a-helical content. Despite any significant sequence homology with lysozymes, structural analysis reveals that family 19 chitinases, together with family 46 chitosanases,
are similar to severallysozymes inc1uding those from T4-phage and from goose. The structures
reveal that the different enzyme groups arose from a common ancestor glycohydrolase
antecedent to the procaryotic/eucaryotic divergence. In general, the family 19 enzymes operate
through an inverting mechanism.
Introduction
Polysaccharide metabolism is a very ancient and widespread biological

activity. Because of its central role in energy flux it is not surprising that a
wide range of enzymes have evolved over time to catalyze these reactions.
Among the enzymes involved in breakdown of sugar-based polymers are
cellulases, chitinases, chitosanases and lysozymes. These have been
cataloged, based on amino acid sequences, into nearly 50 families of
glycohydrolases [1].
Chitinases are found primarily in two ofthe families of glycohydrolases,
18 and 19. Family 18 contains several runs of conserved amino acids, as
shown below for the enzymes from Coccidioides immitis [2, 3], Trichoderma harzianum [4], Aphanocladium album [5], and Serratia marcescens
[6]; the sequence numbers correspond to Coccidioides immitis. These signature sequences are found in the active site, and include a Glu residue,
171, crucial to the catalytic mechanism.
130
Chi-ci
Chi-th
Chi-aa
Chi-sm
LSIGGWTYSPNF
LSIGGWTWSTNF
LSIGGWTWSTNF
PSIGGWTLSDPF
170
FDGIDIDWEYPED
FDGIDIDWEYPAD
FDGIDIDWEYPAD
FDGVDIDWEFPGG
Family 19 contains primarily enzymes from plant sourees, although there

are some representatives from bacterial sourees. A section of signature
126
1. D. Robertus and A. F. Monzingo
sequence for the family 19 chitinases is shown as represented by barley,

Hordeum vulgare [7]; potato, Solanum tuberosum [8]; Arabidopsis thaliana [9]; and pea, Pisum sativum [10]. The numbering refers to the barley
sequence; as will be developed below, Glu 67 and Glu 89 are important

residues in the mechanism of action of family 19 chitinases.
60
70
80
90
100
Chi-hvKREVAAFLAQTSHETTGGWATAPDGAFAWGYCFKQERGASSDYCTPSAQWPCAPGK
Chi-stKRElAAFFAQTSHETTGGWASAPDGPYAWGYCFLRERGNPGDYCPPSSQWPCAPGR
Chi-atKKEVAAFFGQTSHETTGGWATAPDGPYSWGYCFKQEQNPASDYCEPSATWPCASGE
Chi-psKRElAAFLGQTSHETTGGWPTAPDGPYAWGYCFLREQNP-STYCQASSEFPCASGK
It is clear that the members of each family are homologs, and that the two
families are themselves quite dissimilar. As will be developed below, the

sequence differences are reflected in differences in tertiary structure as weIl.
Structure of family 18 chitinases
The three-dimensional structures of several family 18 chitinases have been

solved, including a bacterial chitinase from Serratia marcescens [11] and
the plant enzyme hevamine [12]. The enzyme core ofthese enzymes is, in
both cases, an eight-stranded a/ barrel. That is, eight strands of parallel
sheet are laid down with an a helix as the "return stroke". The eight strands
of the sheet bend into barrel structure with the helices forming a ring
toward the outside. The X-ray structure of the chitinase cloned from the
pathogenic fungus Coccidioides immitis [2, 3] is presently being refined in
our laboratory and exhibits the same a/barrel configuration. A backbone
representation of the Serratia enzyme is shown in Figure 1. It should be
noted that the bacterial enzyme contains 561 residues. The amino (N)
terminal 147 residues form a distinct chitin anchoring domain, while
residues 148 to 561 form the chitinase a/barrel domain.
Structure of family 19 chitinases
The only member of this family for whieh an X-ray structure has been
solved is Hordeum vulgare (barley). The structure was originally solved at
2.8 Aresolution [13] and laterrefined to 1.8 A [14]. Figure 2A shows aribbon drawing of the protein revealing a mixture of secondary structure, including 10 a-helical segments, and one three-stranded sheet. An analysis
of the protein shows that many hydrophobie residues conserved in the
family 19 family form a core for the protein. In a similar fashion, those
polar residues conserved within the family tend to line the large cleft in the
enzyme which is presumed to be the substrate binding and catalytic site.
The nature of these conserved residues justifies the notion that the barley
chitinase is a reasonable model for the other enzymes of family 19 where
127
N
Figure 1. A ribbon drawing of the chitinase from Serratia marcescens. This enzyme is a representative of the farnily 18 chitinases. It is an a / barrel structure with eight parallel strands
of sheet and eight return helices. The view is down the core of the barrel, which is filled with
side chains. The active site is on the top of the barrel, and Glu 315 is the catalytic acid. The
N-terminal 147 residues of the Serratia enzyme form a chitin-anchoring domain; this appears
at the lower left of the chitinase barrel domain, like the handle on a mirror. Many, but not all,
farnily 18 chitinases have such an anchoring domain.
nonpolar residues in the core control folding and those residues responsible
for enzyme activity are conserved in the active site.
Structure of family 46 chitosanases
In addition to chitinases, there exists a family of enzymes called chitosanases, which hydrolyze chitosan. Chitosan resernbles chitin except that a
fraction of the sugar residues, say 20-60%, are glucosamine, that is
deacetylated chitin. The presence of the charged amino groups renders
chitosan a polycationic polymer. Glycohydrolase family 46 is a small one,
containing to date only a chitosanase from Streptomyces NI74 [15] and one
from Bacillus circulans [16]. These protein sequences are related to one
another but show no significant sequence homology to other glycohydrolases. The chitosanase frorn Streptomyces has been crystallized, and the
X-ray structure solved to 2.4 Aresolution [17]. The overall folding ofthis
128
J. D. Robertus and A. F. Monzingo
protein resembles that of the family 19 chitinases in the sense of having a

pronounced bilobal structure with a pronounced substrate binding c1eft. It
bears no structural resemblance to the family 18 chitinases.
A comparison of enzyme structures
Our analysis ofbarley chitinase, family 19 [13, 14], and chitosanase, family 46 [17], led us to the hypothesis that the overall folding of these two
glycohydrolases bore some resemblance to that of three other glycohydrolases [18]. These were the lysozymes from hen egg white (HEWL) [19],
bacteriophage T4, T4L [20] and GEWL, from goose [21]. These enzymes
are representatives of glycohydrolase families 22,24 and 23, respectively.
The folds ofHEWL and T4L had been compared previously, and it was
initially proposed that the two molecules were unrelated, consistent with
their lack of obvious amino acid sequence similarity [20]. It was later proposed, based on structural comparisons, that despite the lack of sequence
similarity HEWL and T4L were almost certainly related by divergent
evolution [22, 23]. Even though the proteins differed in size and complete
folding pattern, they shared several major secondary structural elements,
and their active sites appeared to bind substrate in a similar fashion. Later,
the comparison was extended to inc1ude GEWL, which also appeared to
have a similar core structure [21].
Our analysis involved a comparison in which a-helical and -sheet secondary structural elements were superimposed in a least-squares sense [18].
For example, a pairwise superposition of chitosanase and T4L, proteins of
238 and 164 amino acids, respectively, revealed that 106 residues in various
secondary structural elements occupied essentially the same relationships
in space and that they differed by a root mean square (rms) distance of only
3.7 A. In our study we compared all possible pairs ofthe five enzymes, and
these comparisons led to the conc1usion that, despite lacking any significant
sequence homology, the five proteins shared a common core structure. The
core consists of a bilobal globular domain with an elongated polysaccharide
binding site between the lobes. It is roughly 100 to 150 amino acids long and
contains a number ofhelices and sheets which occupy the same position and
orientation in space. The larger cores in enzymes like chitinase have inserts
in loop regions, but the folding pattern is the same for all the cores.
Our analysis also showed that the ancient core protein, containing the
substrate binding and catalytic site, had been added to during the evolution
toward modem glycohydrolases. The three eukaryotic members of our
comparison possess an N-terminal domain, of around 50 residues, while
the prokaryotic members of this superfamily lack such a structure. The
eukaryotes also have aC-terminal domain of about 40 residues, containing
a single a helix. The prokaryotic enzymes have a larger C-terminal domain,
inc1uding three a helices in its length of roughly 80 residues.
129
This analysis led us to conc1ude that the 150-residue conserved core of

the glycohydrolases represents an ancient glycohydrolase which has
existed since before the prokaryotic/eukaryotic split. After the split,
prokaryotes modified the core hydro lase by adding a relatively large Cterminal domain. The eukaryotes modified the core protein by adding a
small N-terminal and a small C-terminal domain. It is not c1ear how these
modifications improve the enzymes, but presumably they increased
stability or allowed a wider range of substrate affinities to evolve.
Figure 2 shows a panel of four ofthe enzymes to illustrate the conserved
cores and added units of these glycohydrolases. It shows that glycohydro-
Figure 2. Representatives of a glycohydrolase superfamily. The top row shows two eukaryotic
enzymes, (A) barley chitinase (family 19) and (B) goose lysozyme (family 23), whereas the
lower row shows two prokaryotes, (C) chitosanase (family 46) and (D) phage lysozyme (famiIy 22). In each case the ancient and conserved central core is shown as a thin gray ribbon; the
added domains are in wider ribbons, the amino terminal domains are shown in dark shading and
carboxy-terminal domains in lighter shading.
130
lase families 19,22,23,24 and 46 are in fact all distantly related. The threedimensional structure of the core is much more conservative than the
amino acid sequence and it has diverged for the different families of
enzymes over time. In fact, the amino acid sequences, although differing in
identity, have retained certain structurally important patterns which maintain the ancestral folding pattern. For example, the secondary structural
elements are anchored by conserved hydrophobic contacts in which a Phe
residue in one protein may be replaced by a Met in another, which serves
the same role; this has been described in detail elsewhere [18]. It may be
that other families of glycohydrolases also belong to this, or to other
ancient superfamilies, but this will only be revealed when enough tertiary
structural information is available to see beyond the linear structures.
Mechanisms of chitinase action
Chitinases act by hydrolytically cleaving the -glycosidic linkages between
GlcNAc residues. In general, this hydrolysis can occur in one oftwo ways,
either with retention of anomeric configuration in the product or with
inversion. This is illustrated in Figure 3. The details of glycohydrolase
mechanisms have been reviewed extensively [24].
The substrate binding cleft ofbarley chitinase is an extensive one, and it
has been hypothesized to contain at least six sugar binding subsites labeled
A-F, from the nonreducing end [14]. The hydrolytic profile for hexasaccharides by barley chitinase suggests the preferred binding of substrates
may be at sites B-G [25], that is, hexasaccharides are cleaved into two
trisaccharides. This binding mode, together with the catalytic residues, is
shown in Figure 5. Two carboxylates were hypothesized to be responsible
for the catalysis, Glu 67 as the catalytic acid and Glu 89 as a base. Hydrolysis would occur between sugars in sites D and E, a convention developed
for hen lysozyme [26, 27]. The importance of these two residues to catalysis has since been confirmed by site-directed mutagenesis [28]. Conversion of either of these acids to the corresponding amide eliminates
measurable activity. The mechanism was hypothesized to be an inverting
one, because the space between the "second carboxylate", Glu 89, and the
susceptible glycosidic bond demanded that an attacking water be interposed [14]. This inverting mechanism was confirmed using nuclear
magnetic resonance (NMR) to follow the anomeric hand of the product
sugars which were a [25]. This result is consistent with similar work
showing that the chitinase from Dioscorea opposita (yam) proceeds
with inversion ofproduct [29]. It is reasonable to assume that the family 19
chitinases all work in this way. As indicated in Figure 4, the inverting
mechanism proceeds through a positively charged oxocarbonium intermediate which has a distorted geometry; it assurnes a roughly "half-chair"
configuration compared with the chair conformation of the other sugars.
131
oy
~~
GluA
GluA
o~
HO
i-.
GluA
o~
- 0
OH
1.
H
Figure 3. Mechanisms of glycohydrolases. The upper path corresponds to a retaining mechanism where the -glycosidic linkage is preserved in the form of a product which initially exhibits the -anomeric configuration. The lower path is an inverting mechanism, in which the
product is the a anomer.
The single displacement mechanism involves Glu 89 acting as a base to

polarize the attacking water moleeule, whereas Glu 67 acts as an acid to
protonate 04 of the leaving sugar.
Barley chitinase, the archetypal enzyme of the family 19 chitinases, has
been examined kinetically [25]. Inherent fluorescence ofthe enzyme was
used to monitor the dissociation constant for binding of natural substrate
polymers. It revealed that the n-acetylglucosamine monomer does not bind
to any measurable degree while the dissociation constants for the dimer,
trimer and tetramer are 43, 19 and 6 mM, respectively. Substrate cleavage
patterns show that (G1cNAc)6 is cleaved into (G1cNAc)3 and also cleaved
asymmetrically to (G1cNAc)4 and (G1cNAcb with almost equal efficiency. (G1cNAc)4 is cleaved almost exclusively into dimers of
(GlcNAc)z. The Km for (GlcNAc)4 is 3 mM, and k cat is 35 min- 1 These
values are consistent with those measured for yam chitinase, another family 19 enzyme [30]. In addition, a fluorescent substrate, 4-methylumbelliferyl -N,N',N"-triacety1chitotrioside was used in a simple and convenient
assay to characterize kinetic parameters of the enzyme [25]; it gave
apparent Km values of 33 mM, and the k Cat was 0.33 min- 1 The pR profile
for the enzyme shows that activity is cQntrolled by a base with a PKa of
l32
t{
OH
ASN
oH
HO
/LYS165
+
H3
./
~,.. ...o 0NH-<-
OoooooHOt
C
)->m
TYR 123
OO~
~OOH
D NH-<"
ASN 199
"\
HO
OJ..-NH2
O(~H;;67
/ Hot
0j-O
GLU89
~NH
i{0W
.0
:'
.'
GLN~NHi
o
HO
NH//
LYS 86
;-J .........,
HNhG211
00",://
.0.H2N~
NH2
ci
NH~
Hot
)-NH
OH
OH
OH
Figure 4. Hypothetical binding of a chitin polymer to barley chitinase. It has not been possible
to observe binding of substrate or analogs crystallographically to this family 19 chitinase. A
model can be constructed based on the observed binding of polysaccharide to hen lysozyme,
and on kinetic data. By convention, hydro lysis is taken to occur between sugar-binding subsites labeled D and E.
3,9 (Glu 89) and an acid with a pK a of6.9 (Glu 67). Analysis ofkinetic and
dissociation constants proves that the mechanism ofbarley chitinase is consistent with aBi-Bi kinetic model for hydrolysis, with (GlcNAc)4 and water
as substrates and two (GlcNAc)2 moieties as products [25].
Family 18 chitinases have not been studied as extensively as those from
family 19. It appears that this family of enzymes operates by a retaining
133
mechanism [31, 32]. As shown in Figure 4, this might generally be expected to invo1ve two catalytic residues and to proceed through a geometrically distorted oxocarbonium intermediate. In the case of glycohydro1ases like lysozyme, the mechanism is thought to be a double
displacement type. First there is bond breaking between the D and E sugars
involving protonation of the leaving group alcohol and stabilization of the
positively charged intermediate by a second carboxylate [19, 33]. This
intermediate is then attacked by a solvent molecule which replaces the
leaving sugar group. It has been suggested, based on site-directed mutations, that Glu 204 and Asp 200 may be the catalytic residues for the
chitinase from Bacillus circulans [34]. Distortion of the D sugar by interactions between the enzyme and substrate is thought to playamajor role in
transition state stabilization [19]. It has also been suggested that the stable
juxtaposition of a carboxylate and an oxocarbonium ion is unlikely and that
a transient covalent intermediate may occur in the double displacement
mechanism [35].
It has also been suggested that the retaining mechanism of family 18
chitinases may involve substrate assistance. That is, the N-acetyl group at
position 2 of the scissile sugar may itself facilitate the reaction via formation of a transient oxazolinium intermediate [36, 32]. This view is supported by the crystal structure of a complex between the chitinase called
hevamine and the inhibitor allosamidin which contains an oxazoline
moiety which mimics the transition state of the reaction [36].
In summary, the family 18 chitinases are basically eight-stranded a/
barrels which operate by a retaining catalytic mechanism, perhaps with
substrate assistance. The family 19 chitinases are bilobal structures with an
ancient core structure of a helices and a three-stranded sheet; these
chitinases act by an inverting mechanism involving action of enzyme-supplied acid and base.
Acknowledgements
This work was supported by grants from the National Institutes of Health (GM 30048), the
National science Foundation and by grants from the Foundation for Research and the Welch
Foundation.
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1999 Birkhuser Verlag BasellSwitzerland
Classification of chitinases modules

Bemard Henrissat
Architecture et Fonction des Macromolecules Biologiques CNRS, 31 Chemin JosephAiguier,
F-13402 Marseille Cedex 20, France
Summary. Chitinases frequently displayamodular structure featuring a catalytic domain
attached to one or several ancillary noncatalytic domains whose function is often chitin binding.
Gene cloning and DNA sequencing have allowed the determination of a massive number of
amino acid sequences of chitinases during the last 10 years. This chapter presents a uni:IYing
classification system of the various chitinase modules that combines specific features of their
sequences, three-dimensional structures and reaction mechanisms.
Classification of chitinases modules based on sequence similarities

Chitinases are the glycoside hydrolases that hydrolyse the -l ,4-glycosidic
bonds between the N-acetylglucosamine residues of chitin. Although
chitinases are classified under a single IUB-MB enzyme number (EC
3.2.1.14), early comparisons oftheir amino acid sequences have revealed
that their catalytic domains could be grouped in two different families [3],
that is families 18 and 19 of the classification of glycoside hydrolases
based on amino acid sequence similarities [4-6]. Since sequence conservation is indicative of structural similarities, this classification system also
correlates with the structural and mechanistic features of these enzymes
[7]. One of the features of the classification of glycoside hydrolases in
families of related proteins is that, once identified for one member, the
catalytic residues can be deduced for the other members as these residues
are invariant in each family [4]. Similarly, it has been shown that the molecular mechanism is conserved within the families [8]. Similarly to their
catalytic domains, when subjected to independent sequence analysis, the
noncatalytic domains of chitinases can be grouped into a number of distinct
families.
Gene cloning and DNA sequencing have allowed the determination of a
massive number of amino acid sequences of chitinases during the last 10
years. The sections below list the various families of chitinase catalytic and
noncatalytic domains known so far.
Catalytic domains
Family 18
This is the largest chitinase family, with about 180 members found in
eukaryotes, prokaryotes and viruses (Tab. 1). In addition to a vast majority
138
B. Henrissat
Table I. Classification of chitinases and related enzymes in families of related catalytic

domains (compiled in February 1998)
Organism
ECno.
Swiss-Prot EMBLI
GenBank
Glycoside hydrolase family 18

chitinase
chitinase
chitinase
chitinase 1
chitinase 2 (3 repeats)
chitinase A
chitinase
chitinase 1
chitinase 2
chitinase 3
chitinaseA
chitinase C
chitinase 1
chitinase 2
Acanthocheilonema viteae
Autographa californica
nuclear polyhedrosis virus
Aedes aegypti
Aedes aegypti
Aeromonas caviae
Aeromonas sp.
Aeromonas sp. IOS-24
Aeromonas sp. 10S-24
Aeromonas sp. IOS-24
Alteromonas sp.
Alteromonas sp.
Anopheles freeborni
Anopheles freeborni
3.2.1.14
3.2.1.14
3.2.1.14
P41684
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
P32823
P20533
P27050
089568
010594
Anopheles gambiae
Anopheles gambiae
chitinase C 1
chitinase 0
Bacillus circulans
chitinase
unknown protein
chitinase
3.2.1.14
Bacillus subtilis
n.d.
P37531
Bacillus thuringiensis
chitinase SE2
putative chitinase
chitinase-like protein 1
chitobiase
Beta vulgaris
3.2.1.14
3.2.1.14
P36910
Bombyx mori
n.d.
Bos taurus
Bos taurus
unknown
3.2.1.30 Q01458
Anopheles stephensi
Anopheles stephensi
Anopheles stephensi
Aphanocladium album
Arabidopsis thaliana
Arabis gemmifera
Arabis glabra
Arabis lyrata
Aspergillus nidulans
Aspergillus nidulans
Bacillus circulans
Bacillus circulans
AF026491
AF026492
U09139
031818
063139
063139
063139
013762
AB004557
AF026495
AF026496
AF008575
AF026493
AF026494
AF026497
AF026498
AF026499
X64104
M34107
AB006070
AB006071
AB006072
D87063
087895
M57601
chitinase 1
chitinase 2
chitinase 3
chitinase 1
chitinase 2
chitinase 3
chitinase 1
chitinaseA
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase AI
Anopheles gambiae
U14638
L42010
L22858
P32470
P19172
U71214
026185
U89796
S66038
U86876
AFOI1373
M95770
139

Table 1 (continued)
oviduct glycoprotein
chitinase
chitinase
chitinase
chitinase CHT I
ORF C04F6.3
ORF C08H9.10
ORF C08H9.12
ORF C08H9.14
ORFC08H9.6
ORFF07GII.9
ORF F15A4.8
ORF M176.8
ORF R09DI.1
ORF R09D1.10
ORF R09D1.11
ORF R09D1.3
ORFR09D1.6
ORFR09D1.7
ORF R09D1.8
ORF R09D1.9
ORFTOIC4.1
ORFTl3H5.3
ORF ZK938.6
(2 repeats)
ORFTl9H5.1
(2 repeats)
concanavalin B
putative narbonin
chitinase 1
chitinase 2
chitinase 3
Organism
ECno.
Bos taurus
Brugia malayi
Brugia pahangi
Brugia pahangi
Caenorhabditis elegans
unknown Q28042
3.2.1.14 P29030
3.2.1.14
3.2.1.14
3.2.1.14
n.d.
QI1174
n.d.
n.d.
Dl6639
M73689
n.d.
n.d.
Z54342
Z54342
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
AF016419
Z81062
Z78412
Swiss-Prot EMBLI
GenBank
U59689
U59690
AF026152
U42835
Z54342
Z54342
Z70035
Z70035
Z70035
Z70035
Z70035
Z70035
Z70035
Z70035
U70858
Z66524
n.d.
Z49913
n.d.
Z47745
Canavalia ensiformis
Canavalia ensiformis
Candida albieans
Candida albieans
Candida albieans
unknown
unknown
3.2.1.14
3.2.1.14
3.2.1.14
chitinase
Chelonus sp. venom
3.2.1.14
Ul0422
chitinase
chitinase
Chironomus tentans
Chironomus tentans
3.2.1.14
Y13233
Y13234
chitinase
chitinase
Choristoneura fomiferana
nuc1eopolyhedrovirus
Geer arietinum
chitinase B
chitinase
chitinase 1
Clostridium paraputrijieum
Clostridum thermocellum
Coecidioides immitis
P49347
P46876
P40953
P40954
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
X83426
Z46802
U36490
Ul5800
Ul5801
Un030
P36908
X70660
ABOO1874
Z68924
P54196
L41663
140
B. Henrissat
Table 1 (continued)
Organism
ECno.
Swiss-Prot EMBLI
GenBank
chitinase 2
Coccidioides immitis
3.2.1.14
P54197
L41662
chitinase
Coccidioides immitis
Cucumis sativus
3.2.1.14
3.2.1.14
P17541
U60807
M24365
chitinase
ORF 1
Cucumis sativus
n.d.
M84214
ORF3
Cucumis sativus
n.d.
M84214
chitinase 1
Drosophila melanogaster
3.2.1.14
AF026500
chitinase 2
chitinase 3
3.2.1.14
3.2.1.14
AF026502
chitinase 4
3.2.1.14
AF026503
n.d.
U13825
Entamoeba dispar
Entamoeba histolytica
Entamoeba invadens
3.2.1.14
3.2.1.14
3.2.1.14
U78318
chitinase-like
chitinase
chitinase
chitinase
chitinase
AF026501
U78319
U78320
Enterobacter agglomerans
3.2.1.14
n.d.
3.2.1.14
P13656
U18997
chitinase
Escherichia coli
Ewingella americana
endo-N-acetylglucosaminidase Fl
Flavobacterium
meningosepticum
3.2.1.96
P36911
M80793
endo-N-acetylglucosaminidase F2
Flavobacterium
meningosepticum
3.2.1.96
P36912
L06331
endo-N-acetylglucosaminidase F3
Flavobacterium
meningosepticum
Flavobacterium sp.
3.2.1.96
P36913
L06332
3.2.1.96
P80036
3.2.1.14
unknown
3.2.1.14
chitinase
Glossina morsitans
Glycin max
Helicoverpa zea nuc1ear
polyhedrosis virus
Hevea brasiliensis
chitinase
Homo sapiens
3.2.1.14
U58514
U58515
chitinase
(chitoriosidase)
Homo sapiens
3.2.1.14
U29615
YHEB pro tein
endo-N-acetylglucosaminidase
chitinase
putative narbonin
chitinase
3.2.1.14
U59304
X90562
YI1391
Z46825
U67265
P23472
chitobiase
Homo sapiens
3.2.1.30
Q01459
M95767
gp-39 protein
Homo sapiens
unknown P36222
M80927
Homo sapiens
unknown Q12889
U09550
YKL-39 precursor
Homo sapiens
U49835
chitinase
chitinase
Hyphantria cunea
Janthinobacterium lividum
unknown
3.2.1.14
Killer toxin
Kluyveromyces lactis
3.2.1.14
3.2.1.14
chitinase
Kurthia zopjii
3.2.1.14
D63702
Macaca mulatta
unknown
U87259
U86877
U07025
P09805
X07127
141
Table 1 (continued)
chitinase
chitinase
chitinase CHIT42
BRP39 protein
ECF-L precursor
secretory protein
YM-l
chitinaseA
Organism
ECno.
Manduca sexta
Mesocricetus auratus
Metarhizium anisopliae
Metarhizium anisopliae
3.2.1.14 P36362
unknown Q60557
3.2.1.14
3.2.1.14
Mus musculus
unknown
Mus musculus
unknown
unknown Q62010
Mus musculus
Mus musculus
Swiss-Prot EMBU
GenBank
unknown
U02270
032218
X89212
AF027497
X93035
D87757
032137
M94584
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
P29060
Orgyia pseudotsugata
multicapsid polyhedrosis virus
n.d.
010363
Oryza sativa
3.2.1.14
unknown Q28542
unknown P36718
3.2.1.14
AB003195
Ul6719
M59903
U42580
Paramecium bursaria
Chlorella virus 1
n.d.
U42580
chitinase
chitinase 1
Parthenocissus quinquifolia
chitinase 2
chitinase 3
chitinaseA
chitinase
chitobiase
chitinase
chitinase I
chitinase II
chitinase III
chitinase
ORFD9481.7
Penaeus japonicus
Penaeusjaponicus
Phaseolus angularis
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.30
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
chitinase
Serratia marcescens
n.d.
3.2.1.14
chitinase
Serratia marcescens
3.2.1.14
chitinaseA
chitinase B
putative chitinase
chitinaseA
Serratia marcescens
3.2.1.14
3.2.1.14
Nicotiana tabacum
chitinase B
chitinase
Nicotiana tabacum
chitinase
chitinase (putative)
Onchocerca volvulus
chitinase III a
chitinase
(GI-1181344)
ORF (GI-1181344)
Onchocerca volvulus
Ovis aries
Papio anubis
Paramecium bursaria
Chlorella virus 1
Penaeusjaponicus
Psophocarpus tetragonolubus
Rattus norvegicus
Rhizopus niveus
Rhizopus oligosporus
Saccharomyces cerevisiae
Serratia marcescens
Sesbania rostrata
Stenotrophomonas maltophilia
n.d.
3.2.1.14
Z11563
Ul4639
Ul4639
L42021
U75930
P23473
D84250
P29024
Q01460
P29025
P29026
P29027
P29028
D89751
AB008027
011335
D49953
M95768
010154
010157
010158
D87894
M74070
U28373
L38484
L41660
P07254
P11797
L01455
X15208
Z48674
AF014950
142
B. Henrissat
Table 1 (continued)
chitinase
chitinase
chitinase A
chitinase C
exo-chitinase
chitinase-63
end-N-acetylglucosaminidase H
chitinase
heparin-binding
glycoprotein
Organism
ECno.
Streptomyces coelicolor
Streptomyces erythaeus
3.2.1.14
3.2.1.14
P14529
Streptomyces lividans
3.2.1.14
3.2.1.14
n.a.
3.2.1.14
3.2.1.96
P36909
Q05638
Pl1220
P04067
Streptomyces olivaceoviridis
Streptomyces thermoviolaceus
Sus scrofa
Sus scrofa
chitinase
chitinase
chitinase
chitinase
chitinase
chitodextrinase
chitinaseA
narbonin
Trichoderma hamatum
Trichoderma hamatum
putative narbonin
putative narbonin
putative narbonin
narbonin
narbonin
narbonin-like
chitinase
chitinase
chitinase
PRM 3 protein
Viciafaba
Viciafaba
Trichoderma harzianum
Trichoderma harzianum
Vibrio furnissii
Vibrio jUrnissii
Vibrio harveyi
Viciafaba
8wiss-Prot EMBLI
GenBank
AL021411
D13775
D12647
X71080
M82804
K02182
3.2.1.14
unknown
Dl4536
U19900
unknown Q28990
3.2.1.14
3.2.1.14
3.2.1.14 P48827
3.2.1.14
3.2.1.14
U43490
n.a.
3.2.1.14
unknown
Z71415
U88560
L14614
X80006
L42548
U41418
U81496
Z46910
Vitis vinifera
unknown
3.2.1.14
3.2.1.14
3.2.1.14 P51614
Zea mais
unknown
Z46827
Q41660
Z46834
Z25536
Z33641
Z46835
X88801
X88802
Z68123
882314
3.2.1.14
3.2.1.14
M94105
M94106
Viciafaba
Vicia narbonensis
Vicia pannonica
Vicia sativa
Vigna unguiculata
Vigna unguiculata
Glycoside hydrolase family 19

Allium sativum
chitinase 1
Allium sativum
chitinase 2
chitinase
unknown
unknown
unknown
unknown
unknown
chitinase
(gene FI8019.27)
3.2.1.14
3.2.1.14
Z25683
ACOO2333
chitinase
(gene FI8019.28)
chitinase
(gene F18019.31;
T01024.32)
3.2.1.14
ACOO2333
3.2.1.14
ACOO2333
143
Table 1 (continued)
chitinase
(gene FI8019.32;
T01024.31)
chitinase
(gene T01024.33;
FI8019.30)
chitinase
(gene T01024.34;
FI8019.29)
chitinase B
chitinase CHIV
chitinase lA
chitinase 1B
chitinase 2
chitinase 2.1
chitinase 2.2
chitinase 3
chitinase 4
chitinaseA
chitinase B
chitinase
chitinase 4
chitinase SP2
chitinase
Organism
ECno.
3.2.1.14
ACOO2333
3.2.1.14
ACOO2333
3.2.1.14
ACOO2333
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Arachis hypogaea
Beta vulgaris
Beta vulgaris
Beta vulgaris
Brassica napus
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.2.1.14
3.2.1.14
chitinase B4
chitinase-25
ORFC08B6.4
ORF C51E3.8
ORF RI0Dl2.l5
ORFT05H4.7
ORFT26F2.1
ORFT26H2.1
chitinase 1B
chitinase
Brassica napus
Brassica napus
chitinase
chitinase
chitinase
Chenopodium amaranticolor
3.2.1.14
3.2.1.14
3.2.1.14
chitinase
Citrus sinensis
3.2.1.14
chitinase
Coix lachryma-jobi
3.2.1.14
chitinase 1
chitinase
chitinase
chitinase
Cynodon dactylon
Daucus carota
Daucus carota
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
Castanea sativa
Daucus carota
Swiss-Prot EMBU
GenBank
P19171
Q06012
Q06013
P42820
Q06209
Q09023
M38240
Y14590
Q06012
Q06013
Q06014
X82329
X82330
Q06015
Q06016
X56890
X56891
X79301
A23392
L25826
U21848
X61488
M95835
Z72502
Z78410
Z81109
AFOI6452
Z82054
Z82054
U48687
D45182
D45183
D45184
D45181
Z70032
P15326
AF020766
U52845
U52847
U52848
144
B. Henrissat
Table 1 (continued)
chitinase
chitinase
chitinase
chitinase
ORFHI1415
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase 26
chitinase 33
chitinase
chitinaseA
chitinase
chitinase
chitinaseA
chitinase B
chitinase C
chitinase D
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
Organism
ECno.
8wiss-Prot EMBLI
GenBank
Dioscorea japonica
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
n.d.
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
P80052
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Haemophilus injluenzae
Helianthus annuus
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Hordeum vulgare
Linum usitatissimum
Lycopersicon chilense
Lycopersicon esculentum
Medicago sativa
Medicago truncatula
Nicotiana
Nicotiana
Nicotiana
Nicotiana
tabacum
tabacum
tabacum
tabacum
Nicotiana tabacum
Nicotiana tabacum
P44187
P1l955
P23951
Q40114
Q05539
Q05540
Q05538
Q05537
P08252
P17513
P17514
P24091
P29059
U60197
U78888
Z68152
U32821
U96640
X15349
U02287
X78671
X78672
L34210
L34211
U94847
Ll9342
U30465
A32906
Z15141
Z15139
Z15140
Z15138
U83591
YI0373
X16938
M29869
M29868
X51599
X64518
844869
Oryza sativa
3.2.1.14
3.2.1.14
3.2.1.14
A16119
A21091
AFOO1500
chitinase
Oryza sativa
3.2.1.14
chitinase
chitinase
Oryza sativa
3.2.1.14
3.2.1.14
AFOO1501
AF013580
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
Oryza sativa
chitinase
chitinase
chitinase
Nicotiana tabacum
Nicotiana tabacum
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
AF013581
ABOO3194
D16222
D16223
L37289
L40338
X56063
145

Table 1 (continued)
chitinase
chitinase
chitinase
chitinase
chitinase 1
chitinase 2
chitinase
chitinase
chitinase
chitinase 4
chitinase 5
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase 2
chitinase 6
chitinase 8
chitinase B
chitinase C
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase
chitinase I
chitinase 2
chitinase 3
Swiss-Prot EMBLI
GenBank
Organism
ECno.
Oryza sativa
3.2.1.14
3.2.1.14
X87109
Z29961
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
U02286
Z29962
X54367
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Persea americana
Petunia hybrida
Phaseolus vulgaris
Phaseolus vulgaris
Phaseolus vulgaris
Picea glauca
Pinus strobus
Pinus taeda
Pisum sativum
Pisum sativum
Pisum sativum
Pisum sativum
Pisum sativum
Populus sp.
Populus sp.
Populus trichocarpa
Populus trichocarpa
Sambucus nigra
Sambusu nigra
Solanum tuberosum
Solanum tuberosum
Solanum
Solanum
Solanum
Solanum
tuberosum
tuberosum
tuberosum
tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
chitinase 4
chitinase ChtA4
Solanum tuberosum
3.2.1.14
3.2.1.14
Solanum tuberosum
3.2.1.14
chitinase C
chitinase
Theobroma cacao
3.2.1.14
3.2.1.14
chitinase
chitinase
Triticum aestivum
Triticum aestivum
3.2.1.14
3.2.1.14
P24626
P25765
P29021
P06215
P27054
P36361
P36907
P21225
P21226
P21227
PI6579
PI6061
P29031
P29032
P05315
P52403
P52404
P52405
P52406
Z78202
X51427
M13968
X57187
S43926
L42467
U57410
U31309
X63899
L37876
L37876
UOl661
M25337
X59995
X59995
Z46948
Z46950
X07130
AF024537
XI4133
X67693
U49969
U49970
U02605
U02606
U02607
U02608
AF024538
ABOO9289
U30324
X76041
X95000
146
B. Henrissat
Table 1 (continued)
chitinase
chitinase
chitinase 1
chitinase 4
chitinase
chitinase
chitinase B
chitinase
chitinase
chitinaseA
chitinase B
Organism
ECno.
Ulmus americana
Urtica dioica
Vigna unguiculata
Vigna unguiculata
Vitis vinifera
Vitis vinifera
Vitis vinifera
Zeamays
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
Zeamays
Zeamays
Zeamays
3.2.1.14
Swiss-Prot EMBLI
GenBank
Pll218
P51613
P29022
P29023
L22032
M87302
X88800
X88803
U97521
U97522
Z54234
L16798
LOO973
M84164
M84165
n. a., EC number not available, n. d., enzymatic activity not determined; unknown, unknown
enzymatic activity; none, no enzymatic activity; ORF, open reading frame. When a particular
module is known to be present in multiple copies in a protein, the number of repeats is indicated.
of chitinases, this family contains enzymes such as chitodextrinase, an

exochitinase, several di-N-acetyl chitobiases (EC 3.2.1.30) and endo-Nacetylglucosaminidases (EC 3.2.1.96). A number ofproteins ofunknown
function are also found in this family, such as mammalian oviduct glycoproteins and several putative proteins found during the systernatic sequencing of the nematode Caenorhabditis elegans (Tab. 1). An interesting case
is the occurrence of several plant proteins such as concanavalin Band
narbonin, which have no catalytic activity [9, 10].
Chitinases offamily 18 have been found to operate with overall retention
ofthe anorneric configuration at the cleavage point, implying a double displacement rnechanism [11-13]. The three-dimensional (3D) structures of
several members of this family have been determined: examples include
Serratia marcescens chitinase A [14], the chitinase frorn Hevea brasiliensis [15, 16] and the endo--N-acetylglucosaminidases from Flavobacterium meningosepticum [17] and Streptomyces plicatus [18]. Although the
sequence similarity between these three proteins is small, they share a
similar (/a)g fold (Fig. 1). For a detailed description ofthe 3D structures
of chitinases, see Robertus and Monzingo, this volume.
Retaining glycosidases usually perform their catalysis utilising two
carboxylic catalytic residues, one protonating the glycosidic oxygen and
the other providing nucleophilic assistance and leading to the formation of
a glycosyl-enzyme intermediate. Chitinases offamily 18, like other glycoside hydrolases, have a carboxylic residue which protonates the glycosidic
oxygen in the first step of catalysis. However, these enzymes are unusual in
that they lack an enzymic nucleophile, and it is instead the acetamido group
147
l hvq
Ihev
laiw
2baa
l ttg
lexg
lctn
Figure I. Ribbon representation of the main fold of the various modules identified in chitinases
or homologous to chitinase modules. G1ycoside hydro lase family 18: I HVQ, chitin ase of Hevea
brasiliensis [15, 16]; glycoside hydrolase family 19: 2BAA, chitin ase of Hordeum vulgare [24,
25] ; fibronectin type III modules: I TTG, module 10 ofhuman fibronectin [38]; chitin binding
domain family 2: IHEY, hevein of Hevea brasiliensis [40] ; chitin binding domain family 3:
IAIW, cellulose binding domain of the cellulase Z of Erwinia chrysanthemi [33]; cellulose
binding domain type II: I EXG, cellulose binding domain ofthe Cex xylanase of Cellulomonas
fimi [35]; module wl: ICTN, N-terminal domain of Serratia marcescens chitinase [14]. The
figure was prepared using the MOLSCRIPT program [41].
of the substrate that provides intramolecular nucleophilic assistance to the

departure of the aglycone, presumably resulting in an oxazolinium ion
intermediate [12, 19- 21]. Interestingly, narbonin and concanavalin B, the
family 18 proteins devoid of catalytic activity, lack the protonating catalytic
residue [9]. Several proteins listed in Table 1 are probably noncatalytic, as
they also lack the catalytic residue.
Although several chitinases of family 18 comprise only a catalytic
domain, most have a modular structure with one or several noncatalytic
domains which can be found indifferently at their N- or C-termini. A number of these noncatalytic domains function as chitin binding domains.
Some members of family 18 display an extremely large and complex
modular structure such as the chitinase 2 of Aedes aegypti with three chitin
148
B. Henrissat
binding domains and three catalytic domains, or the YHEB protein of

Escherichia coli, which bears no less than five putative polysaccharide
binding domains and one catalytic domain. The largest modular sequence
(2025 residues) in this family is that encoded by the F07G 11.9 gene of the
nematode C. elegans (Tab. 1). Besides its catalytic domain, this huge protein
contains 12 copies of a module found only in the nematode genome.
Family 19
This family counts more than 130 members, which are all from plant origin
except for a chitinase from Streptomyces griseus [22], a putative protein
from Haemophilus injluenzae and several putative proteins found in the
genome of C. elegans (Tab. 1). Family 19 chitinases have been found to
perform their hydrolytic reaction with inversion of the anomeric configuration at the site of cleavage [13,22,23], indicating majordifferences in the
geometry of their active sites compared with family 18 enzymes.
The 3D structures ofthe chitinase from barley has been determined [24,
25]. Its global fold is completely different from that observed in family 18,
as the structure is essentially composed of a-helices (Fig. 1). A detailed
description of the 3D structures of barley chitinase is found in Robertus
and Monzingo, this volume. Although the first examination ofthe structure
had led to the proposal that the enzyme worked by a retaining lysozymetype mechanism [24], it is now clear that this enzyme, like all other studied
family 19 chitinases, operates by inversion of configuration [26].
Catalysis by inverting glycosidases involves a single nucleophilic
substitution by a water moleeule. The catalytic machinery is made of two
carboxylic catalytic residues separated by a distance greater than that of
retaining enzymes [27]. One of these residues protonates the glycosidic
oxygen, while the other activates the water molecule. The two catalytic
residues of barley chitinase are strictly invariant in family 19.
Several chitinases of family 19 are made of a single catalytic domain
with no ancillary domain. However, many family 19 chitinases displaya
modular structure with an N-terminal chitin binding domain homologous
to wheat germ agglutinin. Contrary to family 18, which contains several
members with a large number of modules, only one enzyme, the chitinase
of Urtica dioica, is made of more than two modules and contains a tandem
repeat of two N-terminal chitin binding domains upstream of its catalytic
domain.
Related enzymes
So far chitinases have been found exclusively in the two families
mentioned above. Enzymes acting on somewhat similar substrates
(N-acetylglucosaminidases, lysozymes and chitosanases) can be found in
other glycoside hydrolase families. For instance, N-acetylglucosaminidases can be found in families 3 and 20, lysozymes in families 22-25 and
chitosanases in family 46 [4-6].
149
Noncatalytic domains
Sequence analysis of cellulases and chitinases has revealed that the catalytic domains of these enzymes form a number of different families [2-6,
28]. Similarly, the cellulose binding domains of cellulases and chitin
binding domains of chitinases have been c1assified in several families
based on sequence similarities [2, 29-32]. Given the resemblance between
the two target polysaccharides, it is not surprising that some cellulose and
chitin binding domains display cross-specificity [31]. The precise function
of polysaccharides binding domains has been carefully evaluated only in a
few cases, and the function of most of these domains has been inferred
from sequence data. There are chitin binding proteins that exist as such
(e. g. not associated with a catalytic domain), and these are reviewed by
Schrempf, this volume.
Chitin binding domains
There are three families of chitin binding domains identified so far
(Tab. 2). 3D structures have been determined for members of the second
family (Fig. 1). Whereas the first two families contain modules found only
in chitinases or in nonhydrolytic proteins such as wheat germ agglutinin or
insect intestinal mucin, modules belonging to the third family have also
been found in several cellulases and a serine protease. In the cellulase Z of
Erwinia chrysanthemi, this module binds strongly to cellulose, and its 3D
structure has been solved [33] (Fig. 1).
Cellulose binding domains
Cellulose binding domains have been c1assified in a number of families [2,
29-32]. Several chitinases contain modules which are c1early homologous to
cellulose binding domains of family II (Tab. 2). This family comprises over
50 other members which are found in cellulases, xylanases, acetyl xylan
esterases and arabinofuranosidases. Interestingly, some family II cellulose
binding domains of cellulases have been shown to bind chitin [34]. The affinity of the corresponding modules of chitinases for cellulose or chitin has
not been investigated. The 3D structure of the cellulose binding domain of
the xylanase Cex of Cellulomonas fimi has been solved [35] (Fig. 1), and the
corresponding modules of chitinases most likely have an identical fold.
Fibronectin type lII-like modules
Several discrete modules displaying significant sequence similarity with
fibronectin type III modules have been identified in a number of bacterial
depolymerases (cellulases, polyhydroxybutyrate depolymerases, pullolanases, exomaltopentaohydrolases), inc1uding chitinases [36] (Tab. 2). It is
like1y that the bacterial modules were initially acquired from an animal
source and were then spread further between distantly related bacteria by
horizontal transfers [36]. In several instances, these fibronectin type IIIlike modules occur as tandem repeats (Tab. 2). The function ofthese modu-
150
B. Henrissat
Table 2. Classification of noncatalytic modules of chitinases in families of related sequences

(compiled in February 1998)
Organism
ECno.
Swiss-Prot EMBLI
GenBank
Chitin-binding domains Family I

chitinase 1
Aedes aegypti
3.2.1.14
AF02649I
Aedes aegypti
3.2.1.14
AF026492
putative chitinase
Bombyxmori
n.d.
U86876
unknown (GI-1049446)
(l0 repeats)
Caenorhabdits elegans
n.d.
U39678
chitinase
chitinase
Hyphantria cunea
3.2.1.14
3.2.1.14
chitinase
Manduca sexta
Penaeus japonicus
chitinase
Penaeus japonicus
P36362
U86877
U02270
3.2.1.14
3.2.1.14
D84250
D98751
tachycitin
Tachypleus tridentatus
unknown
D85756
inscct intestinal mucin

IIMI4 (5 repeats)
Trichoplusia ni
unknown
AFOO0605
Chitin-binding domains Family 2

chitinase B
3.2.1.14
hevein
unknown P43082
chitinase SP2
chitinase 25
Beta vulgaris
3.2.1.14
P42820
L25826
Brassica napus
chitinase B4
Brassica napus
3.2.1.14
3.2.1.14
Q09023
Q06209
M95835
X61488
ORF F07G 11.9

(2 repeats)
n.d.
AF016419
n.d.
U70858
Z66524
U48687
D45181
D45182
ORF TOl C4.1 (2 repeats) Caenorhabditis elegans

ORF Tl3H5.3 (3 repeats) Caenorhabditis elegans
chitinase I B
Castanea sativa
chitinase
chitinase
n.d.
3.2.1.14
3.2.1.14
3.2.1.14
chitinase
3.2.1.14
PI9171
M38240
UOl880
D45183
D45184
chitinase
chitinase
Dioscorea japonica
3.2.1.14
Gossypium hirsutum
3.2.1.14
chitinase
Gossypium hirsutum
3.2.1.14
U60197
hevein (major)
Hevea brasiliensis
M36986
chitinase
3.2.1.14
P80052
U78888
hevein (minor)
Hevea brasiliensis
unknown P02877
unknown P80359
chitinase
Hordeum vulgare
3.2.1.14
P1l955
XI5349
root-specific lectin
(4 repeats)
Hordeum vulgare
none
P15312
M29280
killer toxin
3.2.1.14
P09805
X07127
chitinase C
chitinase
Q05538
ZI5140
Medicago sativa
3.2.1.14
3.2.1.14
chitinase
Medicago truncatula
3.2.1.14
Nicotiana tabacum
3.2.1.14
chitinase
U83591
P08252
YI0373
XI6938
151
Table 2 (continued)
chitinase
chitinase
agglutinin (4 repeats)
chitinase
chitinase
chitinase
chitinase
chitinase 1
chitinase 2
chitinase
antifungal chitin-binding
protein
chitinase
Organism
ECno.
8wiss-Prot EMBLI
GenBank
Nicotiana tabacum
3.2.1.14
3.2.1.14
none
3.2.1.14
3.2.1.14
P24091
P29059
Pl1219
X51599
X645 18
P24626
X54367
Nicotiana tabacum
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
Oryza sativa
3.2.1.14
3.2.1.14
3.2.1.14
Persea americana
3.2.1.14
3.2.1.14
Pharbitis nil
unknown
Phaseolus vulgaris
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
chitinase 4
chitinase 5
chitinase
chitinase
chitinase
chitinase
chitinase 6
chitinase 8
Phaseolus vulgaris
Phaseolus vulgaris
chitinase B
chitinase C
chitinase
chitinase 1
chitinase 2
chitinase 3
chitinase 4
wound-induced protein
wound-induced protein
chitinase
Populus trichocarpa
Populus trichocarpa
Picea glauca
Pisum sativum
Pisum sativum
Pisum sativum
Populus sp.
Populus sp.
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
M24504
Dl6223
L37289
X56787
X56063
P25765
Z78202
L41872
P06215
P27054
P36361
P21226
P36907
P21227
P16579
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.l.l4
3.2.1.14
3.2.1.14
3.2.1.14
unknown
unknown
3.2.1.14
P16061
P29031
P29032
P05315
P52403
P52404
P52405
P52406
P09762
P09761
M13968
X57187
843926
L42467
L37876
X63899
U01661
M25337
X59995
X59995
agglutinin 1 (4 repeats)
Triticum aestivum
none
P10968
X07130
U02605
U02606
U02607
U02608
X13497
X13497
U30324
M25536
Triticum aestivum
none
Triticum aestivum
none
P02876
P10969
M25537
J02961
chitinase
chitinase
Triticum aestivum
3.2.1.14
X76041
Ulmus americana
3.2.1.14
L22032
chitinase (2 repeats)
chitinase 1
chitinase
Urtica dioica
chitinase
chitinase
Vitis vinifera
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Theobroma cacao
Vigna unguiculata
Vitis vinifera
Vitis vinifera
Pl12l8
P51613
M87302
X88800
Z54234
U97521
U97522
152
B. Henrissat
Table 2 (continued)
chitinase A
chitinase B
Organism
ECno.
Swiss-Prot EMBLI
GenBank
Zea mays
Zea mays
3.2.1.14
3.2.1.14
P29022
P29023
M84164
M84165
P32823
U09139
D31818
D13762
AB004557
D63139
Chitin-bin ding domains Family 3

Aeromonas caviae
chitinase A (2 repeats)
Aeromonas sp.
chitinaseA
Alteromonas sp.
chitinase C
Alteromonas sp.
chitinase AI
Bacillus circulans
chitinase D
Bacillus circulans
chitinase
chitinase B
Clostridium paraputrijicum
YHEB protein (5 repeats) Escherichia coli
Janthinobacterium lividum
chitinase
Kurthia zopjii
chitinase B
chitinase C
chitodextrinase
chitinase A
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
n.d.
3.2.1.14
P20533
P27050
P13656
D63139
D63139
M57601
Dl0594
U71214
ABOO1874
U18997
U07025
3.2.1.14
3.2.1.14
3.2.1.14
Vibrio jurnissii
Vibrio harveyi
n.a.
3.2.1.14
AB009289
U41418
U81496
3.2.1.14
3.2.1.14
U89796
U42580
Serratia marcescens
Cellulose-binding domains type 11

chitinase
chitinase (GI-1181344) Paramecium bursaria
Chlorella virus 1
chitinase C
chitinase
Fibronectin type III-like modules
chitinase AI (2 repeats) Bacillus circulans
Bacillus circulans
chitinase D
D63702
X15208
3.2.1.14
3.2.1.14
P36909
Dl2647
AL021411
P20533
M57601
Dl0594
U71214
chitinase
chitinase
3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
chitinase 63
chitinaseA
chitinase A
chitinase C
exo-chitinase
Kurthia zopjii
3.2.1.14
3.2.1.14
Stenotrophomonas maltophilia 3.2.1.14
3.2.1.14
3.2.1.14
3.2.1.14
Streptomyces olivaceoviridis
n.a.
P11797
P27050
U89796
PI1220
P36909
Q05638
D63702
M82804
AF014950
D13775
Dl2647
AL021411
X71080
153

Table 2 (continued)
Moduleswl
chitinaseA
chitinaseA
chitinase
chitinase
chitinase
chitinase
Organism
EC no.
Aeromonas caviae
3.2.1.14
3.2.1.14
3.2.1.14
Alteromonas sp.
Autography californica
nuclear polyhedrosis virus
Choristoneura jitmiferana
nucleopolyhedrovirus
Enterobacter agglomerans
chitinaseA
Helicoverpa zea nuclear

polyhedrosis virus
Orgyia pseudotsugata nuclear
polyhedrosis virus
Serratia marcescens
Modules xl
chitinase C
Alteromonas sp.
chitinase (putative)
chitodextrinase
chitinaseA
Swiss-Prot EMBLI
GenBank
P32823
P41684
U09139
013762
L22858
3.2.1.14
U72030
3.2.1.14
3.2.1.14
U59304
U67265
n.d.
010363
U75930
3.2.1.14
P07254
LOl455
3.2.1.14
AB004557
n.a.
Stenotrophomonas maltophilia 3.2.1.14
U41418
AF014950
Vibrio jitrnissii
n. a., EC number not available; n. d., enzymatic activity not determined; unknown, unknown
enzymatic activity; none, no enzymatic activity; ORF, open reading frame. When a particular
module is known to be present in multiple copies in a protein, the number of repeats is indicated.
les in depolymerases is not known. Watanabe et al. [37] have shown that the
deletion ofthe fibronectin type III-like module of B. circulans chitinaseAI
significantly reduces the activity of the enzyme on chitin, but not on
soluble substrates. The 3D structure of the human fibronectin type III
module is known [38] (Fig. 1).
Other modules
An N-tenninal module was identified during the detennination of the 3D
structure of chitinase A of Serratia marcescens [14]. This module shows some
distant structural similarities with fibronectin type III domains albeit with
significant differences [14] (Fig. I). It does not bear any sequence similarity
to typical type III domains offibronectin or with the fibronectin type III-like
domains described in the preceding section, and is found in several other
chitinases (Tab. 2, module wl). There are probably several other types of
chitinase modules left to identify. An example is a stretch of- 80 residues conserved in several chitinases which is found in two copies in a cellulase from
Clostridium thermocellum (Tab.2, module xl). Similarly, genes FIOG2.5,
F07G 11.9 and TO 1C4.1 of the nematode C. elegans (Tab. 1) have a family 18
module preceded by multiple repeats which are found only in the nematode
genome. The function of all these modules remains to be discovered.
154
B. Henrissat
Concluding remarks
The classification of chitinase modules based on the similarities of their
amino acid sequences combines the structural features of enzymes with
their 3D structures [39]. Another advantage ofthis classification is that it
allows enzymes of different specificity (e. g. chitodextrinase, exochitinase,
di-N-acetyl chitobiases and endo-N-acetylglucosaminidases) to be grouped
in a single family (family 18), and offers insights into the divergent evolution of enzyme families [4]. Inversely, the occurrence of chitinases in two
clearly different families (families 18 and 19) reflects convergent evolution
[4].
The highly modular structure of some chitinases, in particu1ar in family
18, shows that some modern chitinases have been subjected to complex
evolutionary events. The classification in families ofrelated protein modules allows evolutionary analysis to be carried out on each independent
module (see for instance [36] for an analysis of the fibronectin type III
modules), an important step in e1ucidating their structure and function.
A great deal of confusion exists in the naming of chitinase genes and the
resulting encoded proteins: for instance, chitinases 1 and 2 of Drosophila
melanogaster belong to family 18, whereas chitinases 1 and 2 of Oryza
sativa belong to family 19. Similarly, while chitinase D of B. circulans
belongs to family 18, chitinase D of Lycopersicon esculentum is a member
of family 19. Class I, 11 and IV chitinases belong to family 19, whereas
classes III and V form family 18. However, chitinases I, 11 and III of
Rhizopus oligosporus all belong to fami1y 18. Chitinase genes and proteins
wou1d be more convenientIy named after their family classification (e. g.
chi18 or chi19 for the genes, and ChiI8 or ChiI9 for the proteins). When
an organism produces multiple genes encoding chitinases from the same
family, these could be named chi18a, chi18b and so on, and the corresponding proteins wou1d be Chi I8a, Chi 18b and so on.
Acknowledgements
I would like to thank Drs. G. J. Davies, B. W. Dijkstra and T. Watanabe for many useful discussions and help throughout the years. The help of Dr. P. M. Coutinho with the preparation of
Figure 1 is gratefully acknowledged.
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21 Tews I, Terwisscha van Scheltinga AC, Perrakis A, Wilson KS, Dijkstra BW (1997)
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ed. by P. Jolles and R. A. A. Muzzarelli
@ , 999 Birkhuser Verlag Basel/Switzerland
Aggressive and defensive roles for chitinases

Graham W Gooday
Department 0/ Molecular and Cell Biology,
University 0/Aberdeen,
Institute 0/Medical Sciences, Foresterhil/, Aberdeen AB25 2ZD, UK
Summary. Chitinases are produced by a wide variety ofpathogenic and parasitic microbes and
invertebrates during their attack on chitin-containing organisms. Examples discussed include
enzymes of insect and algal viruses, of yeast killer toxin plasmids, of bacterial and fungal
pathogens of fungi and insects, and of parasitic protozoa. These chitinases play roles in
penetration of fungal cell walls, and of exoskeIetons and peritrophic membranes of arthropods.
Salivas of some invertebrate predators have chitinolytic activity which may be involved in their
attack on their prey. Chitinases playamajor defensive role in all plants against attack by fungi,
and perhaps also against attack by insect pests. The plant chitinases form a very large and
diverse assemblage of enzymes from two families of glycosyl hydrolases. At least some
vertebrates, incIuding fish and humans, also may utilise chitinases in their defence against
pathogenic fungi and some parasites.
Introduction
Chitinases and related enzymes have many roles in an increasingly wide

range of different biological systems. This account concems their aggressive and defensive roles. Pathogens and predators of chitinous organisms
characteristically produce chitinases [1, 2], whereas hosts to chitinous
pathogens, notably plants but perhaps also vertebrates inc1uding humans,
Table 1. Roles for chitinases
Group
Chitinase family Nutritional
Plasmids
Viruses
Archaea
Eubacteria
Protists
Plants
Fungi
Nematodes
Arthropods
Molluscs
Other invertebrates
Fish
Amphibians/reptiles
Birds
Mammals
18/19
18
?
18*
18
18,19
18
18
18
18
18
Morphogenetic Defence Aggression

2
>8
2-10%
many
few
many
many
many
many
many
aIl?
aIl?
many
some
?
many
all?
all
all
all
all
many
?
?
?
?
all
?
?
?
?
* An exception is the family 19 chitinase of Streptomyces griseus HUT6037.
some
some
few
some
few
some
few
?
158
G.W Gooday
produce chitinases to defend themselves [2, 3]. Chitinases fall into two
unrelated families of glycosyl hydrolases, 18 and 19, distinguished by their
amino acid sequences [4]. Chitinases ofboth families are discussed in this
chapter (Tab. 1). The two families have different hydrolytic mechanisms,
leading to retention or inversion, respectively, of the anomeric configuration [5]. As a result, family 19 chitinases are unable to hydrolyse some
model substrates, such as 4-methylumbelliferyl glycosides, and are
resistant to inhibition by allosamidin, a potent inhibitor of most family 18
chitinases.
Chitinases encoded by plasmids and viruses
Viruses and plasmids are mobile packages of genes, and in several cases
they encode chitinase genes. Plasmids of some strains of yeasts encode
toxins that kill other strains. Killer toxins of some strains of Kluyveromyces lactis and Pichia acaciae have chitinase activity [6, 7]. In K. lactis the
toxin is a trimeric protein, with the intracellular y-subunit responsible for
killing a susceptible cell of Saccharomyces cerevisiae, while the a-subunit
has exochitinase activity essential for the action of toxin, shown by the inhibition of activity by allosamidin. The significance of this chitinase activity remains unclear, but may involve its binding to, and/or localised
weakening of, the susceptible yeast cell surface to facilitate the uptake of
the y-subunit. Chitin-deficient yeast cells are resistant to the toxin. The
amino acid sequence of the a-subunit has striking simi1arities to other
chitinases in two regions, one corresponding to the catalytic domain of
family 18 chitinases and another corresponding to the cysteine-rich chitin
binding domain offamily 19 chitinases and lectins [6], which suggests that
it has arisen by fusion of portions of two genes of different origins.
Autographa californica nuclear polyhedrosis virus (AcMNPV) is a
baculovirus that infects some lepidoptera and is used for biological control
of insect pests. The original rationale for looking for a chitinase activity in
this baculovirus was that, if produced, this enzyme could aid to penetration
of the host caterpillar's peritrophic membrane. The insect cell cultures
(Spodoptera jrugiperda, fall armyworm) used to grow the virus produced
their own chitinases, at a low activity, but on infection an enormous increase in chitinase activity was observed [8]. This enzyme is encoded by
the virus genome, and its amino acid sequence shows high homology to
those of some bacterial chitinases, expecially Serratia marcescens
chitinase A. Comparative DNA sequence analysis suggests that there has
been lateral gene transfer relatively recently, perhaps from S. marcescens as
it is itself an insect gut pathogen. Astrain of A. californica NPV from
which the chitinase gene and the adjacent cathepsin gene had been disrupted was less pathogenic to larvae ofthe cabbage looper, Trichoplusia ni,
but the insects still died. A dramatic difference, however, was that after
159
Figure I. Larvae of cabbage looper, Trichoplusia ni, photographed 7 days after in feet ion with
Autographa californica nucleopolyhedrovirus: M, mock-infected; W, infected with wild-type
virus; D, infected with virus with chitinase and cathepsin genes disrupted. Caterpillar M is
healthy; W has died and liquefied; D has died and dried up. Photograph from R. D. Possee (cf.
[9]).
death the insects infected by the chitinolytic virus were totally liquefied,
whereas those infected by the mutant strain were dry cadavers [9] (Fig. 1).
Thus, although this baculovirus chitin ase may aid penetration of the
peritrophic membrane ofthe insect host, as with some bacterial, protozoan
and microfilarial chitinases discussed later, its major significance is in
aiding release of viruses from the dead host.
Two viruses ofthe green al ga Chlorella also produce chitinases. Analysis
of the genome of Chlorella virus PBCV-l revealed the sequences of three
genes with strong resemblance to microbial chitinases, and one to bacterial
chitosanases [10], whereas several proteins with chitinase activity and
several with chitosanase activity were identified in disrupted particles of
Chlorella virus CVK2 [11]. These enzyme activities presumably are associated with entry and/or eventual lysis ofthe algal cell wall, which contains chitin.
Microbial attack on fungi
A number of chitinolytic mycoparasitic fungi are being investigated for

their potential as biocontrol agents against fungal plant pathogens [12]. The
most investigated, Trichoderma harzianum, produces six inducible chitinoIytic enzymes when grown on chitin, four endochitinases and two -I ,4-Nacetylglucosaminidases acting in exo-type fashion to hydrolyse chitin to
160
G.W Gooday
N-acetylglucosamine [13]. There is good evidence that these enzymes are

directly involved in mycoparasitism, leading to the lysis of the cell walls of
host fungi. Different host fungi induce different patterns ofthe enzymes by
T harzianum. When grown with Rhizoctonia solani, both endochitinase
and exo-type enzyme activities were induced, compared with only the exotype activities when grown with Sclerotium rolfsi. The antifungal activities
of chitinases of mycoparasitic fungi, such as T harzianum and Gliocladium
virens work synergistically with those of other lytic enzymes, notably glucanases [14], and in the case of G. virens, with its toxic secondary
metabolite, gliotoxin [15]. A range of other antifungal chemicals also act
synergistically with these chitinases [14].
Chitinolytic bacteria can be pathogens of fungi, such as Ewingella
americana, the causative agent ofinternal stipe necrosis ofthe commercial
mushroom Agaricus bisporus. A mushroom-derived strain of this bacterium
appeared to produce only one chitinase, but produced this constitutively, and
could not grow with chitin as sole carbon source [16]. Chitinolytic bacteria
such as Enterobacter agglomerans are also being investigated as biocontrol
agents against fungal plant pathogens. Isolates of this bacterium produced
four chitinolytic enzymes when grown on chitin as sole carbon source [17].
Strains of the strongly chitinolytic bacterium S. marcescens from soils
harbouring plant pathogenic fungi also have potential as biocontrol agents
[18]. The direct involvement of chitinase activity in the antifungal activity
is suggested by the demonstration of control of plant disease caused by R.
solani and S. rolfsii by application to infected plants of Escherichia coli
bearing a plasmid expressing the gene for S. marcescens chitinase A, or of
the chitinase itself, but not of a strain of E. coli with a plasmid lacking the
promoter for expression of the gene [19] (see Herrera-Estrella and Chet,
this volume).
Chitinolytic attack on invertebrate exoskeletons
Many fungi are pathogens of chitinous invertebrates, and there has been
much interest in their use as biocontrol agents, in particular against insect
pests [20, 21]. Most of these pathogens produce chitinases. These chitinases can have three roles: they can aid the penetration of the host exoskeleton; they can provide nutrients both directly in the form of amino
sugars and indirectly by exposing other host material to enzymatic digestion; and they can aid the release of progeny pathogens. Examples incIude
the Oomycete Aphanomyces astaci, a pathogen of crayfish [22]; Paecilomyces lilacius, a pathogen of nematode eggs [23]; entomopathogens,
Metarhizium anisopliae, Beauveria bassiana, Aspergillus flavus and
Nomuraea rileyi [24, 25]; and acaropathogens, Hirsutella spp. [26]. The
direct roles of chitinases in the case of the entomopathogens and acaropathogens remains uncIear. They are usually induced by chitin and its
oligomers, and sometimes by N-acetylglucosamine, and so are likely to be
161
produced by the fungus on the arthropod cuticle. In the case of N rileyi,

two virulent isolates had high chitinase activities, whereas an avirulent
isolate had low activity [25]. Often, however, there appears to be little or no
correlation between chitinase activity and virulence. It seems likely that
when they are produced in high activity, chitinolytic enzymes would aid
proteolytic enzymes and mechanieal pressure in penetration of the cuticle.
A bacterial example of a chitinolytic bacterium eroding a chitinous exoskeleton is Photobacterium sp. causing lesions ofthe tanner crab [27].
Chitinolytic attack on the peritrophic membrane

As weH as being a component of insect exoskeletons, chitin also has a
major structural role in the ephemeral protective lining ofmany insect guts,
the peritrophic membrane and of other internaiorgans. Treatment of
isolated peritrophic membranes with chitinases leads to their perforation
[28]. There are examples of bacteria, protozoa and filarial nematodes
where the pathogen's endogenous chitinolytic activities appear to aid
penetration of the peritrophic membrane.
That the chitin of the peritrophic membrane is a site of attack by pathogenie bacteria is suggested by experiments with Drosophila melanogaster
[29]. Flies with mutations in two genes, cut and miniature, are more
susceptible than the wild type to infection via the gut by the bacterium
S. marcescens. That the cut and miniature mutations lead to defieiencies in
chitin content was demonstrated by showing that pupal shells from the
mutant strains were more readily digested by Serratia chitinase, and especially by synergistic action of chitinase and protease, than those of other
strains. Also a mutant bacterial strain, deficient in chitinase and protease,
was much less pathogenic to the flies. Addition of exogenous chitinase aids
the pathogenesis ofinsects by Bacillus thuringiensis [30]. Most isolates of
B. thuringiensis, however, are chitinolytic, and there is a positive correlation between chitinase activity in different strains and host mortality (M. N.
Sampson and G. W Gooday, unpublished observations). Further evidence
for the direct involvement of the bacterial chitinases and virulence comes
from the observation that feeding of aHosamidin, the chitinase inhibitor,
markedly increased the dose of B. thuringiensis required to kill caterpillars
and midge larvae [30]. The sugar-beet root maggot, however, has turned
the chitinolysis by Serratia to its advantage by developing a symbiotic relationship with S. liquefaciens and S. marcescens [31]. These bacteria
become embedded in the inner puparial surface and aid the emergence of
the adult fly by their digestion of the chitin of the puparium. The symbiotic
bacteria are present in all deve10pmental stages, including the eggs.
There is evidence that malarial and trypanosome parasites use chitinases
to penetrate the chitin-rich peritrophic membranes formed around the
blood meals oftheir insect vectors [32]. Thus chitinase is formed during the
162
G.w. Gooday
maturation of ookinetes, the invasive form of Plasmodium gallinaceum

which penetrates the peritrophic membrane of the host mosquito [28, 33,
34]. The involvement of chitinase in this development was shown by its
inhibition by allosamidin, which consequently blocked malaria parasite
transmission [34]. In addition, mosquito gut proteases potentiated malarial
chitinase activity. Cultures of the trypanosomatids, Leishmania species,
also produce chitinases [35, 36]. The precise nature ofthe involvement of
chitinases in infection is less clear, as the parasites undergo a more complex developmental process in the insect, involving invasion of the cardiac
valve, and it may be at this stage that the chitinase plays a role, rather than
in degradation of the peritrophic membrane. This suggestion is supported
by the observation that chitinase secretion was inhibited by incorporation
of haemoglobin or blood in the growth medium of Leishmania major, as
this correlated with the lack of damage to the cardiac valve in blood-fed infected flies [36]. A secretory chitinase has been cloned and characterised
from L. donovani [37]. This gives the possibility of constructing adeletion
mutant lacking chitinase activity to determine the role of the enzyme in
infection. A more complicated involvement of chitinase with infection of a
vector is seen with the implication of chitinase from maternally inherited
bacteria in susceptibility oftsetse flies to infection with trypanosomes. The
susceptible flies are infected with these intracellular bacteria. Resistance of
refractory tsetse flies (lacking the bacterial infection) is ascribed to killing
of the trypanosomes in the gut by a lectin. It is suggested that bacterial
chitinolysis releases amino sugars that inhibit the lectin-trypanosome
binding and thus results in survival of the trypanosomes [38], but additonally the chitinolytic bacteria may weaken the peritrophic membrane,
aiding penetration of the trypanosomes.
As discussed for protozoan parasites, invertebrate parasites may use
chitinases to aid penetration of chitinous hosts [28]. The filarial nematode,
Brugia malayi, has mosquitoes as its vectors between mammalian hosts.
Microfilariae, produced during infection of the mammal, are covered by a
chitin-rich coat which is formed by stretching of the original eggshell. In
model infections in gerbils a major antigen of the B. malayi microfilariae
is a nematode chitinase [39]. The microfilariae are the infective form for
the insect vector, and they produce a range of chitinase isozymes, but the
roles for the enzymes are unclear [40,41]. They may playa role in the regulation of stretching of the chitinous sheath, or they may aid the penetration
ofthe mosquito gut peritrophic membrane.
Chitinases in saliva and venom
As soon as the octopus Eledone cirrhosa has captured a crab, it injects
saliva via the salivary papilla into its prey at one point, sometimes one of
the eye sockets. This saliva, from the posterior salivary glands, contains a
163
potent mix of chitinases and proteinases [42], which rapidly diffuse

throughout the crab and serve very specifically to digest the musculoskeletal attachments. This mimics what occurs during moulting, and allows
the octopus to remove the "meat" cleanly from within the carapace. The
meat is chopped up with the beak, and collects in the octopus's crop. In this
way the octopus reproducibly recovers about half of the fresh weight of the
crab (M. S. Grisley et al., unpublished results). The saliva contains at least
two chitinases, as resolved by isoelectric focussing, with pI values of > 9
and 5.6, with very low molecular sizes, < 12.400 Da as estimated by size
exclusion chromatography [42]. This small size may aid the passage ofthe
enzymes throughout the body of the prey. A chitinase has also been
characterised as a major component in the nonparalysing venom of the
endoparasitic wasp ehelonus sp. [43]. The venom is injected into the eggs
of lepidopteran prey, but its functional significance is unclear, as the eggs
do not appear to contain chitin.
Plant defensive chitinases
All plants produce a range of chitinases which form at least four structural
groups: classes I, 11, and IV of family 19, and class III of family 18 [3].
Chitinases are major components of plant "pathogenesis-related proteins"
induced following attack by potential pathogens or treatment with ethylene
[3, 44]. Some of these chitinases have direct antifungal activity, causing
rapid lysis offungal hyphal tips and germinating spores [45-47]. This is a
consequence of most potential pathogenic fungi having chitin as a key
structural component of their hyphal walls, with nascent chitin being very
susceptible to enzymic lysis. The antifungal activity of the plant chitinases
is synergistically potentiated by -glucanases, another group of pathogenesis-related proteins that attack the glucans cross-linked with chitin in
the fungal cell wall [46]. The plant's chitinolytic activities will also have an
indirect defence action by releasing oligosaccharide elicitors from fungal
chitin. Plant cells are very responsive to specific oligosaccharides, including those of N-acetylglucosamine. Thus exposure to chito-oligosaccharides can elicit a variety of responses, including synthesis of antifungal phytoalexins, induction of antifungal chitinases and induction of
K+ and er release, extracellular alkalinization and H 2 O2 synthesis
[48-50]. A putative receptor, very sensitive to N-acetylchito-octaose, has
been characterised from rice cells [51].
Some plant chitinase molecules are bifunctional, with other defensive
activities in the same molecule as the chitinase. Three class III endochitinases produced by cell cultures of Trichosanthes kirilowii also have
ribosome-inactivating activity [52]. They have the specific 28S ribosomal
RNA (rRNA) N-glycosidase activity characteristic ofribosome-inactivating
proteins. An endochitinase with insect a-amylase inhibitor activity has
164
G.W Gooday
been characterised from seeds of Job's tears, Coix lachryma-jobi [53].

Most attention has been paid to the antifungal activity of plant chitinases,
but they mayaiso act against arthropod pests. Pest attack, like fungal
attack, causes induction of pathogenesis-related proteins. Thus chitinases
were induced to different extents in roots of a range of citrus hybrids when
they were fed on by a root weevil, Diaprepes abbreviatus [54]. Roots with
the highest induced chitinase activities tended to be more resistant to attack
by the weevil. Root protein extracts degraded the insect's peritrophic
membrane, and extracts from more resistance strains showed greater
damage, which suggests that this may be the site of action of the plant
chitinases. Transgenic tobacco plants expressing a chitinase from tobacco
homworm, Manduca sexta, showed increased resistance to attack by
tobacco budworm, Heliothis virescens, as assessed by feeding damage and
growth ofthe pest larvae [55]. Insects feeding on the transgenic plants were
also more sensitive to the effects of B. thuringiensis toxin. Some insectivorus plants produce chitinases which aid in digestion of their prey
[56]. This presumably is an adaptation of the ubiquitous production of
chitinases as defence enzymes in plants. Many plant chitinases have
lysozyme activity [3], which suggests that they may have an antibacterial
role. This has been questioned [57], as the activity is usually only weak and
confined to the plant cell's vacuole, but even a weak activity may synergistically potentiate other antibacterial activities.
Most plant roots are infected by mycorrhizal fungi, which have to exist
in the presence ofthe plants' defence mechanisms [58]. These fungi may
have walls that are resistant to the hosts' chitinases, or they may locally suppress expression ofthese enzymes. Experiments with spruce cells have led
to the suggestion that the host's defensive responses are attenuated by
degradation of chito-oligosaccharide elicitors by host chitinases [50]. It has
also been suggested that changes in fungal wall composition resulting from
host chitinolysis may aid in the exchange of nutrients between the
symbionts [59].
There has been much interest in the possibility of increasing the natural
defences of crop plants against fungal and pest attack by creating transgenic plants expressing chitinase genes from bacteria, fungi, plants and
insects [3, 21, 44, 47]. Results to date have been mixed, as might be expected - we are trying to improve on complex multilayered and tightly
regulated defence systems that have developed during plant evolution.
Nevertheless, some ofthe results look promising and some early field trials
are taking place, so we can expect to see major developments in this area.
Vertebrate defensive chitinases?
There is increasing evidence that chitinases may have defence roles against
pathogenic fungi and parasites in animals. Blood of many vertebrates has
165
Figure 2. Cytochemical localization of chitinolytic enzymes in blood cells of turbot, Scophthalmus maximus. (a) Chitinase activity shown as fluorescence after incubation in 4-methylumbelliferyldiacetylchitobiose. (b) Same cells viewed with brightfield optics. Note activity
throughout cells in Iymphocytes (L) and thrombocytes (T), but associated with nuclei in red
blood cells (R). (c) N-Acetylglucosaminidase activity shown as fluorescence after incubation in
4-methyl-umbelliferyl-N-acetylglucosamine. (d) Same cells viewed with brightfield optics.
Note activity associated with distinct cell inclusions (shown by arrow, probably lysosomes) in
red blood cells (R), but more dispersed throughout cytoplasm in white blood cells CL). Fluorescence is also apparent as haloes around red blood cells and their nuclei. Scale bar represents
10 llm. From [60].
chitinolytic activities. In turbot, Scophthalmus maximus, there are chitinase

activities in white and red blood cells and in plasma, with leucocytes being
especially rich [60] (Fig. 2). This chitinolytic activity of leucocytes may
have a defence role in digestion of ingested chitinous pathogens of fish
such as microsporidia. The turbot plasma chitinase also has antifungal
activity. Serum of ruminants and some other mammals is rich in chitinase
activity [61], which may have a defence role against potential fungal
pathogens.
Human blood also has a chitinase activity which may have a defence
function. Activity has been detected in serum [62] and also in granulocyte-rich homogenates of leukocytes [63]. This enzyme is a marker for
166
G.w. Gooday
Gaucher's disease, as most patients have a greatly elevated activity secreted

by their macrophages [64]. Chitinase has been purified from sampIes from
these patients, leading to the cloning ofits gene [65, 66]. As yet, there is no
evidence for a direct defence role for this enzyme, as opposed to the alternative suggestion, that of processing glucosaminoglycans. Some evidence
in favour, however, comes from experiments with a similar enzyme in
guinea pigs. Upon infection with the fungal pathogen, Aspergillus
fumigatus, the serum levels of chitinase in circulation of pathogen-free
guinea pigs increased about threefold over 4 days [67]. The authors
attributed this increase to the host's enzyme, as its substrate hydrolytic
activities were those of the animal rather than those of the fungus. Treatment with antifungal drugs diminished the increase.
ConcIusions
Work with diverse biological systems over the past 20 years has led us to a
realisation of the very wide occurrence of the enzymes classified as
chitinases by their potential for hydrolysing various substrates, ranging
from chitin itself, and derivatives such as glycolchitin, to N-acetylglucosamine oligomers and their fluorogenic and chromogenic glycosides. These
enzymes have been put to a remarkably wide range of uses [1, 56, 68].
Chitin was originally thought of as being just an inert structural component, but we are beginning to realise that it and its oligomers are used in
a variety ofways throughout biology that require precise enzymic tailoring
ofthe saccharide chains. Thus we can look forward to an exciting future for
chitinase research.
References
1 Gooday GW (1994) Physiology of microbial degradation of chitin and chitosan. In: C
Ratledge (ed): Biochemistry ofmicrobial degradation. Kluwer, Dordrecht, 279-312
2 Gooday GW (1996) Aggressive and defensive roles for chitinases. In: RAA Muzzarelli (ed):
Chitin enzymology, vol 2. Atec, Grottammare, 125-134
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233-243

ed. by P. Jolle5 and R.A.. Muzzarelli
1999 Birkhu5er Verlag Ba5el/Switzerland
Chitinases in biological control

Alfredo Herrera-Estrella 1 and Han Chet2
Centro de Investigacion y Estudios Avanzados. Unidad Irapuato. Apartado Postal 629.
36500, Irapuato, Gto., Mexixo
2 The Hebrew University of Jerusalem, Otto Warburg Centerfor Agricultural Biotechnology,
Faculty ofAgriculture, P. 0. Box 12, Rehovot 76100, Israel
Summary. The public concern over the harmful effects of chemical pesticides on the environment and human health has enhanced the search for safer, environmentally friendly control
alternatives. Control of plant pests by the application of biological agents holds great promise
as an alternative to the use of chemicals. It is generally recognized that biological control agents
are safer and more environmentally sound than is reliance on the use of high volumes of
pesticides. Due to the importance of chitinolytic enzymes in insect, nematode, and fungal
growth and development, they are receiving attention in regard to their development as biopesticides or chemical defense proteins in transgenic plants and microbial biocontrol agents.
In this sense, biological control of some soil-borne fungal diseases has been correlated with
chitinase production. Fungi- and bacteria-producing chitinases exhibit antagonism against
fungi, and inhibition of fungal growth by plant chitinases has been demonstrated. Insect
pathogenic fungi have considerable potential for the biological control ofinsect pests. Entomopathogenic fungi apparently overcome physical barriers of the host by producing multiple
extracellular enzymes inc1uding chitinolytic enzymes, which help to penetrate the cutic1e and
facilitate infection.
In this chapter, the role of chitinases in biological control and their potential use in the improvement of biocontrol agents and crop plants by genetic engineering is analyzed in view of
recent findings.
Introduction
In modern agriculture, monoculture is the norm, providing large numbers

of generally near-identical plants in one vicinity. Such agricultural practice
enables us to continue to provide foodstuffs for the world's ever-increasing
population. It is, however, an ecologically unnatural situation, which is
inherently unstable and offers considerable opportunity for the invasion of
crops by plant pests, weeds and diseases [1]. Pesticides are applied in
agricultural systems for the purpose of protecting plants from injury by
insects, disease and so on which today still destroy almost 33 % of all food
crops. The use of pesticides is considered effective if they achieve the
desired biological result, and economic ifthere is a crop yield and a quality
response above and beyond the cost of the chemicals and their application.
Yet the use of pesticides has also resulted in significant costs to public
health and the environment. In general the amount of pesticides released
into the environment has risen about 1900 % in the 50-year period between
1930 and 1980 [2]. It is clear that this sort of agriculture cannot be
sustained if the price for this success is unacceptable destruction of the
172
A. Herrera-Estrella and I. Chet
environment [3]. However, since there are currently not many alternatives
to agricultural practices such as monoculture, evenjust to maintain current
human populations the need remains for scientists to continually seek for
new, effective and environmentally friendly ways of controlling pests,
weeds and diseases [1]. Biotechnology, in conjunction with conventional
breeding programs, could make significant contributions to sustainable
agriculture. In this regard, there has been intensive research in agricultural
biotechnology aimed at plant protection. This includes, among others,
disease-free clones of fruits, vegetables and ornamental crops; plants
resistant to insects and microbial pathogens; herbicide-tolerant cultivars;
and biopesticides to use as biological control agents [4].
Control ofplant pests by the application ofbiological agents holds great
promise as an alternative to the use of chemicals. It is generally recognized
that biological control agents are safer and more environmentally sound
than is reliance on the use of high volumes of pesticides and other antimicrobial treatments. Besides, there is an equally great or greater need for
biological control of pathogens that presently go uncontrolled or are only
partially controlled by these "traditional" means [5].
Different meanings have been given to the term "biological control".
Representing two extreme views of the concept, we find the following
definitions: "Biological control [of plant pathogens] is their control by one
or more organisms, accomplished naturally or through manipulation ofthe
environment, host, or antagonist, or by mass introduction of one or more
antagonists" [6]. This definition provides us with a broad concept that
includes such notions as cultural practices and disease resistance. On the
other hand, we have the classical concept: "Biological control is the
deliberate use of one organism to control another" [7]. The term "biological
control" will be used in this more restricted sense throughout this text.
Classical biocontrol, when effective, is an outstanding method of pest
control not only because it eliminates the use ofpowerful, environmentally dangerous pesticides, but also because ifthe introduced biocontrol agent
becomes properly established, it is long lasting and further investments in
control are not necessary. In this way, it differs from the use of pesticides,
which require repeated application. This has led to a renewed interest in the
discovery, development and refinement of biological control agents. Such
efforts have followed classical plant pathology screening strategies but
have also begun to utilize the methodologies made available through molecular biology. We can now look at micoorganisms with inhibitory activity
against pathogens as potential sources of genes for disease resistance.
Physiological role of chitinases
Chitinases have been detected in a great variety of organisms, including

those that contain chitin, such as insects, crustaceans, yeasts and fungi, and
173
also organisms that do not contain chitin, such as bacteria, higher plants
and vertebrates. In arthropods, chitinases are involved in molting and
digestion. Insects periodically sheed their old cuticles and resynthesize
new ones. This process is mediated by the elaboration of chitinases in the
molting fluid that accumulates between the old cuticle and the epidermis.
The products ofhydrolysis are recycled for the synthesis ofthe new cuticle.
Often larvae will ingest the old cuticle. Apparently, chitinases found in the
gut have a digestive function in addition to their role in breaking down
chitin present in the gut lining [11].
The model offungal cell wall growth proposed by Bartnicki-Garcia [12]
envisages the role played by lytic enzymes in maintaining a balance
between wall synthesis and wall lysis during hyphal apical growth, providing plasticity to the apex and permitting insertion of nascent chitin into
the wall. Evidence for the association of chitinases and chitin synthases
comes from parallel behavior of the two activities during spore germination
in Mucor mucedo [13], during exponential growth in Mucor rouxii [14] and
Candida albicans [15], and from the finding ofa chitinase activity in the
same cell fraction as chitin synthase in M. mucedo [16, 17]. Sahai et al.
[18] showed that chitinase is present during spore swelling, germination,
sporangium formation and response to mechanical injury in Choanephora
cucurbitarum and four other Zygomycetes fungi. Failure to localize
chitinase at the hyphal tips suggests its possible lack of involvement in
apical growth.
The process of autolysis of mature fruiting bodies of Coprinus lagopus
is accomplished by the action of chitinases which are formed shortly before
spore release begins [19]. Demonstration of lysosomal chitinases was
based on sedimentation studies. Chitinase activity was localized intracellularly in vacuoles together with other lytic enzymes. Chitinases had no
apparent function in intracellular digestion since they were synthesized
shortly before auto lysis in gills [19]. This enzyme is passively released into
the wall when metabolic activity stops in senescing cells. It has been described that some autolytic enzymes including chitinases are bound to
subapical walls of Neurospora crassa and Aspergillus nidulans [20, 21].
These data led to the suggestion that chitinases are associated with hyphal
branching rather than autolytic wall turnover. Thus, fungal chitinases have
been implicated in apical growth, spore swelling and germination, liberation of spores, cell separation and budding.
Considerable interest in the physical, chemieal, kinetic and biocidal
properties of chitinases has been stimulated by their possible involvement
as defense agents against chitinous pathogenic or pestiferous organisms
such as fungi and insects. Resistance to organisms can be imparted by the
degradation ofvital structures such as the peritrophic membrane or cuticle
of insects, the cell wall of fungal pathogens or by liberation of compounds
that subsequently elicit other defense responses [22].
174
Chitinases in insect control
Insect pathogenic fungi have considerable potential for the biological

control of insect pests of plants. The majority of these fungi OCCUf in the
Deuteromycotina and Zygomycotina. Many attempts have been made to
exploit the Deuteromycotina fungi Metarhizium anisopliae, Bauveria spp.,
Nomurae rileyi, Aschersonia aleyrodis and Verticillium lecanii, as weH as
some Entomophtorales for insect control. In this sense, the peritrophic
membrane and exoskeleton of insects act as physicochemical barriers to
environmental hazards and predators. However, entomopathogenic fungi
apparently overcome these kinds of barriers by producing multiple extracellular enzymes, including chitinolytic and proteolytic enzymes that help
to penetrate the cuticle and facilitate infection [23 - 26]. Some insect
venoms also contain chitinolytic enzymes that might serve to facilitate the
entry of venomous components into prey [27]. Similarly, the nematode
Brugia malayi utilizes a chitinase to break down a protective chitinous
extracellular sheath and/or the peritrophic membrane to gain entry into the
mosquito host [28, 29]. Baculoviruses also contain genes for chitinases, but
their precise role(s) in host infection is unclear [30]. Nevertheless, hydrolytic enzymes used by insects, fungi and other organisms for molting or
barrier penetration are potentially useful in pest management because their
physiological action is to destroy vital structures such as the exoskeleton or
peritrophic membrane of insects.
A Manduca sexta chitinase has been shown to increase the killing rate of
a recombinant baculovirus [31]. In that study a recombinant nonocc1uded
baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV)
carrying the M sexta chitinase complementary DNA (cDNA) under the
control of the polyhedrin gene promoter, expressed the chitinase. This
enzyme was secreted into the medium when insect celliines were infected
with the virus. When the recombinant virus was injected in M. sexta larvae,
chitinase was found in the hemolymph, where it does not normally OCCUf.
The recombinant baculovirus expressing the chitinase killed larvae of fall
armyworms (S. jrugiperda) in approximately three quarters of the time
required for the wild-type virus to kill the larvae [31].
Chitinases have also been used in mixing experiments to increase the
potency of entomopathogenic microorganisms. Bacterial chitinolytic enzymes have been used to enhance the activity of microbial insecticides
including Bacillus thuringiensis and a baculovirus. Larvae of spruce budworm, Choristoneura fumiferana, died more rapidly when exposed to a
chitinase-Bacillus mixture than when exposed to the enzyme or bacterium
alone [32-34]. In another study, mortality of gypsy moth (Lymantria
dispar) larvae was enhanced when chitinase was combined with B. thuringiensis compared with treatment with the bacteria alone, and this effect was
correlated with enzyme levels. The larvicidal activity of a nuclear polyhedrosis virus toward gypsy moth larvae was increased fivefold when it
175
was coadministered with bacterial chitinase [35]. In that case, chitinases

were supposed to cause perforations in the gut peritrophic membrane,
facilitating entry of the pathogens into the hemocoel of susceptible insects
[36].
Chitinases appear to be involved in the penetration of host cuticle by
entomopathogenic fungi [25, 26, 37]. In this regard, chitinases and -Nacetylglucosaminidases are secreted when the entomopathogens M. anisopliae, B. bassiana and Verticillium lecanii are grown on insect cuticles
[24]. Virulent isolates of N. rileyi exhibit substantially higher levels of
chitinase activity than avirulent strains at the time of cuticle penetration
[23]. Chitinase gene expression in entomopathogenic fungi is believed to
be controlled by a repressor-inducer system in which chitin or the oligomeric degradation products serve as inducers [24]. However, bacterial chitinases were ineffective in assays in which insects were fed a diet containing
the enzymes. No mortality of the nymphal stages of the rice brown plant
hopper, Nilaparvata lugens, occurred when 0.09% w/v Streptomyces
griseus chitinase was added to an artificial diet [38]. Similarly, Serratia and
Streptomyces chitinases at 1-2 % levels in the diet of the merchant grain
beetle, Oryzaephilus mercator, caused no mortality.
Chitinases in the control of phytopathogenic fungi
Even with intensive fungieide use, the destruction of crop plants by fungal
pathogens is a serious problem worldwide that annually leads to losses of
about 15% [3]. Hence, any development aimed to diminish this problem
will be useful, especially if at the same time it helps to decrease the strong
fungieide application.
Biological control of some soil-borne fungal diseases has been correlated with chitinase production [39]; bacteria-producing chitinases
and/or glucanases exhibit antagonism in vitra against fungi [40, 41]; inhibition of fungal growth by plant chitinases and dissolution of fungal cell
walls by a streptomycete chitinase and -(1,3)-glucanase have been
demonstrated [42, 43]. The importance of chitinase activity was further
demonstrated by the loss of biocontrol efficacy in Serratia marcescens
mutants in which the chiA gene had been inactivated [44].
Molecular techniques have also facilitated the introduction ofbeneficial
traits into rhizosphere competent and model organisms to produce potential biocontrol agents. A recombinant Escherichia coli expressing the chiA
gene from S. marcescens was effective in reducing disease incidence
caused by Scleratium rolfsii and Rhizoctonia solani [45, 46]. In other
studies, chitinase genes from S. marcescens have been expressed in
Pseudomonas sp. and the plant symbiont Rhizobium meliloti. The modified
Pseudomonas strain was shown to control the pathogens F. oxysporum f
sp. redoiens and Gauemannomyces graminis var. tritici [47,48]. The anti-
176
A. Herrera-Estrella and 1. Chet
fungal activity of the transgenie Rhizobium during symbiosis on alfalfa

roots was verified by lysis of R. solani hyphal tips treated with cell-free
nodule extracts [49].
The use of mycoparasites is a promising alternative for disease control
by biological means. Mycoparasitism is defined as a direct attack on a
fungal thallus, followed by nutrient utilization by the parasite [50]. According to Barnett and Binder [51] mycoparasites can be divided into
biotrophic and necrotrophic. Necrotrophic mycoparasites are those that kill
the host cells before, or just after, invasion and use the nutrients released.
These mycoparasites tend to be more aggressive and destructive than
biotrophs, have a broad host range extending to wide taxonomic groups,
and are relatively unspecialized in their mode of parasitism. The antagonistic activity of necrotrophs is due to the production of antibiotics,
toxins or hydrolytic enzymes in such proportions as to cause death and
destruction of their host [52]. Instead, in biotrophic parasitism the development of the parasite is favored by a living rather than a dead host
structure [50]. Biotrophic mycoparasites tend to have a more restricted host
range and in many cases produce specialized structures (haustoria) to
absorb nutrients from their host [52].
There are a number of examples of fungi that parasitize plant pathogens.
Of these only a few have been studied to any extent with the aim of
biological control. Trichoderma species and Gliocladium virens have
probably been studied more extensively. Other mycoparasites reported to
have some potential for biocontrol are Ampelomyces quisqualis, Coniothyrium minitans, Laetisaria arvalis, Pythium nunn, Talaromyces jlavus
and Sporidesmium sclerotivorum [5,50,53].
The potential for the use of Trichoderma species as biocontrol agents
was suggested 67 years ago by Weindling [54], who was the first to demonstrate the parasitic activity ofmembers ofthis genus toward pathogens such
as Rhizoctonia solani [54, 55]. Several species of Trichoderma spp. have
been tested as biocontrol agents; among them Trichoderma harzianum has
proved to be more effective [56], and it has been shown to attack a range of
economically important soil-borne plant-pathogenic fungi. The parasitic
process by Trichoderma apparently includes (i) chemotropic growth, (ii)
recognition of the host by the parasite, (iii) secretion of extracellular enzymes, (iv) hyphae penetration and (v) lysis of the host or their combination. Penetration of the host mycelium takes place aparently by partial
degradation ofits cell wall [57, 58]. Microscopic observations [59, 60] lead
to suggest that Trichoderma spp. produced and secreted mycolytic enzymes responsible for the partial degradation ofthe host's cell wall. Results
supporting this hypothesis have shown that indeed Trichoderma produces
extracellularly a complex set of -(1,3)-glucanases, chitinases, lipases and
proteases when grown on cell walls of R. solani [61, 62].
The level of hydrolytic enzymes produced differs for each host parasite
interaction analyzed. This phenomenon correlates with the ability of each
177
Trichoderma isolate to control a specific pathogen. However, the specificity of Trichoderma cannot be simply explained by a difference in enzyme activity, since the nonantagonistic Trichoderma isolates produce
lower but significant levels of lytic enzymes [63]. This observation supports the idea that recognition is an important factor in the mycoparasitic
activity of Trichoderma. The effect ofthe cell wall-degrading enzymes on
the host has been observed using different microscopy techniques. Interaction sites have been stained by fluoresceinisothiocyanate-conjugated
lectins or calcofluor. The appearance of fluorescence indicated the presence of localized cell wall lysis at points of interaction between the antagonist and its host. Electron microscopy analysis has shown that during the
interaction of Trichoderma spp. with either S. rolfsii or R. solani the parasite hyphae contacted their host and enzymatically digested their cell walls
[57].
The purification and characterization of three chitinases from T. harzianum was reported by De la Cruz et al. [64]. They reported the isozymes to
be 37, 33 and 42 kDa, respectively. Only the purified 42-kDa chitinase
hydrolyzed Botrytis cinerea purified cell walls in vitro, but this effect was
heightened in the presence of either of the other two isoenzymes [64].
However, the chitinolytic system of T. harzianum was recently found to be
more complex [65], consisting of six distinct enzymes. The system is apparently composed of two -(1,4)-N-acetylglucosaminidases of 102 and
73 kDa, respectively, and four endochitinases of 52, 42, 33 and 31 kDa,
respectively. All the chitinolytic enzymes were induced and secreted during
growth of Trichoderma on chitin as the sole carbon source.
The complexity and diversity of the chitinolytic system of T. harzianum
involves the complementary modes of action of six enzymes, all of which
might be required for maximum efficiency against a broad spectrum of
chitin-containing plant pathogenic fungi. Probably the most interesting
individual enzyme ofthe system is the 42-kDa endochitinase because ofits
ability to hydrolyze B. cinerea cell walls in vitro. Since the report of the
purification of this enzyme the corresponding gene has been cloned [66].
Expression of the gene (ech42) is strongly induced during fungus-fungus
interaction. Its expression is apparently repressed by glucose and may be
affected by other environmental factors such as light and nutritional stress
and may even be developmentally regulated [66]. A second endochitinase
and a -(1,4)-N-acetylglucosaminidase encoding genes from Trichoderma
have been cloned [67, 68].
Recently we have analyzed the role of the T. harzianum endochitinase
Ech42 in mycoparasitism by genetic manipulation of its coding gene ech42
[69]. Several transgenic T. harzianum strains carrying multiple copies of
ech42 as weIl as the corresponding gene disruptants were generated. The
level of extracellular endochitinase activity when T. harzianum was grown
under inducing conditions increased up to 42-fold in multicopy strains as
compared with the nontransformed strain, whereas gene disruptants
178
A. Herrera-Estrella and I. ehet
showed practically no activity. In greenhouse experiments, no differences

in the efficacy of the gene disruptants to control Rhizoctonia solani or
Sclerotium rolfsii were observed, as compared with the nontransformed
control strains. However, multicopy transformants allowed about 10%
lower disease incidence. Furthermore, 30% higher degradation ofthe chitin content in the R. solani cell walls was observed during interaction with
the overexpressing Trichoderma than with the wild type, when quantified
by transmission electron microscopy.
In an attempt to increase its effectiveness, T. harzianum was transformed
with plasmid pSL3chiAII containing a bacterial chitinase gene from S.
marcescens under the control of the CaMV35S promoter. Two transformants showed increased constitutive chitinase activity and expressed a
protein of the expected size (58 kDa). When evaluated in dual cultures
against the phytopathogenic fungus S. rolfsii, both showed higher antagonistic activity, as compared with the nontransformed control [70].
Other necrotrophic mycoparasites also secrete chitinases. An extracellular chitinase produced by Myrothecium verrucaria inhibits germination
and germ tube elongation of the groundnut rust fungus Puccinia arachidis .
Similarly, Acremonium obclavatum produces and secretes a chitinase in
vitro which inhibits germination ofuredospores ofthe peanut rust [71].
Penetration of fungal hosts by the biotrophic mycoparasite Piptocephalis virginiana [72] occurs by both mechanical and enzymatic mechanisms.
Light and scanning electron microscopy studies have shown inpushing of
the susceptible host cell wall and enzymatic erosion of the resistant host
cell wall by the advancing infection hyphae [73]. Interestingly, culture
filtrates of P. virginiana contained only negligible levels of chitinases and
chitosanases, thus indicating strict regulatory control of these lytic enzymes, which is characteristic ofa biotrophic mycoparasite [74]. Manocha
[72] has proposed that metabolic shifts favoring chitinase occur in the
susceptible host, Choanophora cucurbitarum and chitin synthase in the
resistant host, Phoscolomyces articulosus when attacked by P. virginiana.
Increased levels of chitinase activity induced in C. cucurbitarum may
culminate in enhanced plasticity of the host cell wall and a limited incorporation of a chitin precursor at the penetration site due to degradation of
nascent chitin by chitinase. By contrast, higher levels of chitin synthase
may be present in P. articulosus because of deposition of chitin and papilla
formation at the penetration sites [72].
Chitinases as defense and transgenes in plants

The role ofplant chitinases in disease resistance is well documented [75].
Numerous plant chitinase genes or cDNAs have been cloned. In a successful case, transgenic tobacco plants were generated which constitutively
expressed a bean endochitinase gene under the control of the cauliflower
179
mosaie virus 35S promoter. The transgenie tobacco plants were less
suseeptible to infeetion by Rhizoctonia solani, and either the disease
deve10pment was delayed or they were not affected at all [76]. In other very
interesting work, the possible functional interactions between two different
hydrolytie enzymes, the riee RCHIO basic chitinase and the aifaifaAGLUI
acidic glucanase, by constitutive coexpression in transgenic tobacco was
analyzed. Hybrid plants were generated by erossing trans genie parental
lines exhibiting strong constitutive expression of CaMV35S enhancerl
RCHI0 and CaMV 35S double promoter/AGLUI gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous
for each transgene, and homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater proteetion against the
fungal pathogen Cercospora nicotianae than either gene. These data led to
the suggestion that combinatorial expression of antifungal genes could be
an effective approach to engineering enhanced crop proteetion against
fungal disease [77].
There are many other examples of the introduction of chitinase genes
into plants under the control of constitutive promoters, resulting in enhancement of resistance of the host plant to fungal pathogens [78-80].
However, not all eases have been sueeessful. When a tobaceo chitinase
gene was expressed in high levels in Nicotiana sylvestris, the transgenic
plants were still as susceptible to C. nicotianae infection as wild-type
plants [81]. Unfortunately, the role ofvarious chitinases in mediating plant
resistanee to inseets is less well understood.
Although the sueeessful use of plant chitinases for controlling fungi is
well documented, no reports of successful use of aplant chitinase in
controlling insect pests are available. In fact, in spite of the substantial
levels of chitinases found in cereal grains (10-100 p.g/g), theyare susceptible to inseet attaek, suggesting that stored-produet inseets have
evolved to overeome plant ehitinases. Furthermore, recently Kramer et al.
[11] found that transgenie rice plants expressing relatively high levels of a
rice chitinase have no detrimental effects on the growth of the fall
armyworm Spodoptera jrugiperda.
In other work, a eDNA eneoding the major molting fluid chitinase of
Manduca sexta was eharaeterized. The M sexta chitinase gene is not
expressed during larval feeding behavior; it is switched on only during a
narrow time frame just prior to larval-larval and larval-pupal molting. The
activity of this gene is apparently tightly regulated by hormones, both
positively and negatively [82]. Based on the tight developmental and
hormonal regulation of the chitinase expression, the authors suggested that
plants eonstitutively expressing it might be resistant to inseets that feed on
them because exposure to this enzyme might damage the gut lining. The
same group generated ehimeric gene eonstructs carrying the M sexta
chitinase under the control of single or double CaMV 35S promoter which
were introduced into tobaeeo and tomato plants. Leaves from transformed
180
and control plants were excised and fed to first instar larvae of the tobacco
budworm. After 3 weeks, the total mass of surviving larvae on control plants
was 966 mg, whereas that on the chitinase transformed leaves was only
177 mg, a reduction of more than 80 % [83].
To determine whether the Manduca chitinase from transgenic tobacco
and several chitinases from other sources were directly toxie to insects, a
beetle feeding study was conducted using purified enzymes. Recombinant
Manduca chitinase from transgenie tobacco and chitinase from Serratia,
Streptomyces and Hordem species were incorporated into a diet at a 1-2 %
level and fed to neonate beetle larvae. Whereas growth and mortality of
larvae consuming the bacterial and plant chitinase-supplemented diets
were the same as those of larvae consuming the untreated diet, all larvae
consuming the insect chitinase-supplemented diet died a few days after egg
hatch [83]. These data led to the suggestion that insect chitinases are potential host plant resistance factors in transgenic plants and might be more
potent than chitinases from other sources.
Concluding remarks
All together, the data summarized in this chapter allow us to envisage

chitinases as an important factor in the development of improved agents
and novel strategies for biological control. Further work on cloning and
characterization of chitinases will provide us the tools and understanding
needed to make better use of these genes. The potential of chitinases is
likely to be enhanced by combining them with other bioactive peptides and
lytic enzymes, such as glucanases, as is found in natural systems [22, 84].
Thus, special emphasis should be made of the use of combinatorial
strategies. The enormous potential of genetic engineering will allow us to
combine the natural responses of plants with transgenes of microbial
and/or insect chitinases, other bioactive peptides and improved microbial
biocontrol agents.
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Host-parasite interactions: elicitation of defense

responses in plants with chitosan
Lee A. Hadwiger
Dept. 0/Plant Pathology, Washington State University, Pullman,
WA 99164-6430, USA
Summary. The plant's defense response against pathogens can be elicited by numerous external
signals. Plant pathogens known to be incompatible on a given plant species can elicit strong
disease resistance responses, whereas an adapted compatible pathogen generates a weaker
response and thus can more readily infect the plant tissue. The plant's response can be manipulated genetically by the transfer of"R" genes (single dominant genes for race-specific disease
resistance) or by treatment with elicitors such as chitosan. Both of these manipulations can
result in the rapid activation of a subset of genes called PR (pathogenesis-related) genes, generally regarded as the genes that functionally develop disease resistance. There appear to be
multiple modes by which chitosan can increase PR gene function, including activating cell
surface or membrane receptors and internal effects on the plant's DNA conformation that in turn
influence gene transcription. A novel strategy for controlling PR gene expression proposes to
transform plants with a chitosan-inducible gene promoter linked in line with a single signal
gene capable of rapid, intense induction of an entire set ofPR genes, thereby enabling the control of disease resistance by external chitosan applications.
Genetics and biochemistry of plant pathogen interactions

To better understand the function of chitosan in inducing disease resistance
in plants, it is useful to state briefly what constitutes natural disease
resistance. Each plant species resists most ofthe fungal, bacterial and viral
pathogens in nature. Pathogens that have ingeniously found a niche for
surviving on a given plant species are certainly the exception to the rule of
incompatibility, which pathologists understand is at the heart of disease
resistance. Thus pathogens that do not signal a rapid, intense incompatibility response in the host are able to tolerate the adversity of the plant environment long enough to reproduce. Genes controlling a qualitative
potential for resistance (R genes) against such pathogens have often been
acquired from the center of origin of the plant species of interest. The
region of origin of aplant species is usually inhabited by plant genotypes
that can still prevent the pathogen/plant species niche. Such genetic traits,
often single dominant genes, have been transferred to commercially suitable crop plants.
The resistance (race-specific resistance) afforded the plant by the single
R genes often provides short-lived proteetion in commercial agriculture
because its function depends on an interaction with a specific gene (avirulence gene) in the pathogen (i.e. a gene-for-gene interaction [1]) and thus
any loss of the avirulence gene function resuits in a susceptible reaction.
186
1. A. Hadwiger
Recently, some ofthese R genes were cloned [2]. The predicted proteins of
these genes provide clues to therr biochemical functions; however, the
understanding of the entrre disease resistance process remains incomplete.
A more complicated disease resistance, nonhost resistance, generated by
the plant against all of the other challenging avirulent pathogens undoubtedly implicates many more interacting genes [3] and is thus very
stable to single gene changes in the pathogen and resembles, biochemically, the resistance induced by chitosan [4, 5].
The dominant R genes controlling race-specific resistance and their
recessive alleles occur within genotypes within a given plant species and
thus can be identified and mapped with the aid of genetic crosses. In contrast, nonhost resistance is so inclusive that any recessive traits remain
hidden, and thus their resistant counterparts are not identifiable with
standard recombinant mapping. The only traits defined in the nonhost
resistance response are those identified in terms of the specific pattern of
gene expression occurring as the plant resists an incompatible pathogen.
Fortunately, many ofthese response genes expressed in nonhost resistance
are the same as those activated as a result ofthe proper gene-fr-gene interaction in race-specific resistance. Because the same response genes are
often activated in a susceptible interaction, however, at a lower intensity
and after an initial delay, the term "pathogenesis related" (PR) has become
accepted to define both the gene and its protein product.
In generating resistance, a reasonable scenario of the interaction between
the challenging pathogen and the host tissue must implicate the release of
a signal, the reception of the signal and the conveyance of the signal to a
target capable of altering transcription. Following the altered transcription,
the host response must in turn signal changes in the pathogen that result in
suppression of growth or replication by the pathogen. Chitosan can elicit
increases in the PR proteins and thus can elicit a defense response resembling nonhost disease resistance. Signals composed of chitin and chitosan
and other elicitor compounds will be reviewed.
Race-specific resistance: its link witb tbe induction of PR proteins
The gene for gene interactions between the R genes and their corresponding Avr genes have been rewarding genetic interactions to study. As
the DNA sequences of the R genes became available, these interactions
were analyzed in the context of their predicted sequences, the visible
symptoms ofthe interaction and the mode by which the products come into
contact with each other. The gene-fr-gene interaction between the Pto
gene from tomato that encodes a serine-threonine kinase and the AvrPto
gene from the Pseudomonas syringe pv. tomato pathogen [6] has provided
some valuable insights. The Pto gene is a member of a clustered gene
family [7]. The other members include (i) Fen kinase, which is 87% similar
Host-parasite interactions: elicitation of defense responses in plants with chitosan
187
to Pto; (ii) Prf, which contains leucine-rich repeats and a nucleotide binding
site, making it resemble the class of R genes discussed above; (iii) Pti I,
which is a serine-threonine kinase possibly acting downstream of Pto. On
the pathogen side, the AvrPto gene product is not endowed with a cell-tocell transport domain; however, Pseudomonads are known to possess a
type III protein secretion system. Components of this system are encoded
by Hrp genes in many bacterial pathogens of plants, and consequently are
usually found to be necessary in the expression of the hypersensitivity
response. The need to confirm the existence of a pathogen cell to plant cell
transport has been bypassed by transferring the PtoAvr gene direct1y to the
host plant. The presence of the host Pto gene and the pathogen-derived
PtoAvr gene in the same plant resulted in the activation of resistance
responses such as phenol accumulations, hypersensitive cell death and
reduced bacterial populations, the symptomology often associated with
resistance against bacterial pathogens. The possibility that at least two gene
products of the clustered Pto gene family can indeed interact was demonstrated in the yeast two-hybrid system [7]. It has been proposed that the
interaction of AvrPto with Pto may stimulate Pto kinase activity and trigger
a phosphorylation cascade. More recently the Pto kinase has been shown to
interact with proteins that bind a cis-element of a PR gene [8] thus possibly
linking the signaling by the avirulence gene to activation of the disease
resistance response. Other R genes that encode proteins with leucine
repeats and or nucleotide binding sites may eventually be found to participate in their own linkage to transcriptional changes. Interestingly, the
predicted proteins of the R genes currently sequenced from plants can be
grouped into potentially related classes of function. The features of these
proteins include leucine-rich repeats, leucine zippers, mammalian interleukin I-like receptors, nucleotide binding sites, membrane anchoring
sequences, kinase domains and some combinations ofthese [9].
Nonhost resistance: investigations of other signals
One might anticipate a myriad of such plant-pathogen signaling when
examining the more complicated and stable nonhost resistance, since the
transcription of the defense response genes in a given plant species is induced by almost every plant pathogen or by any foreign cell. In an effort to
examine the earliest detectable changes that occur in the plant/nonpathogen
interaction, interest has centered on the events of anion flux, oxidative burst
and targeted protein phosphorylation [9, 10]. Such processes have been
examined in the incompatible interaction between parsley cells in culture,
and an elicitor, prepared from the mycelium of the Phytophthora sojae,
a pathogen of soybeans [10]. This elicitor stimulated Ca++ uptake and
hydrogen peroxide increases in the parsley cells and culture solution,
respectively, within 2-20 min. Inhibitors of the elicitor-stimulated ion
188
1. A. Hadwiger
fluxes blocked the oxidative burst (O~) which was proposed to be the
essential element ofthe signal cascade leading to phytoalexin (small antifungal compounds induced in the plant) production. This 0; is proposed
to be generated by an NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase. The details of such a cascade in plants have
not been resolved; however, in animal systems DNA damage can be a direct
effect ofO; action [11].
Examples of other components signaling defense responses
Proteins
In the interaction between barley and the fungus, Rhynchosporium secalis,

a small phytotoxic protein is secreted by R. secalis strains carrying the nip 1
gene (an avirulence gene) when in the presence of barley lines with the R
gene, Rrsl [12]. This interaction is associated with a resistance reaction.
The transfer of the nip 1 gene to a "virulent" race of the pathogen converted
that pathogen to an "avirulent" strain. Single nucleotide exchanges in nip 1
alleles were sufficient to dictate the difference between virulent or avirulent phenotypes.
Enzymes
A plant can also be a target for a fungal enzyme. Endopolygalacturonases

have been shown to activate plant defense responses [13], an activation that
results from the enzymatic release ofresponse-eliciting oligogalacturonide
fragments from the plant's own cell wall [14]. These fragments must be
10-14 sugar units long to be elicitors [15]. Altematively, the smaller fragments, especially mono-, di- and trigalacturonic acid may even assist parasitism by providing nutrition to the pathogen. To further complicate the
interaction, the plant produces a polygalacturonase-inhibiting protein
(PGIP) [16] which is also involved in determining the accumulation of
elicitor active oligogalacturonide. PGIP has been found in walls ofvarious
plants and may serve as a receptor for fungal polygalacturonases [16, 17].
Enzymes of the plant host can also function to release from the fungus
chitin, chitosan fragments capable ofinducing defense responses [18, 19].
Pure preparations of pea chitinase and -glucanase can combine to release
chitosan fragments from Fusarium solani, some ofwhich are large enough
(heptamers) to induce both phytoalexins and PR proteins as weIl as inhibit
the growth of the pathogen. These fragments were also recovered from the
pea-Fusarium interaction [19].
Another example of a fungal enzyme serving as an elicitor of the plant
defense response is a DNase from the fungal pathogen Fusarium solani f
189
sp. phaseoli (Fsph). This DNase adequately induces the nonhost resistance
response of peas to immunize the tissue against its true pathogen, F solani
f sp. pisi. Within 6 h after inoculation with Fsph the catalytic activity of the
fungal enzyme is detected inside the pea nuclei. Also, pea tissue treated
with the enzyme accumulates RNA homologous with PR genes within 3 h
[20].
Other fungal proteins called elicitins are secreted by Phytophthora
species [21]. These proteins induce cell death and tissue necrosis, primarily
in tobacco and radish. In tobacco it appears that elicitin plays an important
role in the determination of avirulence for those species of Phytophthora
for which tobacco is a natural host. Recently, tobacco has been transformed
with the gene encoding the elicitin, cryptogein, which provided the
recipient plant with an increased level of resistance to the tobacco pathogen Phytophthora parasitica var. Nicotianae [22]. The transformed plant
was normal in growth even though the elicitin accumulated in the plant tissue and would have been expected to interact with the cell membrane. The
cell membrane is the hypothesized initial point of contact that sets off a
signal transduction cascade leading to PR protein production. Importantly,
there was an increase in the levels of some PR proteins in the transformed
plants that were not detected in nontransformed plants.
Glycoprotein elicitors
Glycoproteins have been divided into two classes [23]. One class consists
of enzymes that degrade the pectin or pectate components of the cell wall.
The second class involves extracellular fungal products in which the protein component can be subjected to denaturation or proteinase digestion
without any apparent effect on elicitor activity. Consequently, the carbohydrate moiety is credited with possessing the elicitor activity.
Glucan elicitors
Glucan elicitors were first derived from Phytophthora megasperma f sp.

glycinea walls by heat or acid extraction. Subsequent evaluations of such
wall fragments produced subfractions with phytoalexin-eliciting activity. A
well-characterized elicitor isolated from fungal cell walls is a branched
hepta--glucoside [24]. This elicitor was purified to homogeneity, the
primary structure was determined and the structure was confirmed by
chemical synthesis. Reports from two laboratories indicate protein(s) exists
in soybean membranes with a high affinity far the hepta--glucoside
elicitor [15, 25]. As with other receptors, this complex may signal a cascade
of events leading to the eventual activation of genes controlling secondary
pathways en route to phytoalexin synthesis.
190
L. A. Hadwiger
Elicitation of defense responses with chitosan
Because the positively charged chitosan was known to be a component of

fungal walls and polyamines were known to increase the template activity
of chromatin and induce the synthesis of the phytoalexin pisatin in peas
[26], chitosan was evaluated as an inducer of defense responses in plants.
Chitosan induced accumulations of the phytoalexin pisatin at levels as low
as 16 p.g/ml [4]. Additionally, chitosan has been found to directly inhibit the
germination and growth of an array of fungi [27]. More chitosan was
recovered from the interaction between pea tissue and the incompatible
pathogen, F. solani f sp. phaseoli (resistance reaction), than between pea
and F. solani f sp. pisi (susceptible reaction) [4]. There was an early indication that chitosan had a potential dual role in inducing a defense
response as weil as directly inhibiting fungi. It was determined, with both
radio and immunolabeled chitosan that this e1icitor entered the plant cell;
however, chitosan was also seen to be residual in the region of the cell wall
and the plasma membrane [28]. The membrane association was c1early
shown by the Kaus laboratory following chitosan treatments to protoplasts
[29].
Pea endocarp tissue treated with radiolabeled chitosan was fractionated
into microsomal membrane, chloroplast and nuc1ear components that contained 6%, 5% and 19%, respectively of the label taken up by the cell within
5 h [30]. The affinity of chitosan with DNA was demonstrated by DNA
retention on chitosan columns, DNAIchitosan precipitations, alterations of
the DNA melting profile, chitosan-altered DNA circular dichroism spectra
and migration of restriction-digested DNA on a chitosan-containing ge1
[4]. Recently, Kashige et al. [31] showed that chitosan oligomers in the
presence ofCu++ cause single strand breaks in pBR322 plasmid DNA. The
potential of chitosan to cause DNA degradation in pea tissue has also been
reported [30]. Taken together, these reports suggest that DNA may be a cellular target of chitosan.
Altemately, Kauss et al. [29] observed by laser scanning microscope
that chitosan is bound to the surface of protoplasts and induces the synthesis of callose. The degree of induction increases with the degree of
chitosan polymerization up to several thousand. Correspondingly, phytoalexin production and fungal inhibition is size-related, with maximum
activity starting with the chitosan heptamer [18]. This induction potency of
chitosan is reduced by N-acetylation. The phospholipid head groups on the
plasma membrane were suggested as a possible point of interaction [29].
The acetylation of chitosan would both reduce its interaction with fatty
acids and its affinity for the P0 4 - within the DNA sugar-phosphate backbone [30, 32]. Thus there are at least two modes of action proposed for
chitosan in living cells. First, in suspension-cultured plants and protoplasts,
plant responses such as callose synthesis can be triggered by the polycationic chitosan as it localizes to the bound surface ofprotoplasts. Second,
191
in intact pea endocarp and/or tobacco leaf tissue, chitosan can induce a
set of genes known as disease resistance response (DRR) or pathogenesisrelated (PR) genes [5, 33] and/or their promoters [34, 35] (Fig. I). A potential application of chitosan may reside in its ability to activate defense gene
promoters. We propose to utilize the chitosan-inducible promoters to express structural genes [20] capable ofinducing immunity to true pathogens
(Fig. I B).
One ofthe PR proteins, the membrane-Iocated enzyme -I ,3-glucan synthase, does not require induction and is activated by chitosan. This synthase
responds to chitosan with a catalytic increase [29]. It has an absolute requirement for Ca++ in the J.lM range, suggesting that Ca++ uptake may lead
to an increase in cytoplasmic Ca++ and thereby trigger callose synthesis [29,
36]. Because a modification ofthe enzyme may occur to increase the catalytic activity and appearance of callose within 20 min of the application of
chitosan, the increase does not appear to result from the de novo synthesis
of -I,3-glucan synthase. It was proposed that the chitosan-promoted cellular increase in -I ,3-glucan synthase was due to an increased influx of Ca++
ions and that the activity increased by a direct and reversible interaction of
this ion with the enzyme. The action of chitosan was also linked to increased
leakage of cell constituents, thereby causing an influx of external Ca++ into
the cello However, increasing Ca++ uptake or plasma membrane depolarization alone appear insufficient for the induction of callose [32]. Chitosan
binds rapidly (within Imin) to protoplasts. Chitosans differing in degree of
polymerization (DP) and N-acetylation differ in their efficiency to induce
callose synthesis. Chitosan fragments up to DP 14 are almost inactive. Callose (microgram pachyman equivalents) increases from 1.06 to 5.43 as the
DP increases from 90 to 2500 DP when the chitosan elicitor is totally
deacetylated. When the chitosan is 23% acetylated, comparable DPs increase callose from 0.82 to 2.85 J.lg pachyman equivalents. A DP of 5000
produced maximum callose increases, suggesting that part ofthe action was
related to the molecule's ability to bind broad areas of the membrane surface. Similar treatments to plant cells required higher concentrations of
chitosan, possibly because of the reductions due to wall binding and exclusions oflarger chitosan polymers [29]. The formation of callose was proposed to be important in plant/pathogen interactions since callose deposition in the walls of surviving cells is an early event in wound healing. Also
the papillae, a morphological defense structure, develops when the plasma
membrane deposits callose-rich accumulations at sites adjacent to sites of
fungal penetration. Another cell surface action of chitosan may be in
eliciting H20 2 as demonstrated with nitrous acid-cleaved chitosan fragments
(DP 26) [32]. Interestingly, fully deacetylated chitosan exhibited only low
H2 0 2 activity. Highly acetylated chitosan (chitin) oligomers required much
higher levels to elicit a lower concentration of H20 2
In addition to the catalytic increases in callose synthesis and H20 2 ,
chitosan is known to induce defense responses associated with the en-
L.A. Hadwiger
192
A. SOME COMPONENTS OF NATURAL DISEASE RESISTANCE

FUNGALPAmOGEN
-1~~::~-ciiiTIi&\N-===:'~ Direct antifungal action

Activation of _
1,3 glucan synthase
CALLOSE
DISEASE RESISTANCE
Transcription of
some PR genes
PLANTCELL
B. PROPOSED CHITOSAN-ASSISTED DISEASE RESISTANCE

Chitosan
Callose induction
DISEASE RESIST AN CE
1---+10- PR PROTEINS
TRANSGENIC PLANT
Indu tion of
neighboring
cells
Figure 1. Some components ofnatural (A) and proposed elements of chitosan-assisted disease
resistance (B). Fusarium so/ani f. sp. phaseoli (Fsph), apathogen of bean, when challenging
pea tissue, releases a DNase and chitosan both of which can induce some PR genes [35].
Chitosan is also directiy antifungal and can activate -l ,3-g1ucan synthase, an enzyme required
for callose formation, providing additional defense potential [36]. In the proposed chitosanassisted disease resistance, chitosan will activate PR gene promoters and thus should activate
the expression of transferred structural genes (e. g. the fungal DNase gene) linked to such promoters. Extemally applied chitosan will proposedly increase DNase production, an enzyme
shown to promote complete immunity in peas to a pea pathogen [20]. The DNase protein can
enter plant cells and mayaiso translocate to neighboring cells. In interactions such as between
Phytophthora infestans and potato, the pathogen does not have a chitinous wall and is unlikely
to release the chitosan elicitor, thus the extemal application of chitosan initiates the expression
ofthe DNase gene.
Host-parasite interactions: e1icitation of defense responses in p1ants with chitosan
193
hanced transcription/translation of DRR or PR plant defense genes [5].

Some of these genes encode (i) enzymes for secondary pathways en route
to phytoalexins (antifungal compounds) [4], (ii) hydrolytic enzymes such
as RNase, chitinase and -glucanase [37-40], (iii) antifungal cysteine-rich
peptides called thionins orplant defensins [41,42] and (iv) peroxidases that
are directly antimicrobial or capable of generating phenolic polymers such
as lignin [10, 30]. All of these can be induced in plant tissue following
chitosan treatments.
Chitosan mode 0/ action
Since chitosan confronts plant cell walls, cell membranes, the cytosol and
the nuc1eus [28], all ofwhich contain some negatively charged compounds,
one must conc1ude that chitosan may have multiple cellular targets. Chitosan's targets within the membrane enable it to alter membrane function
[36]. Organelles ofthe cytosol capable ofreplicating such as mitochondria,
chloroplasts and nuc1eus possess polyanionic DNA. Chitosan's mode of
inducing defense responses is not solely a product of its cationic nature,
since polygalactosamine when compared with polyglucosamine (chitosan)
is a poor elicitor of pisatin accumulation [30]. Computer-modeled chitosan
octamer is a straight rod that easily fits in the minor groove of DNA,
whereas the distorted rod of a galactosamine octamer does not. Furthermore, chitosan oligomers less than six sugar units long progressively lose
their pisatin-eliciting activity [43]. Other shorter polyamines such as
spermidine, putrescine and cadaverine require much higher concentrations
to elicit pisatin production [26]. Chitosan heptamers have the size and
positive charges capable of interacting with nuc1ear DNA in several ways.
Nuclear proteins such as histones and transcription factors would likely
compete with the foreign chitosan molecule for sites on the DNA. Especially vulnerable to such competition would be histone H1 molecules that
are essential for the intranuc1eosomal assembly of chromatin and histone
H5 that suppresses transcription. Because of the complexity of chromatin
and the assembly of regulatory proteins on the polymerase complex, the
predicted effect of chitosan on the "poised states of chromatin" [44],
enhanceosomes [45], and aggregation of the positive elements of the gene
promoters, is difficult to decipher. Our current knowledge is that chitosan
reaches the nucleus [30], is associated with limited DNA degradation and
induces defense gene promoters in many plant species [35]. Chitosan
is a poor mutagen; thus the DNA alteration is probably both weak and
repairable.
194
L. A. Hadwiger
Practical aspects of chitosan as an agricultural entity

Chitosan has been approved by the Food and Drug Administration of the
USA as a wheat seed treatment [34]. Applications of chitosan to wheat
seeds have been shown to influence the level of lignin in nearly mature
wheat plants, suggesting that the benefits of chitosan applied to seeds can
be translocated to other parts of the developing plant. Seedlings developing
from chitosan-treated wheat seeds have been observed to possess increases
in stern diameter and plumper roots. Further, both seedlings and plants
approaching maturity contain slightly elevated levels oflignin. Under conditions that promote a wheat stem-breaking disease, caused by the fungal
pathogen Pseudocercosporella herpotrichoides, plants from chitosantreated seeds have been observed to suffer less extensive lodging. Lodging
is the toppling over of mature wheat that can significantly reduce recoverable yield, an adversity that could be lessened with larger stern diameters
and lignin-reinforced structural compounds.
Benhamou and Theriualt [46] found that chitosan (0.5 - 2.0 mg/mI) applied
to tomato leaves or roots markedly reduced the number of root lesions
caused by a vascular wilt pathogen, Fusarium oxysporum, and increased the
formation of physical barriers in infected root tissue. EI Ghaouth et al. [48]
have reported a potential use of chitosan in the postharvest preservation
of fruits and vegetables. Specifically, chitosan has activity in controlling
Pythium aphanidermatum on cucumber plants. Chitosan also possesses
antifungal activity against Rhizopus stolonifer, which is also a postharvest
fungal pathogen [48]. The action of chitosan on inhibiting the germination
and growth of plant pathogenic fungi was first reported on an array of
fungi that did not inc1ude those with a predominance of chitosan as a
natural component of the fungal cell wall [27]. More recently, additional
plant pathogenic fungi have been added to the chitosan-inhibited list [34].
The antifungal action of chitosan has also been utilized to reduce the
production of aflatoxin on maize [49].
Chitin as an elicitor
Pearce and Ride [50] have observed that both chitin and chitosan effectively elicit the lignification response in wounded wheat leaves. Glucosamine
or polymers of N-acetylglucosamine less than six sugars in length were
ineffective in inducing this response. Chitin from fungal walls or crab
shells induced a heavy lignification response at 2 p.g/ wound compared
with chitosan that required 5 p.g/wound, suggesting that the induction
response may not be exclusively based on the density of positive charges.
Oligomers consisting of3-5 N-acetylglucosamine residues with variations
via sulfur, sugar and acetyl derivations play vital roles in the nodulation
signaling between Rhizobia and plant hosts [51]. Oligomer size plays a
195
definitive role in the induction ofplant defense responses [15, 18, 19,43].
Chitosan can be prepared in molecular weights exceeding a million. Interestingly, all of these large molecules are capable of eliciting defense
responses to levels comparable to heptamer or octamer size elicitors. The
larger molecules may be digested to smaller oligomers by hydrolysis ofthe
N-acetylglucosamine segments of chitosan, since chitinases appear to be
present throughout plant tissue. Chitosan oligomers are generated in interactions between plants and fungi with chitinous walls. As indicated above,
oligomers with less than five sugars do not substantially elicit phytoalexin
accumulations [18]. Since challenged plants were first examined for
glycosidic enzymes such as chitinase, chitosanase and -glucanase [52]
capable of generating chitinous fragments [19], there have been many
attempts to manipulate oligomer release by engineering plants with the
genes encoding the respective hydrolytic enzymes linked with constitutive
promoters. These efforts have improved the plant's resistance to some
pathogens [53, 54] but not others [55]. The increased level of resistance,
when obtained, was short of immunity. Immunity may require combinations ofhydrolytic enzymes.
Purified chitinases [56] have been shown to be potent inhibitors of
fungal growth [57]. Only a few fungi are extremely sensitive to chitinases
alone [58]. The role of chitinases is variable in the overview of all plantpathogen interactions. For example, chitinases from thorn apple, tobacco
and wheat have similar antifungal properties. The wheat enzyme did not
produce the same oligosaccharide pattern when digesting chitin. All three
enzymes inhibited the germination of Trichoderma hamatum and Phycomyes blakesleeanus spores at 8 - 32 Jlgml- 1, whereas Botrytis cinerea hyphal growth was not affected by concentrations of pure enzyme as high as
320 Jlgml- 1. It is not known if comparable amounts of chitinase are generated at the host-parasite interface. The two simplest explanations of this
antifungal action are that the enzyme either weakens the hyphal tip and the
exposed plasma membrane can burst, or the enzyme generates oligomers
with antifungal properties. The molecular basis ofthe antifungal activity of
chitosan may reside in its ability to inhibit RNA and DNA synthesis in
Fusarium solani [4], since the chitosan-mediated inhibition of F. solani
germination and growth is not accompanied by visible hyphal tip lysis.
Commercial development of chitosan
Chitosan is one of the few natural microbial inhibitors/defense gene inducers that can be produced in quantities that enable economic applications
to crop plants [34]. Applications of chitosan to wheat seeds have been
shown to influence yield and the accumulative levels of lignin in the adult
wheat plants [30]. Some direct benefit may be attributable to chitosan's
antimicrobial properties at the site of the coated, germinating seed as it
196
L. A. Hadwiger
contacts pathogen-infested soil. Although radioactivity from radiolabeled

chitosan applied to seeds can be detected in the upper portion of the
developing plant, it is difficult to obtain accurate determinations of the
final oligomer size transported. Thus it is not clear ifthe chitosan oligomer
is the primary signal inducing defense responses in distal parts ofthe plant.
Wheat grown under optimal conditions of moisture and mild temperature
can incur a wheat stem-breaking disease caused by the fungal pathogen
Pseudocercosporella herpotrichoides, which often causes some obstruction of water transport in the plant. When the wheat seeds are treated with
chitosan (500 p.g g-l of seed) prior to planting, the toppling of the plants
(lodging) is markedly reduced, a benefit that consequently improves yield
[34]. Chitosan treatments of wheat seeds also induce resistance to
Fusarium graminearum [59]. Chitosan at 4 mgml- 1 significantly improved
seed germination and seedling vigor. The occurrence of seed-bome
F. graminearum was reduced by more than 50%. Leaves ofthe developing
seedling increasingly accumulated lignin precursors, and lignin as the
chitosan concentration was increased. Interestingly, there was a wheat
varietal difference in response to the chitosan seed treatment. The treatment
of tomato roots via cut stern openings of decapitated plants with native
chitosan and -glucan elicitors caused a significant decrease in R OXYsporum f sp. radicis-lycopersici infection of roots [60, 61]. A restriction
of fungal growth to the epidermis and the outer cortex, a decrease in pathogen viability and the formation of wall appositions at sites of attempted
penetration were observed. These barriers contained callose, pectin and
phenolic compounds (probably lignin). Chitosan at concentrations of 12
and 37 mg I-I reduced plant mortality, root rot symptoms and yield loss
attributable to Fusarium oxysporum f sp. radicis in greenhouse-grown
tomatoes [60]. Although chitosan is directly antifungal to this pathogen, the
ultrastructural observations suggested that chitosan 's mode of action was to
sensitize the plant to respond more rapidly and efficiendy to attack.
Chitosan elicits other defense responses
Chitosan and its derivatives, especially nitrous-cleaved chitosan, induce the

synthesis of proteinase inhibitor I in detached young tomato plants [62].
Cleaved chitosan was three times more potent than the other oligosaccaride
inducers. The proteinase inhibitor, a factor in the plant's defense against
insects, also accumulates in tomato in response to wounding and insect
damage. Chitosan (125 p.gml- I ) can activate a 48-kDa myelin basic protein
kinase to levels 2.5 times the control when applied to cut ends ofthe tomato
stern [63]. Likewise, chitosan triggers the release of linolenic acid from
membranes and its conversion to jasmonic acid. Further, chitosan elicits
a defense reaction in lodgepole pine that is expressed as monoterpene accumulations in the phloem [64].
197
Conclusions
Chitin and its derivatives, primarily chitosan, constitute part of the natural
signaling in host-pathogen interactions. Chitosan can induce immunity in
the plant to its true pathogens and all or portions of the nonhost disease
resistance response. Thus the importance of chitin and chitosan signals in
agriculture is becoming obvious despite gaps in our understanding of their
individual modes of action.
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ed. by P. Jolles and R.A. A. Muzzarelli
@ , 999 Birkhuser Verlag BasellSwitzerland
Inhibitors of chitinases
Klaus-Dieter Spindler 1 and Margarethe Spindler-Barth 2
I Universitt Ulm, Abteilung Allgemeine Zoologie, Albert-Einstein-Allee 11-13,
D-89069 Ulm, Germany
2 Heinrich-Heine-Universitt flsseldorf, Lehrstuhlfiir Hormon- und
Entwicklungsphysiologie, Universittsstr. 1, D-40225 Dsseldorf, Germany
Summary. In this review we describe inhibition of chitinases from bacteria, fungi, plants and
animais by allosamidin and its derivatives, cyc1ic peptides, styloguanidin and divaient cations.
Most information is available for ailosamidin, whose important structural features necessary for
inhibition are known. At least one N-acetylailosamine sugar must be present, and the spatial
arrangement of the allosamizoline moiety are important for inhibition. Less complex compounds are therefore possible as lead structures for the development of agents interfering with
chitinase. There is a pronounced species specificity in chitinase inhibition by ailosamidin: halfmaximal values are often in the range ofO.l-l pM (e.g. in all arthropods), being lower in
nematodes (0.048, 0.0002 pM, respective1y) and amoeba (0.002-0.01 pM) and quite divergent
in fungi (0.01-70 pM). These differences cannot be caused by the catalytic centers of family
18 and 19 chitinases.
Introduction
Interest in chitinase inhibitors focuses mainlyon two aspects: First, inhibitors are useful tools to elucidate the mechanism of enzymatic catalysis
[1, 2] as shown, for example, by the X-ray crystallographic analysis of
hevamine/inhibitor complexes by Scheltinga et al. [3, 4]. Second, since
chitin does not occur in vertebrates, interference with chitin metabolism is
considered as a suitable target for pesticides [5 - 7], which has been demonstrated successfully by the application of chitin synthesis inhibitors as
insecticides for nearly 30 years [5, 7].
Due to the widespread occurrence of chitinases, a broad range of applications for inhibitors seems possible as agents against fungi [8, 9],
insects and ticks [5-7, 10] or nematodes [10, 11] not only for agricultural
pests but also for the prevention of insect-transmitted diseases [12] and as
antiparasitic drugs [5-8, 10-15]. However, if chitinase inhibitors are used,
therr many essential physiological functions such as defence mechanisms,
morphogenesis and reproduction in plants [16-19], nutrition and molting
in arthropods [20-22] and hatching in nematodes [13] must be taken into
account. In addition, chitinases are present in vertebrates [23], including
humans. In this case chitinases may be involved in defence mechanisms
against pathogens [24-26]. A role in glycoprotein and glycolipid metabolism is also discussed, since chitinase activities are markedly increased
in patients with Gaucher's disease [27].
202
K.D. Spindler and M. Spindler-Barth
Allosamidin
The most prominent and best-characterized chitinase inhibitor, allosamidin

(Fig. 1) was isolated 10 years aga from the culture broth of Streptomyces
sp. by Sakuda et al. [28]. The compound is active in a broad variety of
different animals, bacteria and fungi [2, 30], although considerable speciesspecific differences in sensitivity are observed (Tab. 1). Despite the fact
that a considerable amount of information on plant chitinases is available,
many enzymes have been isolated and the corresponding genes c10ned
[31], it is unknown in most cases whether these enzymes are inhibited by
allosamidin or not. Until recently it was assumed that only members of
family 18 chitinases, which are characterized by an a,p.-barrel structure of
the catalytic centre [32-34] are affected. But this is not strictly true, since
some subtypes of family 19 chitinases are inhibited by allosamidin, for
example the chitinase isoenzyme Ib from Phaseolus, whereas the isoenzyme type Ia from the same species is not inhibited [10]. The highly
susceptible enzymes from root extracts from Pinus and Eucalyptus [35]
might also be family 19 chitinases, because this type of chitinase is predominant in plants [31]. Recently, an enzyme of the family 19 enzyme was
also found in Streptomyces griseus [36], where chitinases ofboth families
N
H 2 N --------#
~N
NH H-
HzN
A.-
N
CI
Styloguanidines 1: R 1 = R 1 = H
2: R 1 = Br, R 1 = H
3:R1 =R1=Br
OH
OH O
OH
HO~~~O~~O~'l
~ ~
OH
Ac
OH
N,2
'AI
Allosamidin: R 1 = R 1 = CH 3
DemethylaUosamidin: R 1 = CH3.R1 = H
DidemethylaUosamidin: R 1 = R 1 = H
Figure I. Structural fonnulae of allosamidin and its demethylated fonns and of styloguanidin
203
Table I. Inhibition of chitinases by allosamidin
Taxon
Species
IC so value (j.1M)
Bacteria
Serratia marcescens
Streptomyces sp.
Fungi
Candida albicans
K i value (j.1M)
Reference
0.1; 0.6
46;44
61; 29
37
3.7; 20.9
0.8
0.3; 0.02
0.23
37
62
0.24
0.49
34.5
3.2; 1.6
0.27; 0.15; 0.29
0.29; 0.48
0.1; 0.01
0.09; 0.045
58
70
63
63
39
11; 48
35
35
35
35
35
35
Plants
Phaseolus vulgaris (type Ib)
Pinus sylvestris
Eucalyptus pilularis
1
0.01; 0.025
0.56; 6
10
35
35
Protozoa
Entamoeba invadens
0.01; 0.002; 0.0011
15
Nematodes
Onchocerca gibsoni
0.0002
11
0.048
10
0.69
10
Trichoderma sp.
Neurospora crassa
Paxillus involutus
Pisolitus tinctorius
Suillus variegatus
Boletinus claviceps
Armillaria ostoyae
Heterobasidion annosum
Haemonchus contortus
Ticks
Boophilus microplus
Crustacea
Artemia salina
Penaeusjaponicus
Insects
Bombyx mori
Chironomus tentans (celIline)
Luci/ia cuprina
Spodoptera litura
0.32
0.2
0.17
0.1
46
64
0.1
0.1; 0.22
65
46;44
10
60
If more than one value per line is given, data from independent investigations are presented or
different substrates were used.
occur simultaneously. Since in this species both an allosamidin-sensitive

and an -insensitive clone has been described [37] it would be interesting to
see whether sensitivity correlates with a certain enzyme family.
Due to the selective inhibition of endochitinases, allosamidin is a lead for
the development of new pesticides. Allosamidin itself is not suitable,
because its structure is too complicated to allow large-scale synthesis, and
204
K. D. Spindler and M. Spindler-Barth
the compound is not stable under field conditions. Tests conducted with a
variety of different allosamidin derivatives, chemically synthesized [8, 29,
30, 38-44] or isolated from the culture broth of Streptomyces [8, 45, 46],
revealed the essential structural features of the molecule necessary for
enzyme inhibition:
1) At least one N-acetylallosamine sugar must be present for efficient inhibition.
2) Glucosamine can replace the allosamine moiety without a negative
effect on inhibitory potential.
3) The spatial arrangement of the allosamizoline moiety is important for
inhibition.
4) If one sugar is omitted and the arrangement of the cyclitol residue is
changed, the inhibitory effect is diminished further.
These essential features were obtained by inhibition studies using an insect
enzyme [44] and can vary in some aspects according to the source of
enzyme used. For example, the insect enzyme is inhibited to the same
extent whether or not an N -acetylallosamine moiety is omitted, whereas the
inhibition of the bacterial enzyme from Serratia marcescens is impaired if
one sugar moiety is lacking [44]. Species-specific variation ofthe inhibition by allosamidin derivatives is also reported from Sakuda [8]. As an
example, the influence of methyl groups at the allosamizoline moiety is
given in Table 2. X-ray analysis ofthe three-dimensional (3D)-structure of
the plant enzyme hevamine [3] and chitinase A from Serratia marcescens
[47] revealed that both family 18 enzymes bind allosamidin in the catalytic groove, which is in line with the competitive type of inhibition which is
Table 2. Influenee of methyl groups at the allosamizoline moiety on allosamidin ehitinase

aetivity
Taxon
Species
Allosamidin
K;
Baeteria
Serratia marcescens [46]
Fungi
Candida albicans [7]
Trichoderma sp. [7]
Saccharomyces cerevisiae [7]
Crustaeea
Artemia salina [46]
Inseets
Bombyx mori [7]
Chironomus tentans (eeIlline) [46]
All data are given in }IM. []
IC so
0.61
Demethylallosamidin
K;
IC so
1.2
1.3
0.5
10.2
1.3
55.6
2.2
0.05
0.1
bibliographie referenee.
K;
n.d.
4.7
12.8
18.4
0.08
2.2
IC so
49
1.6
0.17
Didemethylallosamidin
1.0
206.7
205
observed in most cases [30]. For the Serratia enzyme, the presence of an
additional binding site was assumed. An indication for differences in the
catalytic centre and presumably also for the mechanism ofhydrolysis is the
fact that sometimes a noncompetitive or mixed-typed inhibition by allosamidin is observed [35], even for a family 18 chitinase [48].
Experiments were performed with diacetylchitobiose or triacetylchitotriose instead of two units of N-acetyl-D-allosamine. Moreover, the allosamizoline ring system was replaced by N-phenylcarbamate, epoxybutyl,
histidineamide or formylpyrrolidine groups. In all these cases, three to four
orders of magnitude higher concentrations were necessary for inhibition
[30].
Cyclic peptides
Two years aga a novel chitinase inhibitor, the dipeptide cyclo(L-Arg-DPro), was isolated ftom the marine bacterium Pseudomonas sp. [49]. The
compound is active against chitinases ftom Bacillus sp. [49], Saccharomyces and Candida [50]. Since the Saccharomyces enzyme is also inhibited
by allosamidin, it would be interesting to test whether chitinases ftom other
species such as insects and nematodes are affected by this dipeptide.
In the case of the Bacillus chitinase, the inhibitory effect is the same
whether L- or D-Pro is present in the moleeule, but a change in the optical
configuration ofL- to D-Arg impairs the efficiency of chitinase inhibition.
In contrast, for the yeast enzyme the inhibitory potential is identical for
both optical isomers of Arg and Pro. The type of inhibition is not yet determined, and the binding site of the two optical isomers is also unknown.
The cyclic dipeptide inhibits cell separation in Saccharomyces, although
less efficiently than aIlosamidin. In the dimorphie yeast Candida albicans,
the change into the filamentous form is prevented, indicating a functional
role of chitinase in the elongation process. This is an important observation, because only the filamentous form is pathogenic, but not the yeast
form. Since the compound has no growth-inhibiting activity as tested on
various microorganisms, and since it is not cytotoxic to various vertebrate
celliines, it seems weIl suited also for medical applications.
Styloguanidin
Three c10sely related chitinase inhibitors were isolated ftom the marine
sponge Stylotella aurantium [51] (Fig. 1). Until now only the inhibition of
chitinase ftom a bacterium, Shewanella sp. (2.5 ]lg/disk) and indirectly by
a bioassay in an arthropod, the bamacle (10 ppm), is reported. In the latter
species the settlement of the ftee-swimming cypris larvae is impaired,
making this inhibitor a putative antifouling agent. Unfortunately nothing is
206
K.D. Spindler and M. Spindler-Barth
known so far about the type of inhibition, the specificity of these compounds, the types of hydrolases being affected and about the stability of
these alkaloids in nature. In addition, due to the complex structure largescale synthesis or mass-isolation from sponges seems impossible.
Other inhibitors
Several divalent ions were tested as chitinase inhibitors with inconsistent
results. Cu z+, Znz+ and Hg z+ inhibit chitinases from bacteria [52,53], some
[54,55] but not all [56] insects and two crustacean species [53, 57] only at
concentrations of 1 mM. If inhibition by these ions is observed in arthropods, it is not due to complex formation with SH groups, since SHblocking agents do not influence chitinase activity [53-55]. Some plant
chitinases are inhibited by divalent ions [58]. A unique feature is the activation of chitinase from the marine bacterium Alteromonas by Na+, K+, Ca2+
and Mg z+ [59].
Conclusions
With the isolation of allosamidin [28], the first potential and sensitive
chitinase inhibitor was available. Its efficient inhibitory action on enzymes
from various taxa, which is restricted mainly to family 18 and some family
19 chitinases, makes this compound a promising lead for the development
of a new group of pesticides. Three approaches are currently being pursued:
1) Investigation of the structure-activity relationship between allosamidin
derivatives and inhibitory action to reduce the complexity of the lead
structure, as already demonstrated successfully.
2) Elucidation of the 3D structure of the catalytical centre of various
chitinases, which supports directed drug design.
3) High-throughput screening for compounds interfering with chitinase
activity obtained either from relevant pests or by recombinant gene
expression, as already demonstrated successfully.
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ed. by ~ Jolles and RA A. Muzzarelli
Mammalian chitinase-Iike proteins

Gilles Bleau, Frederic Massicotte, Yannick Merlen and Chantale Boisvert
Departement d 'Obstetrique-Gynecalagie, Centre Haspitalier de 1'Universite de Mantreal,
Hopital Saint-Luc, 264, baul. Rene-Levesque est, Mantreal, Quebec, Canada H2X 1P 1
Summary. Mammals express genes coding for proteins that show significant similarity to
chitinases of family 18 glycosyl hydrolases. These chitinase-Iike proteins have no chitinase
activity due to changes in critical residues in the putative active center. One of these is oviductin,
a high molecular weight glycoprotein most likely involved in fertilization and protection ofthe
tubal epithelium owing to its mucin character. Another is YKL-40 (HCgp39) produced in association with tissue remodeling. Such proteins could have a general function in morphogenesis.
Introduction
An intriguing concept is emerging for a newly discovered group of mammalian proteins characterized by sequence similarity to chitinases. While
chitin is not a polysaccharide found in mammals, all of the species studied
were shown to express chitinase-like proteins. In contradistinction to
family-18 glycosyl hydrolases to which they are related, these proteins are
devoid of chitinase activity due to amino acid substitutions in the region
corresponding to the active site in chitinases. However, their phylogenetic
conservation and their expression under specific conditions predict significant biological roles that need to be fully elucidated.
An impressive literature has rapidly accumulated describing a high
molecular weight glycoprotein secreted by the mammalian oviduct; we will
refer to this protein by the trivial name oviductin, also designated as
oviductal glycoprotein, oviduct specific protein and restrus-associated
oviductal glycoprotein [I]. The other most studied chitinase-like protein
will be conveniently referred to as YKL-40. It inc1udes human HC gp-39,
porcine gp38k, mouse BRP39 and bovine chitinase related protein-I
(CRPI). A different but c10sely related human protein, YKL-39, was
recently described; only sequence information is available on the murine
YM-l and eosinophil chemotactic factor (ECF-L precursor).
In this chapter, we will review the general structure of these proteins, the
striking similarity of their primary sequence as deduced from complementary DNA (cDNA) analysis, their tissue localization in relation to
control of biosynthesis and most interestingly their putative biological
functions.
212
G. Bleau et al.
General structural features of chitinase-like proteins
YKL-40 andYKL-39 are ofrelatively smaH apparentmasses (40 and 39 kDa,

respectively) [2]; they are much smaHer proteins than oviductins, owing to
their shorter polypeptide sequences as weH as major differences in glycosylation. 1t was shown that human YKL-40 is N-glycosylated [3], whereas
YKL-39, which has one consensus N-glycosylation site near the N-terminal,
is not a glycoprotein [2]. By analogy to members of farnily-18 glycosyl
hydrolases, these two proteins would adopt the structure of an (a)8 barrel.
Oviductins, basicaHy the same protein with variations in structure from
one species to the other, are more complex molecules. While the core polypeptide contributes for 57-76 kDa, they are secreted as high molecular
weight glycoproteins (reviewed in [4]). They exist as monomers, since they
do not form intermolecular disulfide bridges. The M r ofthe secreted forms
range from -75-100 kDa in the rabbit and the porcine, ovine and bovine
species [5-9], to -120-130 kDa in the human, baboon, rhesus monkey
[10-12] and cat (G. Bleau, unpublished), and up to -160-350 kDa in the
hamster and mouse [13 -15]. The very large differences in M r mainly result
from the extent ofN- and mostly O-glycosylation, some 40-60% ofthe M r
consist of carbohydrate chains. The presence of numerous sialic acid
residues and of sulfate groups on O-linked sugars contribute to the charge
heterogeneity of these negatively charged molecules (as detected by isoelectric focusing). The absence of intermolecular disulfide bridges and the
charges on the molecule likely prevent the formation of mucus gel; this
should cause the protein to take an open, highly hydrated conformation.
Chitinase-like domain
The region of the proteins oviductin and YKL-40 corresponding to their

chitinase-like domain is presented in Figure 1. Conserved residues in the
deduced sequences of six weH-characterized oviductins from as many
species indicate a high degree of identity (65%) distributed over the entire
sequence, with stretches that are particularly weH conserved and that are
unique to oviductins in some cases. For example, residues 139FFL 141 are
conserved within the region corresponding to the catalytic center of
chitinases, but are not encountered in YKL-40 nor in any of the other
chitinase-like proteins. Also noteworthy are residues 201LGR203 and
237KSSA240. These stretches of conserved residues, found only in oviductins, could be involved in specific interactions with structures such as
the zona peHucida, to which they firmly bind. Even though the biological
function of oviductins is still uncertain, the high percentage of similarity
over this region, which ranges from 76% to 99% upon pairwise species
comparisons [12], as weH as their absolute tissue specificity ofexpression,
indicate that they are homologous proteins.
Mammalian chitinase-like proteins
213
Oviductins
YKL-40 prot .
Identity
M--LLL--GL -L--K---G- A-KLVCYF-N WA-SRP--AS I-P-DLDPFL

MG-----TGF --L-LLQ-C- AYKLVCY-TS WSQYREG-GS --PDA---FL
M-------G- ---------- A-KLVCY--- W---R----S --P-----FL
50
50
Oviductins
YKL-40 prot .
Identity
CTHL-FAFAS M---QIV--- --DE---YPE FN-LKERN-- LKTLLS-GGW 100

CTH-IYSFAN IS------T- EWND---Y-- LN-LK-RN-N LKTLLSVGGW 100
CTH----FA~ ~-------- -------Y-- -N-LK-RN-- LKTLLS-GGW
Oviductins
YKL-40 prot.
Identity
NFGT-RFT-M LST---RE-F --S---- LR- H-FDGLDLFF LYPGLRGSP- 150

-FG--RFS-I AS-T--R--F --SV-PFLR- -GFDGLDLAW --P--R . . . . 146
-FG--RF--- -S----R--F --S----LR- --FDGLDL-- --P--R----
Oviductins
YKL-40 prot .
Identity
-DRW-F-FL- EEL--AF--E A-LT--PRLL LSAAVS--P- -----Y---- 200

.DK----TL- KE--AEF.-- ----G---LL LS-A--AGK- -IO--YDIA- 194
-D------L- -E----F--- --------LL LS-A------ -----Y----
Oviductins
YKL-40 prot.
Identity
LGR-LDFI-V LSYDLHGSWE --TGHNSPLF SL--O ..... -KSSA--M-Y 245

I--HLDFI-- -TYDFHG-WR ---GHHSPLF -GQ-D---DR -SN- -YAV-Y 244
----LDFI-- --YO-HG-W- ---GH-SPLF ----0----- ---------Y
Oviductins
YKL-40 prot.
Identity
WR-LG-P--K L-MG-P-YGR -F-LL--S-N -L-----GPA SPGKYTKQ-G 295

--RLGA-A-K L-MGIPTFG- S.-TLASS-- --GAP-SG-G -PG-FTKE-G 293
---LG--- - K L-MG-P--G- ----L--S-- - - -----G-- -PG--TK--G
Oviductins
YKL-40 prot.
Identity
FLAY-E-C-F ---A-K-WI- -QYVPYA--G KEW-GYD--- SF-YKA---- 345

-LAYYEICDF L-GA---R-- ---VP-ATKG NQWV-Y-D-E SVK-K---LK 343
-LAY-E-C-F ---A------ ---VP-A--G --W--Y---- S---K-----
Oviductins
YKL-40 prot.
Ident it y
-EHFGGAMVW TLD-DD-RG- FCG-GPFPLV --LN--L--- E

---LAGAMVW -LDLDDF--- -C----FPLT -AIKD-LAA-----GAMVW -LD-DD---- -C----FPL- ------L--- -
386
383
Figure I. Comparison of conserved residues found in mammalian chitinase-Iike proteins.

Chitinase-Iike region, corresponding to residues 1- 386 for oviductins and complete protein for
YKL-40, were aligned using the program Pileup with invariant residues deduced using Pretty.
Aligned sequences for oviductins include human (U09550), mouse (032137), bovine (016639,
we assumed M at position 1), porcine (U43490) ovine (UI6719; U17988) and hamster
(032218; U15048) proteins; the sequences utilized for YKL-40 proteins include human
(M80927), mouse (X93035), bovine (AFOI1373) and porcine (U\9900) proteins. Oashes indicate lack of identity; dots represent gaps introduced for optimal alignment of individual
sequences; asterisks identifY the conserved cysteines, and arrow indicates the N-terminal ofthe
secreted forms. The region corresponding to the catalytic center in chitinases is in bold, and the
conserved N-glycosylation site is underlined.
YKL-40 proteins from four species are also weil conserved (Fig. 1), with
63% identical residues. There is no stretch of conserved residues specific
for YKL-40, which suggests that this protein would interact with the same
type of oligosaccharides as YKL-39 and chitotriosidase. The open reading
frame for human YKL-39 codes for a protein of385 amino acids with 61 %
similarity to YKL-40 [2]. As described by many authors, the similarity of
these proteins with members of family-18 glycosyl hydrolase [16] is
214
G. Bleau et al.
striking: the residues that make up the identity sequence (Fig. 1) are most
often conserved in chitinases of this family. For example, this partially
applies to the active center of chitinases, where a major nonconservative
substitution (replacement of one of the aspartic acid residues) in chitinaselike proteins (Fig. 2) results in a total lack of enzymatic activity. The deduced sequence of porcine YKL-40 contains a region, GRRDKR, which
corresponds to a heparin binding site (X _r B-B-X+ 1-B-X+ 3 , where X and
B are hydropathie and basic residues, respectively) reported in a variety of
heparin-binding proteins [17]; despite the presence of an acidic residue at
Oviductins
Human
SVISLLRTHDFDGLDLFFLYPGLRGSPMHDRWTFLFLI
Mouse
SVISFLRIHGFDGLDLFFLYPGLRGSPPHDRWNFLFLI
Bovine
Porcine
Ovine
SVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL
SAIGLLRTHGFDGLDLFFLYPGLRGSPRRDRWNFLFLL
SVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL
Hamster
SVVSFLRTHGFDGLDLFFLYPGLRGSPINDRWNFLFLI
Identity
S----LR-H-FDGLDLFFLYPGLRGSP--DRW-F-FL-
Human
SVPPFLRTHGFDGLDLAWLYPGRR ..... DKQHFTTLI
Mouse
SVAPFLRSYGFDGLDLAWLYPRLR . .... DKQYFSTLI
Bovine
Porcine
Identity
SVPPFLRTHGFDGLDLAWISPGRR ..... DKRHLTTLV

SV-PFLR--GFDGLDLAW--P--R ..... DK----TL-
YKL-39
Human
SIILFLRNHNFDGLDVSWIYPDQK ..... ENTHFTVLI
YM-l
Mouse
SVIRFLRQYNFDGLNLDWQYPGSRGSPPKDKHLFSVLV
Chitinases
S. marcescens
S. cerevisiae
ASERPFDSAVVDGFDFDIENNNEVGYSALRTKLRTLFA
YKL-40
SVPPFLRTHGFDGLDLAWLYPGWR . . ... DKRHLTTLV
VKEFLQTWKFFDGVDIDWEFPGGKGANPNLGSPQDGET
B. malayi
Human
SAIAFLRKNNFDGFDLDWEYPVGVAEEHAKLVEAMKTA
Identity
-----------DG-D-D-E-------------------
SAI RFLRKYSFDGLDLDWEYPGSQGSPAVDKERFTTLV
*
Figure 2. Alignment of a region in chitinase-like proteins encompassing the putative active site
of chitinases. Sequences for oviductins and YKL-40 proteins are as in Figure 1; YKL-39
(U49835) and YM-I (M94584) are also presented. The regions encompassing the putative
active site in chitinases include those ofthe bacterium Serratia marcescens (L38484), the yeast
Saccharomyces cerevisiae (M74069; M74070), the nematode Brugia malayi (M73689) and
human chitotriosidase (U29615). The Asp and Glu residues essential for the glycohydrolytic
activity ofthe active center of chitinases (bold caracters) are marked by asterisks. The conserved
heparin binding site is underlined. Dashes represent the lack of identity, and dots identifY gaps
in the alignment.
Mamma1ian chitinase-like proteins
215
the + 1 position, porcine YKL-40 binds heparin strongly. Among oviductins

[5] and members ofYKL groups (Fig. 2), only porcine oviductin and human
YKL-40 contain a similar consensus sequence with an acidic residue at + 1,
but they both lack the additional basic residue at the position + 3 present in
the porcine YKL-40. The latter residue, which contributes to the basic
character of this region, may counteract the effect of the acidic residue.
Porcine oviductin and human YKL-40 might not bind heparin.
Of particular interest is the presence of four cysteine residues highly
conserved in aIll 0 proteins from the six species (Fig. 1), as weIl as in YKL39, YM-l and ECF-L. These conserved cysteines are also present in
chitinases such as human chitotriosidase, and nematode and insect
chitinases. It is not known whether these sulfhydryl groups are free or
whether they form intramolecular disulfide bonds, information that would
be useful in predicting the overall conformation of the proteins.
Mucin-like domain in oviductins
The C-terminal portion of all known oviductins is rich in threonine and
serine residues (27.1-37.6% T/S depending on the species) compared with
the N-terminal portion (10.7-11.8% T/S); it also contains more proline
residues (9.2% as part ofthe P/T/S region). This PTS region ofthe molecule, starting at position 366 of the mature secreted human protein,
accounts for the differences in the M r of core proteins and is coded by a
single exon [12]. Such a domain is absent in the other mammalian chitinaselike proteins. In addition to its core polypeptide sequence, the sugar
residues in the PTS region contribute significantly to the M r of the fully
glycosylated proteins, since oviductins with the shortest C-terminal portions have lower Mr(~90 kDa) than those with the longest C-terminal
portions (M r of ~ 120 to up to ~ 350 kDa).
Interestingly, the hamster and human proteins contain tandemly repeated
sequences of 15 residues (see Tab. 1), and allelic polymorphism in the
number of tandem repeats was demonstrated in both species [13, 18]. High
numbers of similar repeats, either perfect or displaying small variations
around a consensus, are found in typical mucins; an extensive allelic polymorphism is also observed, for example, in human MUCI and MUC2 but
not in mouse Muc 1 (reviewed in [19]). Some chitinases such as those of
Bombyx mori (see Tab. 1) and Entamoeba histolytica [20] also contain
repeated sequences; length alleles with three or four imperfect repeats were
observed in the case of Brugia malayi [21].
The hamster and human oviductins share the characteristic structural
features that define epithelial secretory mucins [4]. They are extensively
O-glucosylated (~40% O-linked oligosaccharides by weight for human,
and more than 60% for hamster), with tandemly repeated sequences rich in
P/T/S (34% in their C-terminal portions, 24% for the whole proteins), and
216
G. Bleau et al.
Table I. Typical or precise tandemly repeated sequences rich in S/T residues

REPEATEDSEQUENCES
No. OF REPEATS
IN EACH ALLELE
Chitinases
Bombyxmori
Brugia malayi
PTTTTTTVK
SETEAYDTDETEET
SSESKHE and SSEVKPD
3
3/4
8
Mucins
MUCI (human)
Muc1 (mouse)
MUC2 (human)
GSTAPPAHGVTSAPDTRRAP
DSTSSPVHSGTSSPATSAPE
PTTPITTTTTVTPTPTPTGTQT
25 to > 125
16
50 to 100
Oviductins
Human
Hamster
Mouse
GEKTLTPVGHQSVTP
GGETMTTVGNQSVTP
SKTTTGI
3/4/5/5.5
5/6/7
21
See text for references and other accession numbers; Bombyx mori (U86876).
allelic polymorphism in the number of repeats. This concept could be of

significance in defining the biological function of oviductins as mucins [4]
or sialomucins [22] possessing a chitinase-like domain.
Lack of chitin ase activity
Using different substrates, many authors have observed that the mammalian chitinase-like proteins are devoid of glycolytic activity [2, 3, 5, 11,
22,23]. This is best explained on the basis ofnonconservative substitutions
of either one or both of the acidic amino acid residues (Fig. 1) shown to be
essential for chitinase activity [24]. The corresponding region in a similar
glycoprotein, DS47 from Drosophila melanogaster, also has such substitutions and does not exhibit chitinase activity [25]. In contrast, human
chitotriosidase, which shows a high degree of sequence similarity to
several chitinases and to the chitinase-like proteins, has conserved these
catalytic residues and can hydrolyze chitin; it was therefore considered to
be a chitinase [26]. This enzyme is responsible for the marked increase in
chitotriosidase activity in the plasma of patients with Gaucher's disease and
could be used in the diagnosis of this condition and to assess therapeutic
modalities [27].
Chromosomallocalization
The single-copy gene for human YKL-40 (HC gp-39) was localized to
chromosome 1 [3], the gene for YKL-39 (HUMCHIT) to chromosome
Ip13.3 (GenBank accession no. U58515) and the human oviductin gene
217
was also mapped to chromosome I p 13 [18]. This suggests a chitinase-like

gene cluster in this region of the chromosome 1. In contrast, the locus for
human chitotriosidase, the enzyme responsible for plasma chitinase activity, was mapped to chromosome 1q in the area of 1q31-1 qter [28].
Tissue specificity and control of expression
Oviductins are tissue-specific, whereas the other chitinase-like proteins are
expressed in diverse tissues. Cloning and functional studies of the promoters should soon explain this major difference. The biosynthesis of oviductins is confined to the epithelial secretory cells ofthe oviduct, mostly in
the ampullary portion (reviewed in [4]). In almost all ofthe species studied,
maximum expression occurs around the time of ovulation with barely
detectable levels during the luteal phase ofthe estrus cycle [5, 10,22]. In
contrast, oviductin messenger RNA (mRNA) levels in the hamster are
fairly constant throughout the estrus cycle [13], and the protein is present
in the oviduct even during early pregnancy [29]. Long-term ovariectomy
invariably results in virtually undetectable levels of oviductin mRNA and
protein. Estradiol replacement is a potent stimulus that increases the
signals, an effect that is antagonized by progesterone in all species except
the hamster (reviewed in [4]). These studies indicate a key role for
estrogens in the control of oviductin production. The possible participation
of luteinizing hormone (LH) in increasing oviductin production would
not be at the promoter level but wou1d rather reflect an effect on mRNA
stability in cultured cells [30]. The physiological importance of this putative control mechanism remains to be demonstrated.
The production ofYKL-40 was originally observed as a heparin-binding
protein secreted by cultured vascular smooth muscle cells ([23] and
references therein) and bovine breast tissue during its involution [31]. Protein and/or mRNA for YKL-40 were also detected in the liver, synoviocytes, chondrocytes, the MG-63 osteosarcoma cell line (a murine mammary tumor) and low or undetectable level were reported for other tissues
(reviewed in [2]). A higher level of expression of porcine YKL-40 was
detected in nodulating smooth muscle cells compared with these cells in
monolayers [23]. Expression ofYKL-39 was also detected in chondrocytes
and synovial cells, as well as lung, heart, infant brain and placenta [2].
Given the cell types in which these proteins are produced, interest focused
on their potential control by cytokines: transforming growth factor
(TGF) can repress expression [3] but interleukin (IL)-l, tumor necrosis
factor a (TNFa), or insulin-like growth factor-I (lGF-I) do not [3, 32].
More recently, YKL-40 (HC gp-39) was shown to be expressed in association with late stages of monocyte to macrophage differentiation [33, 34];
interestingly, the levels of expression correlate with the degree of morphological differentiation of a promyelocyte leukemia cell line induced
218
G. Bleau et al.
by phorbol myriotyl acetate (PMA) and vitamin D3 treatments [34]. The

deduced protein YM-l (M94584) would also be expressed by activated
murine macrophages, and ECF-L precursor (D87757) by T lymphocytes.
There is a paucity ofinformation on promoter ftmction for chitinase-like
proteins. 5' -Flanking sequences are now available for the hamster oviductin
gene (GenBank accession no. AF026552) and for CHI3L1 [33]. For the
hamster oviductin gene, the presence of an atypical TATA box is noteworthy, and an almost perfect estrogen responsive element (ERE) could
most likely account for the estrogen-dependent expression of the protein
(Y. Merlen and G. Bleau, unpublished). The 5' -flanking region of the
gene CHI3L1 (coding for the human YKL-40) contains two transcription
initiation sites, with the major one at - 82 relative to the first in-frame start
codon and 28 nucleotides downstream of a consensus TATA box [33]. Promoter analyses will be critical to better understand the regulation of tissue
specificity and temporal expression of chitinase-like proteins under diverse
physiological and pathological conditions.
Biological functions
The search for the biological roles of chitinases-like proteins is surely the
most important issue in this area. The anatomical sites and the particular
conditions under which the proteins are expressed provided the basic clues
in formulating hypotheses. Evidence supports an implication ofYKL-40 in
tissue remodeling in cartilage [3], morphological reorganization of vascular smooth muscle cells [23], tissue involution after cessation oflactation
[31] and late macrophage differentiation [33, 34]. In most species, oviductins are produced and secreted in large amounts around the time of
ovulation, which coincides and may be related with the profound changes
in the morphology and secretory activity of the oviductal epithelium. A
role for oviductins in fertilization is a logical assumption, since these
proteins associate with the zona pellucida and/or the perivitelline space of
ovulated oocytes. This is substantiated by the increase in sperm binding and
penetration of the zona pellucida of ovarian oocytes exposed to the protein
[35, 36 and reviewed in 12]. Interestingly, the association of ovine oviductin with F-actin in areas of blastomere-blastomere contact in earlyembryos suggests a role in early embryonic development [37]. Intemalization of the glycoprotein by the blastomeres occurs in many species
(reviewed in [4]), but the importance of this intriguing phenomenon
remains obscure. While a facilitating role for oviductins in normal in vivo
fertilization and embryonic development is possible, the success rate of in
vitro fertilization in humans, a technique that completely bypasses any contribution by the oviduct, challenges the paradigm. The proposed role for
oviductins in protecting the oviduct against invasion by pathogens [13, 18]
is consistent with the antiadhesion properties of certain carbohydrate-
219
binding proteins as weH as with the protective role generally attributed to

mucins.
The lack of chitinase activity led authors to propose functions and mechanisms based on a sugar-binding activity. The identification ofthe ligands is
a key objective in attempting to unravel the physiological role of chitinaselike proteins. For oviductins, a general strategy ought to be targeted at the
zona pellucida, which is the natural ligand par excellence. Its biochemical
composition is now well known, and the structure of the sugar residues was
elucidated in some species. As for YKL-40 and the other similar proteins,
the problem stands at a higher level of complexity due to the diverse cell
types and conditions under which the proteins are secreted, as well as to the
ligand-containing structures that they recognize. In arecent publication, it
was e1egantly demonstrated that human YKL-40 is a chitin-specific lectin
[38]. These authors proposed that "He gp-39 may interact with so far
unknown endogenous compounds with chitin-like motifs". We suggested
that the zona pellucida, areminder of an ancestral protective chitinaseligand structure such as the shell and the bacterial cell wall, could bind oviductin via its oligosaccharide moieties [4]. Given the likely role ofYKL-40
in tissue remodeling, we suggest that the oocyte and most interestingly the
preimplantation embryo could be used as models to expand our concept of
the physiological role of chitinase-like proteins in morphogenesis.
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14 Suzuki K, Sendai Y, Onuma T, Hoshi H, Hiroi M, Araki Y (1995) Molecular characterization of a hamster oviduct-specific glycoprotein. Biol Reprod 53: 345-354
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ed. by ~ Jolles and R.A.A. Muzzarelli
Chitinases of human parasites and their

implications as antiparasitic targets
Mohammed Shahabuddin 1 and Joseph M. Vinetz 2
1
Medical Entomology Section, Laboratory ofParasitic Diseases,

National Institute ofAllergy and Infectious Diseases, National Institutes of Health,
4 Center Drive MSC 0425, Bethesda, MD 20892-0425, USA
WHO Collaborating Center for Tropical Diseases, Department ofPathology and Division
of Infectious Diseases, University of Texas Medical Branch,
Keil/er 2.138, 301 University Blvd., Galveston, TX 77555-0609, USA
Summary. Pathogens causing a number ofhuman and animal diseases use chitin and chitinases
in their life cycles. Most of these diseases are caused by protozoan or metazoan pathogenic
parasites. Some ofthese parasites contain chitin coats that protect them from the harsh conditions in the animal body or the environment. Some pathogens use chitinase to invade or exploit
the chitin-containing structures of their host to establish successful infection or to be transmitted from one vertebrate to another via insect vectors. Recent studies indicate that each of these
organisms has evolved to use chitin and chitinases differently and in a developmental stage-specific manner. Genes ofmany ofthese pathogenic parasites have been isolated, and the predicted amino acid sequences show a great deal of diversity. In this chapter we will discuss the roles
chitin and chitinases play in several animal diseases, the strategies used to clone the chitinase
genes from various parasites and the usefulness of chitinases as preventive or therapeutic
agents.
Introduction
Chitin and chitinases play important roles in the life cyde of several
protozoan and metazoan parasites that infect humans. In some parasites,
chitin is a structural component of these organisms, while in others the
parasites interact with the chitin-containing struetures that they encounter
during development. In the latter, the parasite may degrade the chitinous
structures of the host to facilitate their further growth and transmission.
The eggshell and the sheath that covers the outer surface of the microfilarial worms contain chitin [1]. Also, chitin is a component of the eyst
wall of the intestinal pathogens Entamoeba [2] and Giardia [3]. These
parasites also make chitinases, perhaps to modulate their chitinous structures. The parasites of leishmaniasis, malaria, and Chagas' disease do not
contain chitin; however, they secrete chitinase enzymes to degrade the
internal chitin-containing structures of their insect vectors [4].
Until now, a considerable number of medically important parasites have
been shown to possess chitinase-like activities (Tab. I). In most ifnot all
cases, the enzyme is produced at a particular stage of the parasite, which
may suggest that chitin or chitinases are important for the function of the
parasite at that stage. This implies that the blocking ofthe chitin production
or the chitinase activity in these parasites may block their development to
224
M. Shahabuddin and 1. M. Vinetz
Table I. List of parasite chitinases detected as enzymatic activity, antigens or gene cloning
Parasite
Brugia malayi
Brugia pahangi
Oncocerca gibsoni
Wuchereria bancrofti
Entamoeba invadens
Entamoeba dispar
Leishmania donovani
Leishmania major
Leishmania braziliensis
Leishmania infantum
Trypanosoma lewisi
T. brucei brucei
Leptomonas seymouri
Crithidia Jasciculata
Plasmodium gallinaceum
References for parasite chitinases as

Enzyme activity
Genecloned
Antigen
[43-45]
[43-45,28]
[43,28]
[31]
[7]
[31]
[13]
[13]
[13]
[34]
[30,6]
[10]
[31]
[13]
[32,33]
[15]
[15,46]
[15]
[15]
[15]
[47]
[15]
[15]
[35,20]
[7]
[31]
1. M. Vinetz et al.,
unpublished results
maturity and their transmission to the next host [4]. In this chapter, we will
ftrst summarize the putative biological roles of chitin and chitinase in some
human pathogens. We will also discuss how the genes for the different parasite
chitinases have been cloned, the current concept of transmission-blocking
vaccines and how a chitinase can be deployed as a vaccine for humans.
Filarial worms
Microfilarial nematodes cause a number of human diseases and are major

health problems in several tropical countries. The worms of Wuchereria
bancrofti and Brugia malayi grow in the lymph glands and cause lymphedema or elephantiasis in humans. Current estimates of the World Health
Organization (1994) suggest that 100 million people are infected with lymphatic filariae of all types. W. bancrofti is distributed throughout the
tropical regions of Asia, Africa, China, the Paeific and isolated locations in
the Americas. The endemie range of B. Malayi is confined to South and
Southeast Asia, from India in the west to Korea in the east. Microfilaria of
Onchocerca volvulus are the primary cause of river blindness, found in
both the Old and New World but with about 95% of all cases in Africa.
Other important foei of 0. volvulus are Mexico, Guatemala, Venezuela and
most ofWestem Africa.
The microfilarial parasites require an insect and avertebrate host to
complete their life cycle. Wuchereria and Brugia parasites use mosquitoes,
Chitinases ofhuman parasites and their implications as antiparasitic targets
225
and Onchocerca parasites use blackfly vectors for transmission from one
infected vertebrate to another. The adult worms in an infected human shed
large numbers of microfilariae, which circulate in the peripheral blood or
wander through subcutaneous tissues. The insects ingest the microfilariae
during the blood mea1. The worm penetrates through the gut wall of the
insect to reach the hemolymph. The parasite then enters the thoracic muscle
and passes through two to three stages of development or molting. Brugia
and Wuchereria microfilariae have a sheathlike structure that surrounds the
parasite. During each molting process, the worm emerges from this sheath
by a process called exsheathment [5].
The sheath of the microfilarial nematodes contains chitin [1]. It has been
proposed that the microfilariae produce a chitinase-like enzyme to facilitate
the exsheathment and further development of the worm [5]. Antibodies
against filarial chitinase apparently block the infection in some endemic
areas [6-8]. The causative agent of cattle filariasis does not have a sheath;
however, the eggshells of Onchocerca gibsoni and 0. volvulus contain
chitin [9]. A chitinase-like activity present in 0. gibsoni is perhaps essential for hatching ofthe eggs [1, 11].
Entamoeba
Entamoeba histolytica is an intestinal protozoan parasite that causes
amebic dysentery in humans. The parasite is found all over the world, but
the infection is more common in the tropics. The parasite colonizes in the
large intestine and, in severe cases, invades the tissue of the colon, causing
hemorrhage in the colon and bloody stoo1. In an adverse environment, E.
histolytica forms cysts. The cysts are shed with feces; in areas with poor
sanitation, they mix with the drinking water.
Cyst formation begins when the trophozoite rounds into a precyst in the
intestine. The parasite secretes an impermeable outer cyst wall containing
chitin [2, 12]. Cysts are sturdy and resist adverse environmental conditions.
After ingestion, in a favorable environment of the small intestine, the cyst
wall is disrupted and the amoeba divides to form trophozoites. A chitinase
gene has been detected in E. histolytica that expresses during the excystment process [13]. It has been proposed that chitinase is essential for the
emergence of the parasite from the cysts and establishment of a new infection.
Leishmania
Leishmaniasis is a major threat in many tropical countries. Two types of

infection caused by the parasites of genus Leishmania are commonly
known: visceralleishmaniasis, caused by L. donovani, and cutaneous leish-
226
M. Shahabuddin and J. M. Vinetz
maniasis, caused by L. tropica. In visceralleishmaniasis, parasites invade

internaiorgans such as spleen, liver and so on, causing enlargement of the
organs. In cutaneous leishmaniasis, the parasite remains at the site of
inoculation and causes severe, ugly-Iooking lesions. The parasites are
transmitted from one host to another by the bites of sandflies.
When an infected sandfly bites a Leishmania-infected human, amastigotes enter the stomach of the insect. Immediately after blood feeding, a
transient saclike structure, the peritrophic matrix (PM), is farmed in the
stomach. The PM gradually surrounds the blood meal [14]. The parasites,
with the blood, are also encased in the endoperitrophic compartment.
Inside the PM, the parasites transform into promastigotes and rapidly
multiply. The anterior ofthe PM begins to break 3 to 4 days after ingestion.
The parasites escape from the blood bolus and attach to the midgut epithelium. As the parasite further matures, it migrates toward the fore gut and
damages the cardiac valve [15]. The cardiac valve regulates the unidirectional flow of the blood to the midgut during feeding and prevents regurgitation of blood from the stomach. When an infected fly feeds on an
uninfected human, due to the damaged cardiac valve, the fly regurgitates
the contaminated blood from the midgut, and the parasite enters the human
blood at the biting site.
The promastigotes (sandfly stage) of Leishmania secrete chitinase when
cultured in vitra. It is proposed that the Leishmania chitinase plays a dual
role in the transmission ofthe parasite. First, it disrupts the PM, helping the
parasite to escape from the blood bolus [15]. Second, it destroys the
chitinous 1ining of the cardiac valve of the sandfly, helping the infected
blood to be regurgitated at the site of biting [16]. In an uninfected sandfly,
the cellular integrity of the valve is maintained by the chitinous lining. If
the sandfly is heavily infected, the lining is damaged and cellular assembly
of the valve is disrupted. The PM of the sandfly appears to be essential far
the initial survival of the parasite in the fly stomach. When PM formation
was disrupted in the stomach, the infection was abrogated [17]. It has been
proposed that the PM protects the parasite from being damaged by the proteases secreted to digest the ingested blood. The developmentally regulated
expression of the chitinase gene in Leishmania promastigotes is therefore
important, because chitinase production at an earlier stage would damage
PM formation in the stomach of the fly and disrupt the establishment of
infection.
Plasmodium
Malaria, the disease caused by the parasites of genus Plasmodium, is the

most devastating insect-borne parasitic disease of humans. It kills more
than 2 million people worldwide every year. The parasite is transmitted
from one human to another by the bite of mosquitoes. In the human, the
Chitinases of human parasites and their implications as antiparasitic targets
227
injected parasites first invade the liver cells and then infect red blood cells
in a cyeIic manner. Plasmodium infection of erythrocytes results in the
eIinical syndrome of malaria. Malaria is associated with lysis of erythrocytes leading to anemia and the adherence of infected erythrocytes to postcapillary endothelial cells (Plasmodium falciparum only) that can cause
neurological, cardiovascular, pulmonary and metabolic complications.
Some parasites develop as gametocytes after invading the red blood
cells. When a mosquito ingests gametocytes, sexual fertilization occurs.
The zygotes then trans form into motile ookinetes. In mosquitoes, as in
sandflies, chitinous PM surrounds the ingested blood. The ookinetes invade
the PM [18, 19]. The ookinete stage ofthe parasite secretes a chitinase that
is activated by the midgut proteases [20, 21]. The activated chitinase allows
the parasite to cross the PM and facilitates further development ofthe parasite. If the chitinase activity in the mosqito midgut is inhibited, transmission of malaria is completely blocked [20].
Trypanosoma
Chagas' disease, essentially limited to Central and South America, is

caused by Trypanosoma cruzi. Trypanosoma are flagellated protozoan
parasites transmitted by the bite of infected kissing bugs such as Triatoma
infestans. The bug feeds on humans as well as on other animals. When the
bug feeds on an infected animal, the parasite enters into the midgut and
gradually become surrounded by the PM. The parasite escapes from the
blood bolus and reaches the hindgut. Once in the hindgut, the parasite
develops to the metacyeIic stage, which is infectious to vertebrate hosts.
When a bug feeds, it simultaneously defecates. The feces of an infected bug
contain large numbers of parasites. After feeding, the parasite enters the
body through skin damaged by the etching and scratching of the biting site.
The role of chitinase appears to be more complicated in the transmission
of Trypanosoma than in that of Plasmodium and Leishmania. The enzyme
has been detected in T lewisi and T brucei, the parasite of cattle, suggesting that chitinase is involved in both parasite maturation and transmission. T brucei is transmitted by black flies. In addition to the procyc1ic
stage of T brucei, a Rickettsia-like organism (RLO) of the tsetse also
secretes chitinase in the midgut [22]. The ability ofthe black fly to establish
T brucei infection appeared to be positively related to its RLO load. It has
been proposed that the lectins secreted in the midgut of the fly in response
to the blood meal are toxic to the newly ingested parasite. The chitinase
secreted by the RLO partially degrades the PM and releases D-glucosamine
into the midgut. The released sugars neutralize the activity of the lectins
and help the parasite to survive [23]. The role of Trypanosoma chitinase in
development and transmission remains to be understood.
228
Cloning of parasite chitinase genes

Chitinase genes have now been identified and cloned from a number of
human parasites such as filarial nematodes (B. malayi, W bancrofli and
Onchocerca volvulus), E. histolytica, L. donovani and, most recently, Plasmodium gallinaceum, an avian parasite phylogenetically related to the
human malaria parasite P. jalciparum (Tab. 1). A variety of approaches
have been used to clone these genes. A discussion of some of these approaches follows.
Filarial chitinases
The best-characterized filarial chitinase is that of B. malayi. This chitinase

was initially defined as the target antigen of a monoclonal antibody named
MFI that recognized two proteins migrating on SDS polyacrylamide gel
electrophoresis (PAGE) at 70 kDa and 75 kDa [24]. Administration ofMFl
reduced microfilaremia in B. malayi-infected jirds. The hatching of the
filarial eggs and the exsheathment of the microfilaria are calcium-dependent processes; MF 1 antigen was later shown to bind calcium, suggesting
that the antigen plays an important role in these mechanisms [25]. In addition, the expression of the gene correlates with the development of the
parasite to the mosquito stage [26]. The appearance of the MFI antigen
correlated with the onset ofthe parasite's ability to infect the mosquito.
Interest in the MF 1 antigen as a vaccine candidate for human brugian
lymphatic filariasis increased when it was found that sera from immunologically protected individuals contained antibodies that reacted with the
same B. malayi 70-kDa and 75-kDa antigens [8,27]. The 70-kDa and 75kDa MFl antigens were partially purified by DE-52 chromatography of
jird-derived microfilarial extracts and analyzed by microsequencing ofthe
amino-termini and internal tryptic peptide fragments. These amino acid
sequences were used to design degenerate oligodeoxynucleotides for
polymerase chain reaction (PCR) amplification, and the resulting PCR
fragments were used to screen complementary DNA (cDNA) library. A
single full-Iength cDNA contained sequences with high similarity to
several chitinases. This finding was confirmed when both parasiteproduced native MF 1 antigens had chitinase activity when tested on a
glycol chitin activity gel [6]. Furthermore, both the 70-kDa and the 75-kDa
proteins had virtually identical profiles on high-pressure liquid chromatography (HPLC) chromatogram of tryptic digests of the isolated proteins,
suggesting three possible relationships between the two proteins: (i) they
were isoform products of two different but closely related genes; (ii) they
were products of differential splicing of coding messenger RNA (mRNA)
from a single gene; or (iii) they arose from differential posttranslational
processing, either proteolysis or glycosylation. Although Southern blot
229
analysis was unable to resolve this discrepancy, it was later found that the
mRNAs encoding transcripts for the 70-kDa and 75-kDa proteins differed
only in an extra copy of a serine/ threonine-rich repeat [28]. The functional
significance of this repeat remains obscure but is likely not relevant to
vaccine research.
A chitinase from the other major causative agent oflymphatic filariasis,
W. bancrofti, was identified as a potentially protective immunogen using
sera from patients from W. bancrofti-endemic areas [7, 8]. It was observed
that 100 % of putatively immune (PI) (i. e. amicrofilaremic but with strong
cellular and humoral responses to parasite antigens) but only 8% of infected microfilaremic subjects in endemie regions of the Cook Islands
recognized a 43-kDa antigen from infective third-stage (L3) W. bancrofti
larvae [29]. The PI sera preferentially recognized a 43-kDa antigen. Purified 43-kDa protein was used to immunize rabbits; the antisera were used
to probe a W. bancrofti genomic expression library. The resulting partial
genomic DNA clone was found to encode a chitinase with 23% identity
(47% similarity) to the B. malayi chitinase. The rabbit antibodies to the 43kDa protein were found to react with intrauterine microfilariae as well as
with tissues ofL3 larvae [7]. The relation ofthe B. malayi L3 larval 43-kDa
antigen to the MF 1 microfilarial antigen has not yet been clearly defined.
A chitinase from Acanthocheilinema viteae, a filarial parasite of rodents,
was identified in a similar way. Irradiated L3 larvae were used to immunize
jirds; the resultant immune sera reacted with an antigen subsequently
shown to be a chitinase [30]. Finally, oligonucleotide primers based on the
A. viteae chitinase were used to amplify a chitinase from 0. volvulus, the
filarial agent of onchocerciasis and river blindness. The function and transmission blocking-inducingltherapeutic potential ofthe 0. volvulus chitinase
has not yet been explored [31].
Entamoeba chitinase
A chitinase was demonstrated to be present in the reptilian parasite

Entamoeba invadens, with its presumed function related to encystation of
the parasite within a cell wall, allowing resistance to environmental conditions [32]. Subsequently, the chitinase from the human enteric pathogen
E. histolytica was cloned using degenerate oligonucleotides derived from
consensus sequences of bacterial, fungal and insect chitinases [13]. This
chitinase has putative substrate binding and catalytic and chitin binding
domains similar to those previously described for bacterial and fungal
chitinases. Additionally, the N -terminus has acidic and hydrophilic
domains composed ofmultiple tandemly arrayed seven-amino acid repeats
similar to the repeats of other Entamoeba serine-rich proteins that displace
the normally N-terminally located catalytic site toward the center of the
open reading frame. Recombinant E. histolytica chitinase was demonstrated to bind chitin and to show endochitinase activity with chito-oligo-
230
saccharide substrates. Of interest, the chitinase of all three Entamoebae

were found to be highly similar, pointing to the close evolutionary relationship of the members of this genus, especially E. histolytiea and
E. dispar.
Allosamidin, a potent inhibitor of chitinase, delays E. invadens encystation in vitra, but the effects of chitinase inhibition have not been studied in
vivo for any of the Entamoebae in their natural hosts. It is also unknown
whether chitinase inhibition has an effect on the morphology, life cycle or
killing of Entamoeba cysts. E. invadens has several chitinase isoforms
[33], but a similar finding has not been made for the pathogenic E. histolytiea or the commensal E. dispar. Finally, while an encystation chitinase
presumably functions as a cell wall-modifying enzyme during chitin
synthesis, no excysting chitinase has yet been demonstrated that would
allow the parasite egress from the cyst.
Leishmania ehitinase
Similar to the E. histolytiea chitinase, an L. donovani chitinase gene. Ld

CHTl, was cloned by use of degenerate oligonucleotide PCR [34]. Compared with other bacterial, fungal and parasite chitinases, the specific
activity of the native L. donovani chitinase seems to be substantially lower.
While typical catalytic and chitin binding domains are encoded by Ld
CHTl, the consensus sequence identified by Shakarian and colleagues as
the substrate binding site differs from other reported chitinases in that a
cysteine substitutes for highly conserved serine or alanine at amino acid
position 131. This substitution could be one reason for the enzyme 's slower
kinetics. The native L. donovani chitinase has both exo- and endochitinase
activity as assessed by the ability of promastigote culture supematants to
hydrolyze both 4-methylumbelliferone-chitobiose and chitotriose.
Plasmodium ehitinase
The chitinase of the avian malaria parasite P. gallinaeeum has been biochemically identified, but cloning of the Plasmodium chitinase gene has
been challenging. A number of groups have unsuccessfully attempted to
use degenerate oligonucleotide PCR to amplify the chitinase gene from
both P. gallinaeeum and the human malaria parasite P. jalciparum. Because
the development of P. jalciparum ookinetes in vitra is difficult, P. gallinaeeum has been mostly used for chitinase studies. P. gallinaeeum is
closely related to P. jalciparum, its ookinetes can be obtained in sufficient
quantities from in vitra cultures of gametocyte-containing chicken blood
and ookinete chitinase activity can be readily detected [35]. Based upon
these characteristics P. gallinaeeum chitinolytic activity has been purified.
231
Two distinguishable peaks of endochitinase activity (as detected by hydrolysis of 4-methylumbelliferone -acetyl chitotrioside) can be separated
by chromatography. Chitinase activity in one peak is associated with an
- 55-kDa doublet, in the other with an - 21O-kDa protein. Protein microsequencing of the - 55-kDa doublet led to the cloning and sequencing of
a single exon open reading frame that contained sequences typical of
family-18 glycohydrolases. The relationship of the two chitinases whether they are products of the same gene with posttranslational
proteolytic modification followed by multimerization, or whether they are
products of two different genes - is not clear at this time (I M. Vinetz et al.,
unpublished data). The predicted amino acid sequence contains a signal
sequence, a putative substrate binding site, a catalytic active site and a
cysteine-rich chitin binding domain.
Chitinase as a target for antiparasitic strategies

The expression of stage-specific chitinases by the medically important
parasites suggests that chitinase could be a potential generalized target
for blocking the development and transmission of these parasites. The
strategies against the chitinase would, however, be dependent on the
parasites. For some parasites, a chitinase inhibitor can act as a drug; for
others, a vaccine can be prepared against the vertebrate stage of the
parasite, while in other cases, a transmission-blocking vaccine against the
vector stage would be more suitable.
For the modulation of the cyst wall in Entamoeba, a drug with chitinase
inhibitory activity may be suitable. The potent chitinase inhibitor allosamidin delays the cyst formation of Entamoeba invadens [32]. Whether
this inhibitor can also block the emergence of the parasite from the cyst
remains to be seen; however, a suitable analog of allosamidin or other
chitinase inhibitor can be a potential drug against Entamoeba infection.
The hatching of filarial eggs and exsheathment of maturing nematodes in
the human also suggests that a chitinase inhibitor would be a potential drug
against microfilaria infection. The effects of chitinase inhibitor analogs
remain to be investigated.
Detection of chitinase antibodies in the sera of immunologically protected individuals is a direct evidence that interfering with the chitinase
activity would block the establishment of filarial infection. This observation suggested that chitinase could be a suitable candidate for an antifilarial
vaccine. This hypothesis has been tested injirds. Passive transfer ofmonoclonal antibodies to the MF I antigen of B. malayi conferred a moderate
degree of protection to infected jirds [24]. Jirds immunized with recombinant B. malayi chitinase also showed partial protection against microfilarial challenge. In addition, when jirds were immunized with recombinant chitinase, they showed protection against filarial challenge.
232
In the cases of malaria, leishmaniasis and trypanosomiasis, the vaccine

would have to be against the insect vector stages, as these organisms apparently express chitinase only at the insect stage. Therefore, only transmission blocking would be possible.
The concept of a transmission-blocking vaccine is relatively new. In this
procedure, vector stage-specific antigens are targeted as vaccine candidates. The vertebrate host is immunized with purified native or recombinant proteins. The immune response in the host theoretically does not
have an effect on the vertebrate and does not cure if the individual is infected. Instead, the immune sera - when ingested by an insect with the
parasite - attacks the parasite when the antigen is expressed in the parasite.
The attack blocks further development and transmission of the parasite. In
malaria, this strategy has already shown potential. When an in vitro-transformed mosquito stage of Plasmodium was used to immunize monkey and
chicken [36, 37], the immune sera blocked development ofthe parasite in
the mosquitoes. Several parasite surface antigens have been subsequently
identified that showed potential as transmission-blocking targets [38].
Because chitinase is essential for parasite development in the mosquito,
and because this requirement transpires within 24 h after blood ingestion,
an activity that blocks antibody against Plasmodium chitinase would block
parasite development. Recent cloning of the P. gallinaceum chitinase has
opened the possibility of testing this hypothesis.
Compared with those ofbacteria, yeast and plants, parasite chitinases are
poorly understood. One potential problem of using parasite chitinases as
drug or vaccine targets is their amino acid sequence and functional
similarity to recently discovered human chitinases [39-41]. The function
of the enzyme in humans is not clear but may be restricted to clearing
fungal chitin from the body. Interfering with this enzyme in humans may
cause unpredicted side effects. In addition, although the overall amino acid
sequence similarity is low, the catalytic active sites of the parasite
chitinases are similar enough to the human chitinases/chitinase-like
proteins that cross-reactive antibodies could be induced by vaccination
[42]. Structural studies to identify conformationally and immunologically
unique regions of the chitinases will be critical for success in using
chitinases as human vaccines. To fully exploit the potential of parasite
chitinases, it is important that the properties of the enzymes and the
structures of the genes by understood in detail. Studies of the recently
cloned parasite chitinases have opened a new vista of antiparasitic drug and
vaccine research.
233
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27 Kurniawan L, Basundari E, Fuhrman JA, Turner H, Purtoma H, Piessens WF (1990) Differential reeognition of mierofilarial antigens by sera from immigrants into an area endemie
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36 Gwadz R (1976) Suceessful immunization against the sexual stages of Plasmodium gallinaceum. Science 193: 1150-1151
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ed. by P. Jolles and R.A.. Muzzarelli
Analytical biochemistry and clinical significance

of N-acetyl--D-glucosaminidase and related
enzymes
University o[Ancona, Faculty o[Medicine, Center tor Innovative Biomaterials,
Via Ranieri 67, 1-60100Ancona, Italy
Summary. Human N-acetyl--D-glucosaminidase, N-acetyl-a-D-glucosaminidase, endo--Nacetylglucosaminidase, hexosaminidase, -N -acetylgalactosaminidase and glucocerebrosidase
have not been so widely studied as the -N -aeetylhexosaminidases in baeteria, fungi and arthro
pods. Their bioehemieal role has been elueidated, however, and their urinary and plasma determination is being adopted for the early detection of diseases before c1inical manifestation, in
particular for hypertension, renal injuries and disorders, depression and lysosomal storage
diseases. The speetrophotometric determinations ofN-aeetyl--D-glucosaminidase, most often
done with 3-eresolsulphone phthaleinyl N-aeetyl--D-glucosaminide, have been reeently
simplified and adapted to automatie instruments.
-N-Acetylhexosaminidase, EC 3.2.1.52, involved in glycolipid and glycoprotein degradation, is widely distributed in animals, higher plants and
microorganisms [1-4]. The enzymes from bacteria, fungi and crustaceans
have been studied in considerable detail. For instance, the N-acetyl--Dglucosaminidase from Escherichia cali is a monomeric protein with four
domains and three disulphide bonds. There are no cofactors, metals or other
ligands in the enzyme. The complex ofthe enzyme with the substrate chitobiose has been crystallized and characterized [5]. The enzyme encoding gene
from Serratia marcescens has been c1oned, sequenced and expressed in E.
cali. Sequencing has revealed an open reading frame encoding a protein of
885 amino acids. Comparison with the members of family 20 of glycosyl
hydrolases revealed that N-acetyl--D-glucosaminidase has a conserved
central region which aligns with almost all members ofthis family and which
comprises the catalytic domain [6]. The reactions catalysed by these enzymes
are shown in Figure 1. The tolerance ofN-acetyl--D-glucosaminidase for
the aglycon moiety R is generally high with carbohydrate chains of glycoproteins and with phenolics, alkaloids and steroids. On the other hand, in
transglycosylation the tolerance for the acceptor structure may be strict.
The present chapter deals with the human N-acetyl--D-glucosaminidase
and related hydrolases, whose importance has been recently recognized.
A few words about nomenc1ature would be essential for understanding
the bibliography and the content ofthis chapter. The term -N-acetylhexosaminidase, E.C. 3.2.1.52, covers today both enzymes known as chitobiase
236
R. A. A. Muzzarelli
(a) Hydrolysis, Rl
HO
~
OH
=H
C>
HO
HO-R l
AcNH
C>
~t
OH
HO-R
AcNH
(b) Transglycosylation, Rl~ H
Figure 1. Reactions catalyzed by -N-acetylhexosaminidase (EC 3.2.1.52). (a) Rydrolytic

activity: R, the aglycone partial structure of the -N-acetylhexosaminide represents a noncarbohydrate residue, a single sugar or an oligosaccharide moiety, or the carbohydrate side
chain of a glycoprotein. (b) Transglycosylating activity: under certain conditions, the enzyme
ftmction as a transferase, with ROR' as the acceptor.
EC 3.2.1.29 and N-acetyl--D-glucosaminidase EC 3.2.1.30 (both outdated names) [7]. These exoglycosidases catalyse the hydrolysis of
terminal nonreducing N-acetylglucosamine residues in N-acetyl--glucosaminides. In the current literature, however, the "old" names are still being
used. -N-Acetylgalactosaminidases, EC 3.2.l.53, are distinctly recognized. The term hexosaminidase is currently used to indicate enzymes that
act on substrate G M2 gangliosides N-acetylgalactosaminyl--l ,4-(N-acetylneuraminyl-a-2,3)-galactosyl--l ,4-glycosyl--I, l-ceramide. Endo--Nacetylglucosaminidase, EC 3.2.1.96, is a distinct enzyme that catalyses the
hydro lysis of the N,N' -diacetylchitobiosyl unit in high mannose glycopeptides containing the [Man(GlcNAc)2]Asn structure.
Enzyme characteristics
Kidney N-acetyl--D-glucosaminidase has been purified from human
[8-13], equine [14], canine [15], murine [16], vaccine [17] and simian [18]
sources. The characteristics of the baboon enzyme [10] are the following
(values in parentheses are for human N-acetyl--D-glucosaminidase):
it exists in three forms, A, Band I,
the A form can be further refined into two forms, A-l and A-2
the carbohydrate content accounts for microheterogeneity, since it varies
from 31 % for A-l [8.5%, human] to 17% for A-2 and the sialic acid is
6%and 1%.
the molecular weight for A-1 is 52.1 kDa [ca. 100 kDa, human], the pH
optimum is 4.55 [4.4] and pI 4.97
the optimum temperature for A and B is 50 and 42C
the Km for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine for
both isoforms is 0.497 and 0.627 mM, respectively [0.5 mM, human]
Ag + and Pb 2+ are the most potent uncompetitive inhibitors, having K;
values of 3.6 and 8.5 mM, respectively. Acetate is a competitive inhibitor [also for the human enzyme].
Analytical biochemistry and c1inical significance ofN-acetyl--D-glucosaminidase ...
237
A total of 17 compounds are known today as true inhibitors. The best inhibitors are structurally and mechanistically glycon-based analogues ofthe
substrate and inc1ude N-acetylglucosaminono-1,5-lactam (K i = 0.67 pM
forbovine kidney enzyme), N-acetylglucosaminono-1,5-lactone oxime (K i
0.45 pM for bovine kidney enzyme); N-acetylglucosaminono-1,5-lactone
(K i = 0.036 pM) and nagstatin (K i = 0.017 pM). Like nagstatin, a number
of inhibitors are N-heterocycles: these bind to the active site in their protonated forms to counteract the negative charges [19] of the catalytic
carboxylate. Lactones and lactams resemble the flattened ring of the
transition state [19]. None ofthese inhibitors shows sufficient antifungal
activity to justify a practical use [7].
The crystals of chitobiase from Serratia marcescens have the shape of
prisms, and the space group is P21 with = 101.00 and unit cell dimensions
a = 63.2 A, b = 133.2 A, c = 55.1 A [20]. Several N-acetylhexosaminidases
have been c10ned and sequenced; for instance from Alteromonas, Vibrio
harveyi and S. marcescens.
The endo N-acetylglucosaminidase from Streptomyces plicatus has
dimensions 31 x 34 x 57 A and is a (aI)8 barrel protein. The putative catalytic residues Asp 130 and Glu 132 are surrounded by several aromatic
residues. The loop area of the c1eft is formed by neutral polar residues,
mostly asparagines. This endo H enzyme is commonly used in the analysis
of glycoproteins and asparagine-linked oligosaccharides. It c1eaves the
bond between the two N-acetylglucosamine units of the chitobiose core
and is specific for high-mannose asparagine-linked oligosaccharides. The
structure of endo H is very similar to that of N-acetyl--D-glucosaminidase secreted by Flavobacterium meningosepticum, the only major difference residing in the structure of one loop that imparts the ability to process glycoproteins having an a(1-6)-linked fucose substituent on the first
N-acetylglucosamine unit [21]. The endo N-acetylglucosaminidase from
Stigmatella aurantiaca has the following substrate specificity towards
glycoasparagines: oligomannoside, high; hybrid, medium; complex, moderate [22].
Mechanism of hydrolysis
The reaction catalysed by glycoside hydrolases is a nuc1eophilic substitution at a saturated carbon. In a first step, a carboxylate group leads to a
glycosyl enzyme and to the removal of the aglycon group. Then, the
nuc1eophilic attack takes place, leading to hydro lysis or transglycosylation.
In the second step, a glutamic acid unit protonates the oxygen to make it a
good leaving group and deprotonates the acceptor [23]. The 2-acetamido group vicinal to the glycosidic bond stabilizes the reaction intermediate through formation of an oxazoline (see Robertus and Monzingo,
this volume).
238
R. A. A. Muzzarelli
Analytical assays
The differences in substrate affinity are minor among the N-acetylhexosaminidases of different origins. The microbial N-acetylglucosaminidase Km
values are between 0.1 and 0.8 mM; in animals they range between 0.2 and
5.7 mM and in particular for humans between 0.4 and 4.5; in plants 0.1-0.9
(all values for substrate 4-nitrophenyl N-acetyl--D-glucosaminide). Besides 4-nitrophenol, other chromophores of test substrates for the assay for
N-acetyl--D-glucosaminidase are 2-chloro-4-nitrophenol, 4-amino-2,6dichlorophenol, 3,3' -dichlorophenylsulfon phthalein (Chromogenic) and 4methylumbelliferone (fluorogenic) and a number of related compounds.
The detection limit as low as 0.005 mU/ml makes 4-methylumbelliferyl-N-acetylglucosaminide the preferred substrate over the other whose sens itivity is 0.1-0.5 mU/mI. A microtiter plate version is the method of choice.
A number of other chromogenic substances have been developed, among
which 4-amino-2,6-dichlorophenyl N-acetyl--D-glucosaminide satisfies
the criteria for a good substrate, that is the product should absorb at visible
wavelengths where absorbance from bilirubin and hemoglobin is negligible, and the absorptivity of product should be high. In fact it produces a
green color (max302 ~ 718 nm, with E=42,000) atpH 5.0. It is also sufficiently stable and soluble [24]. N ew chemiluminogenic substrates, 4'-(6'diethylaminobenzofuranyl) phthalyl hydrazide-N-acetyl--D-glucosaminide and o-aminophthalylhydrazido-N-acetyl--D-glucosaminide, have
been examined as substrates [25,26].
The literature contains much information on the analytical assays being
used to characterize and quantify N-acetyl--D-glucosaminidase. The
most popular substrates are 4-methylumbelliferyl-N-acetyl--D-glucosaminide, 4-nitrophenyl-N-acetyl--D-glucosaminide and 3-cresulsulphone
phthaleinyl-N-acetyl--D-glucosaminide.
Two protocols for the determination of N-acetyl--D-glucosaminidase
with the two most popular substrates are the following. Details are to be
found in [27].
Fluorimetric procedure
For instance, milk sampies (10 ].11) with added 4-methylumbelliferyl-Nacetyl--D-glucosaminide are incubated for 15 min at 25 oe under shaking.
Stopping buffer (pH 10.4) is then added. The amount of N-acetyl--Dglucosaminidase is proportional to 4-methylumbelliferone released from
the substrate per unit time, which is determined by fluorimetry at 355 nm
(ex) and 480 nm (ern) [17].
Analytical biochemistry and clinical significance ofN-acetyl--D-glucosaminidase ...
239
Spectrophotometric procedure
Enzyme sampie (5 p.l) in citrate buffer (25 p.l, 100 mM, pH 4.5) at 37C
is incubated with 4-nitrophenyl-N-acetyl--D-glucosaminide in citrate buffer (25 p.l, 100 mM) for 20 min. The reaction is stopped at pH 10, and
readings are made at 405 nm. One unit ofN-acetyl--D-glucosaminidase is
defined as the amount of enzyme liberating I mmol of 4-nitrophenol
(A 450 nm) in 1 min at 25C [18]. In the case ofurine sampies, the urinary
creatinine is also measured, and the N-acetyl--D-glucosaminidase levels are
indexed to creatinine to minimize the effects of diuresis [14]. The 3cresolsu1phone phthaleinyl N-acetyl--D-glucosaminide, currentlyavailable
as a test kit, also lends itselfto spectrophotometric determinations at 570 nm.
The methods indicated above generally require gel filtration of urine to
eliminate interfering and inhibitory substances, expecially urea. This
methodological disadvantage has in the past been the essential reason for a
limited appreciation of N-acetyl--D-glucosaminidase activity. New synthetic substrates that obviate these problems should be easily soluble in order
to saturate the enzyme, making unnecessary gel filtration of urine to eliminate urinary inhibitors, and release a stable chromophore with a pK a value
near the pH optimum of the enzyme. The substrate 3,3'-dichlorophenylsulfonphthaleinyl N-acetyl--D-glucosaminide releases the chromophore
chlorophenol red at pH 6.25, which is a compromise between the enzyme
optimum (5.20) and the maximum for chromogenesis. The test has been
adapted to automatic analysers, and therefore the N-acetyl--D-glucosaminidase can be done routinely in view of its diagnostic significance [28].
Increased activity ofN-acetyl--D-glucosaminidase
Increased activity of N-acetyl--D-glucosaminidase may be indicative of
acquired and genetic diseases and various forms of stress. Lysosomal
enzymes are synthesized as precursors ofhigher molecular weight and subsequently processed to mature forms. In fibroblasts and some other cell
types (endothelial cells, lymphocytes, hepatocytes, smooth-muscle cells)
no mature forms of lysosomal enzymes are secreted; macrophages, however, are known to release mature lysosomal enzymes upon stimulation.
Another source of mature forms are damaged cells.
Human lysosomal N-acetyl--D-glucosaminidase is known to exist in at
least five isoenzymic forms termed A (acidic), B (basic), 11 and 12 (intermediate), and P (pregnant plasma). Lysosomal enzymes are common in
human body fluids, such as serum, urine and tears. Increased serum acivity is found in thyroiditis [29], diabetes mellitus [30, 31], leukemia [32],
acromegaly [33] and cancer [34]. In diabetes, the increased enzymatic
activity results from metabolic derangements hut is independent of the
development of microvascular complications [35]. However, liver disease
(including alcohol intoxication) [36, 37], pregnancy [36, 38] and hypertension [39] appear to be the conditions mainly responsible for the increase in
240
R. A. A. Muzzarelli
serum N-acetyl--D-glucosaminidase achvlty: the serum intermediate

isoenzymes land P are the main cause of the increase in total activity. The
increased serum N-acetyl--D-glucosaminidase activities might be due to
a decreased clearance of this enzyme by nonparenchymal cells of the liver
and/or increased production of lysosomal enzymes, possibly by activated
liver macrophages. Serum N-acetyl--D-glucosaminidase was clinically
evaluated as a liver-function test [40]. In urine, the major form normally
excreted is tissuelike N-acetyl--D-glucosaminidase A, with traces of B
and I, and is normally accepted as indicative ofkidney disease or damage.
However, it is important to keep in mind that urinary N-acetyl--D-glucosaminidase may originate in macrophage-type cells [41]. Secreted enzymes
probably are involved in the physiological process of inflammation [42].
Bourbouze et al. [43] characterized N-acetyl--D-glucosaminidase isoenzymes released from monocyte-derived macrophages when cultures were
supplemented with an inflammatory agent such as zymosan, a yeast cellwall product chiefly made oftwo carbohydrate polymers, -D-glucan and
a-D-mannan.
Clinical significance
The determination of N-acetyl--D-glucosaminidase is most relevant to

pediatrics, nephrology and urology. This is a lysosomal enzyme widely distributed in human tissues, released to serum from cells by exocytosis or due
to breakdown of cells. Because of its high molecular weight it does not
filter through the glomerular membrane. In the presence of a glomerular
lesion or tubular damage, urinary N-acetyl--D-glucosaminidase activity
increases. The diagnostic value of the urinary N-acetyl--D-glucosaminidase activity measurements was reviewed [44, 45].
Hypertension
Elevated serum N-acetyl--D-glucosaminidase acttvlty is an effective

indicator of future hypertension and may be related to functional and structural changes in the cardiovascular system [46]. Since hypertension is
increasingly recognized as beginning in childhood, urinary N-acetyl--Dglucosaminidase changes with increasing blood pressure may start early in
life and mayaiso be evidence ofthe existence of early hypertensive disease.
The urinary N-acetyl--D-glucosaminidase changes in 980 young adults
aged 18-32 was analysed in relation to age, race, sex, and systolic and
diastolic blood pressure: a significant association between urinary Nacetyl--D-glucosaminidase and blood pressure exists in normal young
adults, and changes in urinary N-acetyl--D-glucosaminidase may be
evidence of early hypertensive disease [47].
241
Nephrology
Tubular renal damage induced by crystals can trigger further stone formation. Thus early detection ofthe initial damage is important. In the case of
increased N-acetyl--D-glucosaminidase excretion in stone patients, there
is a positive correlation between N-acetyl--D-glucosaminidase excretion
and phosphate, sulphate, uric acid, oxalate and creatinine excretion [48].
Elevated urinary N-acetyl--D-glucosaminidase has been reported in
glomerulonephritis, tubulointerstitial diseases, renal allograph rejection,
toxic renal injury and diabetes. In the diabetic patients, the N-acetyl--Dglucosaminidase/creatinine ratios are higher than in controls, this ratio
being indicative of one or more complications [49]. The increase in the Nacetyl--D-glucosaminidase index or the changes in the isoenzyme pattern
are also valuable early signs of an incipient rejection crisis after kidney
transplantation [50-52].
Raised urinary excretion ofN-acetyl--D-glucosaminidase is associated
with proteinuria; however, remission of the disease restores normal concentrations, which are 10.2 2.9 U/I and 4.4 2.1 U/I for N-acetyl--DglucosaminidasesA and B, respectively. Urinary N-acetyl--D-glucosaminidase and proteinuria are weIl correlated at all levels of renal function in
patients with glomerulonephritis, hypertensive nephrosclerosis and chronic pyelonephritis, but not in those with diabetic nephropathy [53].
Numerous studies have demonstrated the deleterious effects of vesicoureteral reflux on the structure of the kidneys, in particular impairment
of renal growth and development of renal scarring. Several renal tubular
enzymes such as a-glucosidase, y-glutamyltransferase and N-acetyl--Dglucosaminidase have been shown to be elevated in urine with obstructive
uropathy as a result of nephrotoxic substances. Once the obstruction has
been alleviated or the nephrotoxic stimulus removed, the enzyme levels
return to normal. Thus, the enzymuria is a means of following tubular
damage and recovery [54]. Renal abnormalities can be detected via the
determination ofthe enzyme in the amniotic fluid [55].
Many ofthe drugs widely used in intensive neonatal treatment are nephrotoxic, and transient hypoxie periods and pathological oscillations in oxygen
tension mayaiso cause tubular damage. The degree of damage is reflected
bya characteristic increase in either the urinary protein content and enzymes in the urine. Among the many enzymes studied, N-acetyl--D-glucosaminidase has proved most useful [56]; the same can be said for aminoglycoside nephrotoxicity, in which case this enzyme proved to be of interest in
assessing renal interference of this kind of antibiotic [57, 58]. In patients
with cystic fibrosis on gentamicin inhalation, a positive correlation betwen
N-acetyl--D-glucosaminidase concentration in urine and cumulative dose
of nebulized gentamicin was found, indicative of a risk of renal toxicity [59].
Most patients with diabetes mellitus display some histological renal
lesions, but only 30-45% of insulin-dependent patients develop diabetic
242
R. A. A. Muzzarelli
nephropathy [50]. It would be important to regularly screen the renal

function ofpatients at greatest risk using a weIl established N-acetyl--Dglucosaminidase or 2-microglobulin assay.
Acute renal failure is a weIl-known complieation of birth asphyxia,
which may result in permanent renal damage in up to 40% of survivors
[50]. Furthermore, transient hypoxie periods and pathologieal oseillations
in oxygen tension mayaIso cause tubular damage. Early reeognition of
acute renal failure is partieularly important in asphyxiated infants because
it facilitates the administration of appropriate fluid and electrolyte replacement.
The detection oftubulopathy caused by hypoxia and the estimation ofthe
efficieney ofthe therapies are equally important. N-aeetyl--D-glucosaminidase index values have been proved to be valuable tools in the diagnosis
of tubulopathies caused by either idiopathie respiratory distress syndrome
in hypoxie preterms [50].
Lithotripsy
Short-term follow-up studies have revealed that shock wave lithotripsy is a
safe and effective treatment for urinary traet stones. However, some degree
of histologieal, morphologieal and funetional renal damage has been
reported in experimental and clinical studies. Sakamoto et al. [60] investigated the faetors that aggravate renal damage after lithotripsy by
measuring the urinary excretion of a tubular-eeIl-exeaped enzyme which is
thought to be a marker for renal impairment. There was a significant
elevation in the urinary excretion of N-aeetyl--D-glueosaminidase immediately after lithotripsy in a group of patients who previously suffered
from urinary traet infeetions. Results indicated that infeetions may be one
of the risk faetors for the aggravation of renal damage after lithotripsy. In
spite of the administration of antibiotics, baeteria hidden in the stones may
appear after destruction of the stones and may aggravate renal damage
caused by exposure to the shoek waves.
Depression
In bipolar patients, levels of urinary N-aeetyl--D-glueosaminidase were
signifieantly associated with severity of depressive symptoms. A significant relationship between cerebrospinal fluid N-acetyl--D-glueosaminidase and serotonin was found [61]. Because the N-aeetyl--D-glucosaminidase aetivities correlate weIl with 24-h urinary free eortisol contents
and less signifieantly with platelet imipramine binding, the authors assumed that urinary N-acetyl--D-glueosaminidase activities were influenced by the density ofthe postsynaptie serotonin receptors.
243
Tay-Sachs and Sandhoff diseases
Deficiency of hexosaminidases causes lysosomal storage diseases. Two

human hexosaminidase genes encode proteins that form dimers A, B and S
of differing substrate specificities. The heterodimer A (a ) has full
activity against G M2 -type gangliosides, accumulation of which cause
blindness, paralysis, dementia and death. Tay-Sachs disease is caused by a
deficient or defective hexosaminidase a-subunit.
Sandhoff's disease involves a deficiency or defect in the -subunit. The
absence of hexosaminidase A causes G M2 accumulation, whereas lack of
hexosaminidase B leads to accumulation of oligosaccharides. The severity
ofthe clinical course ofboth diseases corre1ates with enzyme activity.
Various factors may cause these pathologies: poor folding, insolubility,
instability within the lysosome, failure to form the heterodimer or to bind
the activation protein. The known mutations have been mapped into the
hexosaminidase structure. The most severe infantile disease is generated by
mutations around the active site [5].
Gaucher s disease
Splenomegaly, hepatomegaly, anemia and deterioration of the skeleton are

the major signs ofthis disease. It is caused by a deficiency in activity ofthe
lysosomal hydrolase glucocerebrosidase, Ee 3.2.1.45, leading to lipidladen macrophages that secrete large quantities of hydrolases and cytokines. Methylumbelliferyl tetra-N-acetylchitotretraoside hydrolase hase
been reported in human plasma; it was also described as a chitotriosidase.
A concentration increase of the enzyme in plasma of patients with
Gaucher's disease was noted. The characteristics of the enzyme are very
similar to those of chitinase [62-64].
Sanjilippo s syndrome
Sanfilippo's syndrome is an inbom error of glycosaminoglycan metabolism characterized by mental retardation, bone deformities and excessive
urinary excretion of heparan sulphate. a-GlycosidicaIly linked N-acetylglucosamine units occur in heparan sulphate as weIl as in heparin. This
genetic disorder is generated by the deficiency of the lysosomal a-Nacetylglucosaminidase [65]. Purification of this enzyme from urine has
been reported [66, 67]: it is a glycoprotein with over 300 kDa molecular
weight, optimum pH 4.5, Km 0.14-0.74 mM for various a-D-glycosides.
Heparan sulphate, heparin and dermatan sulphate are competitive inhibitors. Multiple forms exist with pI values 3.3-6.0 that differ in their
recognition and endocytosis by skin fibroblasts. The fluorimetric deter-
244
R.A.A. Muzzarelli
mination of N-acetyl-a-D-glucosaminidase is made with methylumbelliferyl-N-acetyl-a-D-glucosaminide [68], in analogy with the protocol
given above, at 360 (ex) and 448 (em) nm.
Wound healing
Recent clinical data indicate that modified chitosans play an active role in
wound healing. The good c1inical results obtained in wound healing with
the aid of modified chitosan dressings can be explained in terms of high
susceptibility of certain modified chitosans to hydrolysis by lysozyme and
N-acetyl--D-glucosaminidase in vivo, and consequent generation of
hydrolysis products having high biological and biochemical significance
forthe repair ofconnective tissues [69-72] (see Tokura et al., this volume).
Aeknowledgements
The present work was carried out with financial support from the National Research Council of
Italy, Progetto Finalizzato Materiali Speciali per Tecnologie Avanzate 11.
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70 Biagini G, Bertani A, Muzzarelli RAA, Rizzoli C (1991) Wound management with Ncarboxybutyl chitosan. Biomaterials 12: 281-286
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Carbohydr Polymers 19: 29-34
Exogeneous chitosans

Biochemistry, histology and clinical uses of chitins

and chitosans in wound healing
Riccardo A.A. Muzzarelli, Monica Mattioli-Belmonte,
Armanda Pugnaloni and Graziella Biagini
Center for Innovative Biomaterials, Faculty ofMedicine, University ofAncona,
1-60100 Ancona, Italy
Summary. Biodegradability, biocompatibility and capacity to promote the synthesis of hyaluronan are main characteristics of chitin-derived wound healing materials, whose biological
significance in the human body depends largely on the actions that certain hydrolases exert on
them. The resulting chitooligomers stimulate various cells, while the released monomers are
phosphorylated and incorporated into hyaluronan, keratan sulphate and chondroitin sulphate,
components of the intracellular matrix and connective tissue. The healing process favoured by
these materials is examined in terms of macrophage activation, cytokine production by macrophages and fibroblasts, antiinflammatory action, angiogenesis stimulation, granulation and scar
formation. Current biomedical applications are illustrated by the treatment ofleg ulcers, the use
of skin substitutes, and the regeneration of bone, nerve and meniscus tissues.
Introduction
Modified chitins have been administered to humans in the form of
dressings for wounded soft and bone tissues, anticholesterolemic dietary
foods, cosmetics and items fr the contrlled delivery f drugs. In addition,
scientists have reported that chitins show antibacterial, antimetastatic, antiuricemic, antiosteoporotic and immunoadjuvant activities, indicating a
large general potential of the polysaccharide in alleviating diseases, preventing sickness or contributing to good health [1-7].
For the purpose of bone and soft tissue healing, it would seem that
the most relevant characteristics of chitins and chitosans are their biodegradability, biocompatibility and similarity to hyaluronan, besides their
capacity to release glucosamine and N-acetylglucosamine monomers and
oligomers.
Biodegradability
Biodegradable and nontoxic materials capable of activating host defences
to prevent infection and to accelerate the healing of the wound are highly
desirable. Chitin is degraded by enzymes such as lysozyme [4] N-acetyl-Dglucosaminidase and lipases [8]. NO mayaiso playa role in the chemoenzymatic degradation process. Evidence has been collected that significant
portions of chitin-based dressings are depolymerised, and that oligomers
252
R. A. A. Muzzarelli et al.
are further hydrolysed to N-acetylglucosamine, a common amino sugar in

the body which either enters the innate metabolic pathway to be incorporated into glycoproteins or is excreted as carbon dioxide [9].
Chitin sutures developed in the early 1980s undergo relatively rapid
biodegradation in vivo: the resistance to traction oftheir knots falls to 74%
after 5 days and 18% after 15 days. Increased knot resistance is cIaimed far
N -acyl chitosans, particularly N -pentanoyl chitosan (degree of substitution
< 0.20) that is expected to last for 2 years or longer [10].
The biodegadability is verified in many kinds of dressings and is independent of the degree of acetylation of the chitins. This last point implies
that lysozyme is not the only enzyme involved in the degradation, or that
the substrate is modified in situ [11].
Hyaluronan synthesis
Hyaluronan, a nonsulphated glycosaminoglycan of D-glucuronic acid and
N-acetylglucosamine, is found in extracellular matrices of various connective tissues. In fresh wounds chitins seem to enhance its production,
resulting in a reduced risk of scar formation and related complications such
as keloids, intraperitoneal adhesion and intestinal strictures [12]. Recent
evidence is found for the presence of DG42 protein (a chito-oligomer
synthase) during embryogenesis, producing chito-oligomers capable of
acting as primers in the synthesis ofhyaluronan. Overexpression ofDG42
in mouse cellS leads to the synthesis of chito-oligomers, and hyaluronan
synthase preparations also contain chitin synthase activities. Thus chitooligomers can act as templates for hyaluronan synthesis [13, 14]. Hyaluronan has been shown to promote cell motility, adhesion and proliferation,
and to have an important role in such processes as morphogenesis, inflammation, wound repair and metastasis that require massive cell movement
and tissue reorganization. Many of the effects of hyaluronan are mediated
through cell surface receptors, three of which have been molecularly
characterised, namely CD44, RHAMM and ICAM-l. The hyaluronanmediated signals are transmitted, at least in part, by activation of protein
phosphorylation cascades, cytokine release and stimulation of cell cycIe
proteins. These are important biochemical aspects that chitosans seem to be
able to promote, based on their similarity to hyaluronan: novel hyaluronanlike chitosans have therefore been developed recently [15].
Biocompatibility
The in vitro biocompatibility of wound dressings in terms of toxicity for
fibroblasts has been assessed and compared with three commercial wound
dressings made of collagen, alginate and gelatin. Methylpyrrolidinone
Biochemistry, histology and clinical uses of chitins and chitosans in wound healing
253
chitosan and collagen were the most compatible materials [16, 17]. The
ranking of chitosan cytotoxicity was established by various laboratories to
be chitosan hydrochloride > chitosan glutamate> glycol chitosan> chitosan lactate> methylpirrolidone chitosan. Chitosan hydrochloride is, however, fourfold less toxic than poly-L-Iysine. Glutaraldehyde cross-linked
chitosans are cytotoxic. Rat erythrocyte lysis was observed after 24 h of
contact with soluble chitosan salts, and most evidently with chitosan glutamate. Berscht et al. [16] observed that after 72 h ofincubation with fibroblasts the chitosan salts inhibited cell growth by 70-80%; in contrast,
methylpyrrolidinone chitosan caused only 35% inhibition [18].
The combination of basic fibroblast growth factor (FGF) and methylpyrrolidinone chitosan has an especially advantageous influence on impaired wound healing because the chitosan prevents excessive scar formation, and basic FGF induces fast wound c10sure [19].
Chitosan has been associated with other biopolymers and with synthetic
polymer dispersions to produce wound dressings. Biosynthetic wound
dressings with a drug delivery capability composed of a spongy sheet of
chitosan and collagen, laminated with a gentamicin sulphate-impregnated
polyurethane membrane, have been produced and clinically tested with
good results. Mosbey proposes a wound-filling gel obtained from chitosan
malate, polypropylene glycol, guar gum and locust beam gum, sterilized in
an autoc1ave [20].
In this chapter we focus on the complex mechanisms by which chitin and
chitosans enhance the wound-healing process. Thereafter, we will discuss
the main medical applications of these versatile polysaccharides.
Wound healing
Wound healing consists of a complex series of biochemical processes

regulated by humoral factors and antiinflammatory mediators, resulting in
the rebuilding of tissue and protection against infection [21, 22]. Regulating
factors inc1ude biochemical substances, growth factors and immunologie
mediators, whose influence can be decisive particularly during the early
phase oftissue rebuilding [23].
Basically, the wound-healing process consists of three stages. First,
inflammatory cells from the surrounding tissue move towards the lesion
site. Subsequently, fibroblasts appear and begin to produce collagen
connective fibres that impart tensile strength to the regenerated tissue.
Simultaneously, numerous capillaries begin to form to supply the site with
nutrients and oxygen; and epithelial cells at the edge of the wound start
filling in the area under the scab. In the third and final phase, the new
epithelium forms and the wound is healed.
Several preclinical studies on chitin and chitosan biomaterials show that
they are endowed with biochemical significance not encountered in cel-
254
lulose, starch and other polysaccharides [24]. In general, chitin-based

products provide improved healing of surgical wounds by first intention
in all cases; the healing process is faster and smooth scars are obtained.
They can be considered a primer on which anormal tissue architecture is
organised. Key factors in the rebuilding of physiologically effective tissues
exerted by chitosans are enhanced vascularisation and a continuous supply
of chito-oligomers to the wound that stimulate correct deposition, assembly and orientation of collagen fibrils, and are incorporated into the
extracellular matrix components [25]. The chito-oligomers are released by
hydrolytic action of lysozyme and N-acetyl--D-glucosaminidase.
According to Qin and Gilding [26] a wound dressing should inelude a
contact layer (carboxymethyl chitosan or its Ag salts) that assists healing,
and an extemallayer (alginate salts, Ca, Zn, Ag and pectin) that ensures
that exudate is removed from the contact layer before it becomes saturated.
Many studies have been performed into the mechanisms of improved
healing by chitins [27]. More details about the complex process by which
chitin and chitosan stimulate a variety of cells, and favour the release of
various proteins and regulatory factors involved in wound healing, are
given below.
Macrophage activation
Macrophage activation is probably the main effect of the administration of

chitin-based dressings. Macrophages obtained from laboratory animals are
activated by chemically modified chitin [28-29]. Chitosan activates
macrophages for tumoricidal activity and for the production of interleukin-l
(IL-l). Moreover, chitosan shows immunopotentiating activity [29]. This
mechanism involves, at least in part, the production ofinterferon-y(IFN-y)
[30). Chitosan also has an in vivo stimulatory effect on both macrophage
NO production and chemiotaxis [31-33). NO production plays a major
role in diverse physiological functions and pathological conditions. Chitosan-based dressings also show the capacity to modulate peroxide production [34].
Polykaryocytes appear in granulation tissue in the early days of implantation of spongelike chitin in animals, and disappear within 4 weeks
after surgery. These cells, derived from macrophages, possess a yet unknown higher level of function. There may be a elose relationship between
the disappearance of granulation tissue and the appearance of polykaryocytes. These data and the absence ofpolykaryocytes in controllesions
suggest that chitin induces the formation of polykaryocytes.
Cytokine production by macrophages
It is weIl known that granulation is accelerated by IL-l, tumour necrosis
factor-a (TNFa) or FGF [35]. In particular, IL-l and TNFa produced by
255
macrophages [36] are known to activate fibroblasts. Peritoneal macrophages obtained from laboratory animals are activated by chitin [28, 29].
The amount of IL-I in the exudate taken from around the areas where
chitin nonwoven fabrics were implanted showed a twofold increase in
comparison with that of control medium [37].
Chitinlchitosan items administered intravenously to mice become bound
to macrophage plasma membrane mannose/glucose receptors that mediate
their interiorisation. They are then degraded enzymatically. Within 3 days,
macrophages give a large oxidative burst when elicited with phorbol
myristate acetate; oligosaccharides are unable to prime macrophages.
The mechanism for macrophage priming by the chitin particles involves
the production of endogenous IFN y by NKl.l + CHi cells. This production
is due to cell-cell interactions, especially macrophages to NK1.1 +. These
responses may be common with microbial particulate components. In mice
preconditioned with a small dose of IFN y, a higher level of priming is
induced by chitin [30].
Cytokine praduction by jibrablasts
Chitins and chitosans do not enhance the rate of fibroblast proliferation in

vitra, but chitosan heparin complex in vivo may be responsible for accelerated wound healing. They do not directly stimulate the production of
IL-l, IL-6 and TNF a by fibroblasts; rather they induce IL-8 release from
fibroblasts in vivo, which leads to angiogenesis and migration of neutrophils. This is further demonstrated by the respiratory distress syndrome
produced by subcutaneous injection of high doses of chitosan in dogs,
generated by increased neutrophils in the lung. IL-8 is a potent neutrophil
chemotaxin [38].
Antiinflammatory action and angiogenesis stimulation
Chitin accelerates the first phase of wound healing where inflammation is

accompanied with infiltration of mononuclear and polymorphonuclear
cells without any uncomfortable side effects such as high temperature and
dolour. In vivo histological studies in animal models also highly support the
fact that the chitosan fibres stimulate migration ofthese cells [39-41].
N-Acetylglucosamine is itself an antiinflammatory drug and is synthesised in the human body from glucose and incorporated into glycosaminoglycans and glycoproteins. It has been administered to human volunteers by intravenous, intramuscular and oral routes for pharmacokinetic
studies [42]. N-Acetylglucosamine diffuses very rapidly in most tissues
and organs, even after oral administration, and accumulates in the articular
tissue and bone [43].
Morphological examination performed at the site of lesion treated with
chitin shows an increase in vascularisation during wound healing [28]. The
256
newly formed granulation tissue around chitin-nonwoven fabrie composite

implants is actively invaded with new blood vessels, a phenomenon not
observed in control experiments [37].
Granulation and scar formation
Granulating tissue can generally be divided into healthy and unhealthy

granulating tissue. The healthy one develops only in the absence of foreign
bodies (bacteria, debris). Chitins can induce healthy granulating tissue in
the early stage of wound healing; however, the effects of chitin on the
mechanism of formation of granulating tissue are still unclear. Chitosan is
superior to chitin in its effect on the acceleration of granulation tissue.
Scar formation depends on the continued synthesis and catabolism of
collagen, and is a serious problem in the wound-healing process. Fibroblast
proliferation increases at the site in contact with chitin sponge. Mild fibroblast activation is observed in the area close to the chitosan implant in experimental animals, and minimum scar formation remains in the wound.
Several reports document an acceleration of tensile strength without an
increase in collagen synthesis in the presence of chitin, suggesting that
N-acetylglucosamine may serve as a sub stratum for reinforcement of
wounded tissues without excessive inflammatory reaction [44]. However,
recent studies show that in chitin-treated lesions many histiocytes are
present and that fine collagen fibres are produced. The presence of histiocytes might be induced by chitin, and might promote the proliferation of
fibroblasts which produce fine collagen. In contrast, litde histiocyte invasion and thick collagen fibres were observed in the controls. Therefore it
is concluded that in chitin-treated wounds synthesis of collagen will accelerate in the early wound healing stage, but synthesised collagen will be
degraded to an appropriate amount in the subsequent healing stages by a
variety of collagenases from granulocytes, macrophages [45], epidermal
cells, neutrophils and fibroblasts [46]. The observed inflammatory accumulation at the site of contact with chitin sponge suggests that the excess
collagen may be degraded by these chitin-induced inflammatory cells.
Evidence has been collected that chitin prornotes keratin production and
facilitates rapid and effective regeneration of oral mucosa. In chitosan
treatment of purulent digit disease, epidermisation is observed with a
minimum of convalescence, without antibiotic administration [47].
Biomedical applications
Leg ulcers
The efficay of chitosan in the treatment of leg ulcers sterns from its antiinflammatory action and stimulation of epithelialization. Chitosans stimu-
Biochemistry, histology and c1inical uses of chitins and chitosans in wound healing
257
C
Figure 1. Patient L.A., aged 84. (A) U1cer, August 1995; (B) u1cer medicated with freeze-dried
methylpirrolidinone chitosan sponge, August 1995; (C) complete healing, October 1996.
late the granulation process and epidermis formation, thus accelerating

healing. Among the many patients treated so far, the case ofL.A., aged 84,
suffering from chronic venous insufficiency is reported in Figure 1. The
treatment with methylpirrolidinone chitosan lasted for 14 months and led
to complete healing [48].
258
Skin substitutes
Reacetylated chitin gels are also used to treat leg and decubitus ulcers in
paraplegie subjects [25]. Selective preparative conditions generated asolid
gel useful for this application. The treatment periods were 63 -182 days,
and complete healing was obtained [49].
Cultured skin from human cells is extremely thin and needs mechanical
support that can conveniently be provided by biopolymer complexes. A typical example is a skin substitute made of type I and type III collagen, chitosan and chondroitin 4- and 6-sulphate (72% collagen, 20% chitosan and 8%
glucosaminoglycan). The cross-linking between the chitosan primary amine and the sulphate groups is essential in imparting insolubility and mechanieal resistance. This skin substitute inc1udes human fibroblasts on the
"dermal side" and keratinocytes on the "epidermal" side. The latter, once
exposed to air during the last stage of culture, generates a skin similar to the
natural one, in which the epidermal layers inc1uding the stratum comeum
are present. The differentiation processes inc1ude the expression offilaggrin
and keratin and the formation of a structure similar to the basal lamina.
Chitosan is indispensable for skin substitutes not only to provide insolubility but also to increase production of collagen and regulatory factors
by fibroblasts. The addition of chitosan also increases the cytotoxicity
levels, and provides good adhesion (better than collagen alone), without
proliferation problems. The dermal substitute does not cause any antigenie
incompatibility and allows controlled vascularisation and fibroblastic
colonisation, resulting in an organised matrix and limited formation of
granulation and hypertrophie scar [9, 50, 51].
Nerve regeneration
Thanks to its expanded structure chitosan is suitable as a matrix for anchorage-dependent mammalian cell encapsulation [52]. For application in
neurosurgery, macrocapsules and hollow fibres made of polyacrylonitrilepolyvinyl chloride (PAN-PVC) were filled with PC12 cell suspension in
chitosan solution. The chitosan prevents extensive cell c1umping and
necrosis, which is known to take place in alginate gels and other encapsulation devices. When microencapsulated with chitosan, the PCl2 cells attached successfully to the precipitated chitosan and respond to exposure to
nerve growth factor (NGF) by extending neuritis. Differentiation of neuronal cells was also supported by the chitosan matrix.
Meniscus regeneration
Chitosan has also been used to assist the spontaneous tissue repair of the
meniscus, whieh is usually extremely difficult. Results of an experimental
259
study indicate that the natural polysaccharide is weH tolerated at the articular
synovial level. It also favours and stimulates the repair processes, which do
not take place spontaneously in the meniscus. Its initial angiogenic action
appears to be effective enough to stimulate the repair ofthe meniscus by providing the latter with the necessary tissue elements and humoral factors [53].
Regeneration 0/ bone tissue
Several studies dealing with reconstruction of periodontal tissue with

chitosan were aprelude to the discovery ofthe osteoinductive properties of
chitosan [54, 55]. Surgical wounds from wisdom tooth avulsions were
treated with freeze-dried methylpyrrolidinone chitosan, which promoted
bone regeneration. The polysaccharide is depolymerised by lysozyme and
is no longer detectable 6 months after surgery. Methylpyrrolidinone
chitosan was found to be useful in apicectomy as well. None ofthe patients
reported adverse effects over 3 years of observation [56].
The existence of osteoprogenitor (bone-producing) cells in the wound
site offers the possibility of regenerating the periodontal, periimplant and
alveolar ridge bone tissue simply with the aid of chemical mediators from
chitosan. It has been found that the number of bone-forming colonies is
almost doubled in the presence of chitosan. Chitosan also stimulates the
differentiation of osteoprogenitor cells and thereby facilitates the formation
ofbone[57].
Bone defects surgicaHy produced in sheep and rabbit models have been
treated with freeze-dried imidazolyl chitosan and methylpyrrolidinone
chitosan [56, 58-60]. Histological examination performed 60 days after
surgery showed a considerable presence ofneoformed bone tissue as compared with control. The cationic nature and chelating ability ofthe methylpyrrolidinone chitosan apparently favoured mineralisation. Endostealperiosteal and bone marrow osteoblast-like precursors, stimulated by
growth factor entrapped in the coagulum-polysaccharide mixture, gave rise
to intramembranous bone formation. Ultrastructural examination showed
that bone osteoid formation was foHowed by mineralisation. Osteoinduction was also observed in rabbit endochondral bones [61].
Experimental studies on rabbit and sheep were performed in order to
evaluate the possibility of improving bone tissue reconstitution with chitosan associated with calcium phosphate. Microscopic and histological
analyses showed the presence of an osteogenic reaction moving from the
rim of the surgical lesion towards the centre. In controllesions, dense
fibrous tissue without the characteristic histoarchitecture of bone was
observed.
The pattern of bone regeneration has been studied in an osteoporotic
experimental model with bone morphogenetic protein (BMP) linked to
chitosan. Biodegradation of chitosan leads to controlled release of BMp,
260
providing a synerglstlc effect on bone fonnation. Morphometric and

morphological analyses show that bone tissue regeneration in a surgical
bone defect is improved using this special chitosan. BMPs are members of
the transfonning growth factor (TGF ) superfamily, and are among the
extracellular signals that can induce the osteoblastic phenotype. This
important result also proves the validity of a biochemie al approach to the
therapeutical correction ofvarious affiictions in the elderly [62].
Effects of oral delivery on wound healing and renal functionality
Exogenous glucosamine is readily incorporated into hyaluronan by

cultured fibroblasts. Exogenous glucosamine can be transported into the
cell and acted on by a kinase which phosphorylates it at the 6 position,
enabling it to enter the pathway of glycosaminoglycan synthesis [63]. It
seems that glucosamine 6-phosphate synthesis is the rate-controlling step.
Enhanced availability of exogenous glucosamine is beneficial for the
synthesis of hyaluronan because endogenous glucosamine synthesis is
insufficient to achieve the high concentration of UDP-N-acetyl glucosamine necessary to optimize synthase activity, especially when rebuilding
extra cellular matrix is necessary. Thus, administration of glucosamine as
wen as chitosan (to be viewed as a delayed-release agent) in an possible
ways is beneficial for wound healing [64].
The nephrotic syndrome is often associated with hypercholesterolemy
and anaemia, that is reduction of serum haemoglobin value, which is difficult to eure. Human erythropoietin is used for treatment of patients with
anaemia nephrotic syndrome undergoing haemodialysis, but its administration must continue long tenn with the risk of many side effects.
The oral administration of chitosan (for 12 weeks) to patients with
chronical renal failure produced elevation of the serum haemoglobin level
(from 58.2 12.1 to 68.1 9.0 g/l). Other expression ofimprovement of
renal function were the reduced serum levels ofurea (from 75 51 to 45 19)
and creatinine (from 1.001 0.509 to 0.875 0.227 m/I), as weil as reduced cholesterollevels [65].
Ophthalmology
N,O-Carboxymethylchitosan is suitable as an excipient in ophthalmic formulations to improve the retention and bioavailability of drugs such as pilocarpine, timolol maleate, neomycin sulphate and ephedrine. Most of the
drugs are sensitive to pH, and the composition should have an acidic pH to
enhance stability of the drug. Therefore, the delivery should be made
through an anion exchange resin that adjusts the pH at around 7, which is
physiologically acceptable. Autoc1aving the carboxymethylchitosan (CMC)
261
improves the ocular retention characteristics [66]. Chitosan solutions do not

lend themselves to thermal sterilisation. A chitosan suspension, however,
can be sterilised at 121C for 15 min and then treated with sterile solution
of a suitable acid (HCI). The presence of mannitol, sorbitol and glycerol
seem to stabilise the sterilised solutions. This preparation is particularly useful for pharmaceutical and ophthalmie compositions [67].
Conclusions
The biological significance of chitosan biomaterials in the human body

depends largely on the actions that certain hydrolases exert on them [68].
The resulting chito-oligomers stimulate various cells, while the released
monomers are phosphorylated and incorporated into hyaluronan, keratan
sulphate and chondroitin sulphate, components of the intracellular matrix
and connective tissue.
Studies performed over the last few years have directed chitins and
chitosans to the forefront of applied medical research, offering truly
valuable medical items in the field of general medication, wound healing,
plastic surgery, drug carriers and immunostimulators.
Aeknowledgments
The authors are grateful to Prof. S. Mancini, University of Siena, ltaly, for providing experimental data and photographs, and to Maria Wecla. for assistance in retrieving the bibliographie material. This chapter was prepared under the auspices of the Italian National Research
Council, Roma (contract no.98.00032.PF34).
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Veterinary practice with chitin and chitosan

Saburo Minamit, Yoshiharu Okamoto 1, Koji Hamada2, Yukio Fukumot0 3,
Yoshihiro Shigemasa4
1
2
3
4
Department olVeterinary Surgery, Faculty 01Agriculture, Tottori University, 4-101 KoyamaMinami, Tottori 680-8553, Japan.
OmuAgricultural MutualAidAssociation, Ohmu, Monbetsu 098-1702, Japan.
Asa Zoological Park, Asa-Kita, Hiroshima 731-3355, Japan.
Department 01 Materials Science, Faculty 01Engineering, Tottori University,
4-101 Koyama-Minami, Tottori 680-8552, Japan.
Summary. Dramatic effects of chitin and chitosan on wmmd healing were demonstrated in field
cases ofmany small animals (dogs and cats), food animals (338 cows) and 142 zoo animals. In
comparison with conventional therapy with irrigation and antibiotic administration to wound,
new treatment with chitin and chitosan permitted a substantial decrease in treatment frequency
with minimum scar formation.
Introduction
In recent years, much attention has been paid to various biological dressings derived from natural products due to their high compatibility with
various organisms. Since 1985, we have been developing chitin and chitosan products for veterinary use, and we have previously reported their
clinical effects on wound healing [1-5], their experimental effects on
wound healing [6-8] and their biological activities [9-24]. The biological
activities of these materials can be summarized as follows: complement
activation [23], polymorphonuclear cell (PMN) activation [15-17, 19,21,
22,24], fibroblast activation [9, 13, 14,20], cytokine production [13, 20],
giant cell migration [6, 11] and stimulation oftype IV collagen synthesis
[18]. Complement activation appeared to be induced by high-dose (200 mgl
kg) administration of chitosan to canine subcutaneous tissue. The dogs
administered chitosan experienced unexpected hemorrhagic pneumonia,
and 70% died within the first 7 days after administration [17]. The pneumonia was induced by the production of C5a, a well-known strong
chemotactic factor for PMN, that was itself induced by the complement
activation by chitosan. Both chitosan and chitin activate complement via an
alternative pathway [23]. The systemic activations induced by 1 mg/kg of
subcutaneous administration of chitosan or 1 mg/kg ofvenous administration of chitin can also be explained by the corresponding systemic
complement activation by these materials [22]. Furthermore, local accumulation of PMN caused by administration of chitosan or chitin is also
attributable to the local complement activation by these materials. On the
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S. Minami et al.
other hand, fibroblasts were shown to produce interleukin 8 (lL-8) by

stimulation of chitin or chitosan [20]. IL-8 is also a fairly strong chemotactic factor for PMN.
We [19] demonstrated that chitin promoted the migration ofPMN more
strongly than chitosan, and the migration ofPMN corresponded to the concentration of chitin and chitosan. This phenomenon means that chitin and
chitosan are direct chemotactic factors for PMN. Furthermore, D-glucosamine and chito-oligosaccharides have much stronger chemotacticity than
chitosan. On the other hand, N-acetylchitooligosaccharides (GlcNAc)
whose degree of polymerization is below 5 are not a chemotactic factor for
PMN. GlcNAc6 showed chemotactic effect on PMN. Chitin and chitosan
are biodegraded in body tissue [10-12], so the chemotactic ability of
chitin would also rapidly disappear consequent to biodegradation. We have
previously investigated prostaglandin E 2 (PGE 2 ) production by these
materials in vivo and in vitro [24], and found a high concentration ofPGE 2
in exudate produced from chitin-implanted subcutaneous tissue. Furthermore, PMNs incubated with chitin or chitosan produced PGE2 [38]. These
data suggest that extended angiogenesis will be induced by the local increase ofPGE 2 level caused by migration ofPMNs. We also observed that
many giant cells appeared in the chitin- or chitosan-implanted areas, and
that large and marginal amounts, respectively, of type IV collagen and type I
collagen were synthesized in the chitin-implanted area [18]. These results
suggested that minimum scar formation would be induced by strong biodegradation of migrated giant cells and acce1eration of type IV collagen
synthesis. In the present chapter, we focus on the c1inical effects of chitin
and chitosan used in surgical treatment of various animal diseases.
Preparation of biomaterials
Chitin products
Chitipack S (Fig. 1): Squid pen -chitin (Nipp on Suisan Co., Ltd., Tokyo)
purified from Neon flying squid (Ommastrephes bartrami) was 9%
deacetylated; it had an average molecular weight of over 100,000 Da. A
1.5% (w/v) dispersion of chitin in water was frozen at - 20C and then
freeze-dried for 24 h, resulting in a spongelike chitin product [25]. The
Chitipack S was a pure chitin product and consisted of 200 mg of -chitin
[commercialized in Japan by Eisai Co., Ltd. (Tokyo) since 1994].
Chitipack P (Fig. 2): Squid pen chitin was disaggregated (dispersed and
swollen) in water at 40C or lower by use of a homogenizer or a mixer. The
mixture was poured into a predetermined amount of water to achieve
0.5-4.0 gl of chitin. The chitin suspension was poured onto a reinforcing
material (a nonwoven fabric of polyethyleneterephtalate (Du Pont Co.,
Ltd., San Francisco) by use of a suction-type paper-making apparatus of
267
Figure 1. Chitin sponge-type commercial wound remedy in Japan (Chitipack S).

Figure 2. Chitin and nonwoven fabric composite commercial remedy in Japan (Chitipack P).
Figure 3. Chitosan cotton-type commercial wound remedy in Japan (Chitopack C).
the batch style, resulting in a composite sheet having a chitin layer. The
thickness of the composite sheet could be adjusted by acting on the concentration of the starting chitin suspension. The Chitipack P contained
144 mg of -chitin in one sheet [commercialized in Japan by Eisai Co., Ltd.
(Tokyo) since 1994].
Chitosan product
Chitopack C (Fig. 3): Chitosan flake (Flonac C, Kyowa Tecnos CO., Ltd.,
Chiba, Japan), which was 82% deaeetylated a-ehitin from erab shells
(average moleeular weight of 80,000, maximum 1.2% ash and 5 ppm heavy
metals as Pb, Cd and As) was pulverized into several grades between 3 and
90 pm with a mill (Ube Industries, Ltd., Ube, Japan, CF-400). The eottonlike ehitosan produet obtained was eomposed offibers 2-20 mm in length,
20-50 }lm in width and 3-15 pm in thiekness, and had an apparent
specifie gravity ofO.l-0.2 g/em 3 Chitosan was dissolved with stirring in
a mixture of water and aeetie acid, and filtered twiee by the applieation of
pressure, and then defoamed by letting the solution stand overnight. The
dope was extruded from a spinneret, having 500 capillaries (0.1 mm diam.)
into a eoagulating bath eontaining a mixed solution of ethylene glyeol, iee
and potassium hydroxide. After eoagulation, the spun threads were passed
into a mixed solution of methanol and water. The resulting threads were
268
s. Minami et al.
stretched 1.15-fo1d in air, washed with water ovemight, treated with hot
water at 70-80C for 3-5 hand immersed in methanol ovemight. The
material was reeled with a reeling machine and dried. The resulting chitosan threads were cut to a length of 1-2 cm, treated with a mixer, and dried,
resulting in a cottonlike chitosan product [commercialized in Japan by
Eisai Co., Ltd. (Tokyo) since 1993; 100 mg of chitosan in one pack].
Application to small animal clinic
Between 1992 and 1998, there were approximately 500,000 c1inical applications of chitosan and 300,000 of chitin in Japan [4]. The methods used
and the results obtained with these materials are summarized below.
Chitipack S
Wound dressing or filling agents: Skin defect with sores, and skin problems
arising as a result of traffic accidents was covered by Chitipack S (Figs.
4- 6). Wound cavity caused by abscess, surgical tissue defect due to oncotomy, hemiorrhaphy or other operations, and various types of trauma
inc1uding bite wound was filled by Chitipack S. Before application of
Figure 4. Tail skin trauma in a cat.

Figure 5. Wound covering performed with Chitipack S.
Figure 6. 2 weeks after first treatment, evident wound contraction was observed.
269
Chitipack S, wounds were rinsed and disinfected with saline and povidoneiodine, and thereafter Chitipack S was exchanged every 4- 5 days until the
trauma and abscess healed. Antibiotic was administered systemically in
some animals which had systemic symptoms such as fever, anorexia and so
on, but was not done locally in all cases. In about 90% of all cases, good
healing developed. In the case of injuries, including traumas and abscesses,
healthy granulation tissue formed within I week after treatment. Skin
defects subsequently reepithelialized without scar formation or any functional disturbances. In cases in which granulation tissue did not develop,
general conditions were serious, and contamination of the wounds was
severe.
ChitipackP
Artificial skin: In the treatment of large skin defects due to skin tumor
resection, in which the resected ends could not be apposed by suturing,
Chitipack P was used as an artificial skin. Chitipack P was cut to about
70% of the wound area, and placed over the wound bed (Fig. 7). The
resected ends and Chitipack P were sutured using a continuous or interrupted suture pattern. The center site of Chitipack P did not fix to the
Figure 7. Large skin defect in a dog upon resection oflarge skin tumor. Chitipack P was placed
in the wound and was sutured to surrounding skin edge.
Figure 8. Ten days after operation, quite good wound contraction was observed after removal
of Chitipack P.
Figure 9. Severe hind leg trauma in a dog with automobile accident. Large skin defect and
severe tissue damage with dislocations ofmetatarsalphalangealjoints (long arrow). Application
of Chitopack C to wound surface (small arrows).
270
s. Minami et al.
wound bed because granulation tissue invaded into Chitipack P. After I

week, good wound contraction by reepithelialization, and granulating tissue formation, were observed (Fig. 8). Chitipack P was removed, and the
wound was closed by suturing, if possible. In the case where it was not possible to appose the wound, a similar treatment was performed until the
wound could be closed by suturing.
Hernia treatment: In reduction of perineal hemia, Chitipack P was used as

a filling agent. Chitipack P was rolled and inserted into the hemia cavity
following reduction ofhemia content, and was fixed to the surrounding tissue by sutures. In the case of large hemias, surgical treatment was reduced
from the usual2-3 h to a mere 30 min.
Chitopack C
There were approximately 1000 clinical applications of chitosan to contaminated skin and subcutaneous tissue trauma or abscess. The methods
used and the results obtained with these materials are summarized below.
All cases showed good response, such as disappearance of purulence,
formation of granulating tissue, and reepithelialization within 3 -1 0 days in
slightly or moderately wounded cases, and within 1 month in severely
wounded ones. Figure 9 shows severe trauma in the hind leg of dog caused
by an automobile accident. The dog was 2 years old and weighed 25 kg.
Figure 9 shows the appearance ofthe leg upon first examination. The skin
of the toes was widely defected, with about half of the digits and their subcutaneous tissue, tendons, vessels and bones damaged with contamination
(three metatarsus bones were visible and were dislocated at the metatarsophalangeal joints). After irrigation ofthe wound with saline, Chitopack
C was applied to the wound surface, and the wound was covered with
sterile bandages and fixed with an aluminum splint. Figure 10 shows a
typical wound reaction to the application of chitosan. Very fresh vascular
granulation appeared in the wound area 12 days after the first examination.
The dog recovered walking ability, with a slight gait in his wounded leg.
Thirty-three days after the initial treatment, the wound rapidly contracted,
and the dorsal skin of the leg was almost regenerated (Fig. 11). Complete
recovery of the wounded leg with no functional disturbance and minimum
scar formation was observed on 60 days post initial treatment (Fig. 12).
Based on the results of many cases of trauma, the characteristic biological reactions under chitosan treatment can be considered to be complete reconstruction of body tissue, including construction of normal subcutaneous tissue and regeneration of skin.
Application to pyothorax: Pyothorax is one of the most frequently encountered diseases in felines. Treatment is usually complicated. Our own
271
Figure 10. Twelve days after first treatment, fresh glanulating tissue regenerated in the
wounded site.
Figure 11 . After 33 days, wound contraction and reepithelialization were observed.
Figure 12. After 60 days, the wound was almost healed.
treatment has usually consisted of simple pleuroclysis and injection of

chitosan into the pleura after irrigation with physiological saline. In the
present study, Chitopack C (0.8 mg) was injected with 5 ml of saline into
the pleural cavity. At 1 week post injection, the cat had recovered vigor
and appetite, and a complete cure was indicated by X-ray findings. We
have treated numerous cases of pyothorax, all cases recovered after either
one, two or three injections of chitosan (3 - 7 days interval by the symptom
ofrecurrence of dyspnea).
Application to large animal clinic
There were 338 clinical applications of chitin or chitosan to bovine surgical

treatment. The methods used and the results obtained with these materials
are surnmarized below.
ChitipackP
Umbilical hernia: Twelve bovine umbilical hernias that appeared to require
reinforcement with artificial material due to their large hernia rings were
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S. Minami et al.
treated. In two of these cases the disease had recurred, resulting in the
rupture of the hemia ring of the suture site by a typical surgical procedure
(simple closure by suturing without reinforcing material). The method of
the typical surgical procedure is as follows: The hemia sac was completely
resected from the hemia ring, and then the hemia ring was closed by the
horizontal mattress sutures. In the present study, after the hemia ring was
closed by suturing, Chitipack P was applied over the suture site and fixed
to the abdominal wall by continuous suturing. Temporary swelling of the
implanted site was observed within 7 days, after which the swelling
gradually disappeared without side effects, that is purulence or rupturing
of the suture site. Because the umbilical suture site locates at the base of
the abdomen in standing cattle, and because cattle generally weigh 500800 kg, the suturing can be expected to undergo considerable stress. Chitipack P is the most suitable reinforcing material as an implant for cattle.
Chitopack C
Foot diseases: Two-hundred-fifty-eight cases of foot diseases were treated
using Chitopack C. The treatment procedures were as follows: To treat foot
disease, after rinsing diseased interdigital skin and surgical debridement of
necrotic tissue, and following disinfection by povidone-iodine solution,
Chitopack C was packed on to the debrided area, covered with cotton, and
bandaged. The bandage was changed, and Chitopack C was reapplied
weekly. Antibiotics were used in cases in which there was infection in the
deeper tissue or in which there were systemic disorders. Sixty out of 63
cases were cured (95%), and the mean treatment frequency was 3.4 times
(2.9 times among successful cases). Six ofthe 63 cases had an interdigital
nodule, and 5 of them were cured by the present treatment (83%). The
mean treatment frequency among the 6 cases was 8.2 times (2.8 times
among successful cases). One-hundred-and-eighty-three out of 195 cases
each having a sole ulcer were cured (94%), and the mean treatment
frequency was 3.1 times (2.7 times among successful cases). Among all
foot diseases, 243 out of 258 cases were cured, and the mean treatment
frequency was 3.1 times (2.8 times among successful cases). The 15 failures were all cases of severe purulence with progressing bone and joint
diseases in the infected foot. Because 2 or 3 days are required for each
retreatment using ordinary antibiotic ointment, the l-week retreatment
period required for Chitopack C represents a substantial improvement, and
this combined with the treatment's success rate makes Chitopack C a
suitable material for treatment of cattle foot diseases.
Other surgical diseases: Sixty-eight cases, 30 with trauma, 20 with
abscess and 18 with arthritis, were treated by Chitopack C. After surgical
debridement and irrigation with an antiseptic agent, Chitopack C was
applied to the trauma surface, and then the wound was covered with
273
Figure 13. Chronic udder trauma near nipple. Wound did not heal with antibiotic ointment
treatment.
Figure 14. The trauma was completely healed with a single application of Chitopack C.
bandages. Abscesses were also treated by direct application of Chitopack C

to the wound after irrigation of the abscess cavity following incision. For
treatment of arthritis or periarthritis, after irrigation of the infected region
Chitopack C was inserted via the vent. Some cases were treated only by
aspirating discharge using a syringe and an 18-gage needle followed by
injection of Chitopack C suspension to the aspirated cavity. Antibiotic was
applied systemically to the severe purulent cases, 11 with trauma, 4 with
abscess, and 4 with arthritis. Among cases with trauma, 29 of 30 (97%)
were cured, and the mean treatment frequency was 5.4 times (5.0 among
successful cases). Among cases with an abscess, 20 were completely cured
(100%), and mean treatment frequency was 3.3 times. Thirteen of 15 cases
with periarthritis were cured (87%), and the mean treatment frequency was
3.7 times (3.3 among successful cases). Two of3 cases with arthritis were
cured (67%), and the mean treatment frequency was 9.3 times (12.5 among
successful cases). Cases of arthritis with severe skin defect or with bone
infection were also cured. Figure 13 shows a case of chronic trauma in the
udder skin. Although this wound was previously treated for about 2 months
by direct application of an antibiotic ointment, it did not heal. After a single
application of Chitopack C, the wound contracted rapidly and was cured
within 2 weeks (Fig. 14).
As described above, many surgical diseases showed good response to
chitosan application. Treatment of purulent bovine diseases could thus be
greatly simplified by utilizing chitosan.
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S. Minami et al.
Applications in wild and zoo animal clinics

Chitopack C
Chitopaek C was applied to the surfaee of wounds, or suppurative or
debrided wounds were paeked with it after rinsing with disinfeetant. As a
rule, the dose of Chitopaek C was not greater than 50 mg/kg of body
weight, and it was applied at intervals of several days to several weeks.
Studies were earried out on 143 animals (121 mammals, 18 birds and
4 reptiles) at 16 zoos and an aquarium aeross Japan, the animals were classified into 3 classes, 23 orders (11 in Mammaria, 10 in Aves and 2 in Reptilia), 37 families and 62 speeies (Tab. 1). The effeets of ehitosan were classified into 4 grades ranging from exeellent (++) to poor (-). It should be
noted that the evaluation was classified as no better than good (+), even
though they were severe wounds whieh healed very quiekly.
The effeet of ehitosan was classified as exeellent (++) in 63 of 143 animals and good (+) in 58 animals. The overall efficaey rate was 85%.
Wounds were not healed in the remaining 21 animals or 15%.
When drug effeets were evaluated aeeording to the classifieation of
animals, a eure was effeeted in 107 of 121 mammals or 88%, but the eure
rate for birds was 61% (11/18). The eure rate for reptiles was 75% (3/4).
The therapeutie results were obtained in six groups of Artiodaetyla,
Camivore, Primates, Rodentia, Marsupialia, and Lagomorpha whieh included 10 animals or more. A eure rate of 95%, the highest among all
Table 1. Classification of case animals *

Class
Order
Mammalia
Marsupialia
Edentata
Chiroptera
Primates
Camivora
Proboscidea
Perissodactyla
Hyracoidea
Artiodactyla
Rodentia
Lagomorpha
Family
2
1
Species
4
No.ofcases
3
1
9
9
1
2
1
10
5
1
10
1
4
19
23
3
5
6
29
12
9
6
4
1
1
1
4
I
I
Subtotal
11
25
44
121
Aves
10
10
16
18
22
37
62
143
Reptilia
Total
* Data were obtained at 16 zoos and an aquarium by 34 veterinarians.
275
Figure 15. A masked palm civet with severe bite wound on his neck.
Figure 16. Application ofChitopack C to the wound.
Figure 17. The wound was contracted and healed with several applications of Chitopack C to
the wound surface.
mammals, was achieved in Primates (21/22). When the effects of chitosan

were compared between suppurative and nonsuppurative wounds, woundhealing acceleration was confirmed in 84% of suppurative wounds and
96% of nonsuppurative ones.
Figures 15 -17 show the dramatic effects of wound healing in a masked
palm civet with a bite wound in his neck. The wound was extremely contraeted without purulence by several Chitopack C applieations (Fig. 17).
When unhealed animals were examined, most were found to have had
severe general emaeiation or severe suppuration eomplieated by periostitis.
Most birds had severe suppuration eomplieated by periostitis or osteomyelitis or both. Chitosan was not effeetive for the treatment of infeetions
with Pseudomonas aeruginosa in birds.
Although animals generally liek or bite their wounds, in our study animals tended to refrain from such aets when treated with ehitosan.
276
S. Minami et al.
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Tatara S, Shimazu M et al (1995) Significance of clinical application of chitosan in zoo and
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world. Wirtschaftsverlag NW, Germany, 402-407
6 Okamoto Y, Minami S, Matsuhashi A, Sashiwa H, Saimoto H, Shigemasa Y, Tanigawa T,
Tanaka Y, Tokura S (1994) Polymeric N-acetyl-D-glucosamine (chitin) induces histionic
activation in dogs. J Vet Med Sei 55: 739-742
7 Okamoto Y, Tomita T, Minami S, Matsuhashi A, Kumazawa NH, Tanioka S, Shigemasa Y
(1995) Effect of chitosan on experimental abscess with Staphylococcus aureus in dogs. J Vet
Med Sei 57: 765-767
8 Okamoto Y, Shibazaki K, Minami S, Matsuhashi A, Shigemasa Y (1995) Evaluation of
chitin and chitosan on open wound healing in dogs. JVet Med Sei 57: 851-854
9 Okamoto Y, Southwood L, Stashak TS, Norrdin RW, Nelson AW, Minami S, Matsuhashi A,
Kato K, Shigemasa Y (1997) Effect of chitin on nonwoven fabric implant in tendon healing.
Carbohydr Pol 33: 33-38
10 Sashiwa H, Saito K, Saimoto H, Mianmi S, Okamoto Y, Matsuhashi A, Shigemasa Y (1993)
Enzymatic degradation of chitin and chitosan. In: RAA Muzzarelli (ed): Chitin enzymology,
voll. Atec, Grottammare, 177 -186
11 Okamoto Y, Mianmi S, Matsuhashi A, Tanioka S, Shigemasa Y (1995) The fate ofN-acetylD-glucosamine (chitin) in canine subcutaneous tissues. Seitai Zairyo 13: 112-116
12 Saimoto H, Takamori Y, Morimoto M, Sashiwa H, Okamoto Y, Minami S, Matsuhashi A,
Shigemasa Y (1997) Biodegradation of chitin with enzymes and vital components. Macromol Symp 120: 11-18
13 Tanigawa T, Tanaka Y, Tomita K, Sasaki T, Sashiwa H, Saimoto H, Shigemasa Y, Okamoto
Y, Minami S, Matsuhashi A (1992) Effect of chitin on the production of interleukin-l in
human blood monocytes. YonagoActa Medica 35: 147-150
14 Minami S, Okamoto Y, Matsuhashi A, Sashiwa H, Saimoto H, Shigemasa Y, Tanigawa T,
Suzuki T, Tanaka Y (1995) Polymeric N-acetyl-D-glucosamine (chitin) induces prostaglandin E 2 in dogs. JVet Med Sei 57: 377-378
15 Usami Y, Okamoto Y, Minami S, Matsuhashi A, Kumazawa NH, Tanioka S, Shigemasa Y
(1994) Chitin and chitosan induce migration ofbovine polymorphonuclear cells. J Vet Med
Sei 56: 761-762
(1994) Migration of canine neutrophils to chitin and chitosan. JVet Med Sei 56: 1215 -1216
17 Minami S, Oh-oka M, Okamoto Y, Miyatake K, Matsuhashi A, Shigemasa Y (1996)
Chitosan inducing hemorrhagic pneumonia in dogs. Carbohydr Pol 29: 241-246
18 Minami S, Okamoto Y, Miyatake K, Matsuhashi A, Kitamura Y, Tanigawa T, Tanaka Y,
Shigemasa Y (1997) Chitin induces type IV collagen and elastic fiber in implanted nonwoven fabric ofpolyester. Carbohydr Pol 29: 295-299
(1997) Influence of chain length ofN-acetyl-D-glucosamine residues on direct and complement-mediated chemotactic activities for canine polymorphonuc1ear cells. Carbohydr
Pol 32: 115-122
20 Mori T, Okumura M, Matsuura M, Ueno K, Tokura S, Okamoto Y, Minami S, Fujinaga T
(1997) Effects of chitin and its derivatives on the proliferation and cytokine production of
fibroblasts in vitro. Biomaterials 15: 947-951
277
21 Minami S, Mura-e R, Okamoto Y, Sanekata T, Matsuhashi A, Tanioka S, Shigemasa Y

(1997) Systemic effect of chitin by intravenous administration in dog. Carbohydr Pol 33:
243-249
22 Minami S, Masuda M, Suzuki H, Okamoto Y, Matsuhashi A, Kato K, Shigemasa Y (1997)
Subcutaneous injected chitosan induces systemic activation in dogs. Carbohydr Pol 33:
285-294
23 Minami S, Suzuki H, Okamoto Y, Fujinaga T, Shigemasa Y (1998) Chitin and chitosan
activate complement via the alternative pathway. Carbohydr Pol 36: 151-155
24 Usami Y, Okamoto Y, Takayama T, Shigemasa Y, Minami S (1998) Effect ofN-acetyl-Dglucosamine and D-glucosamine on canine polymorphonuc1ear cells in vitro. Carbohydr
Pol 36: 137-141
25 Tanioka S, Okamoto Y, Minami S, Matsuhashi A, Tokura S, Sashiwa H, Saimoto H, Shigemasa Y (1993) Development of chitin and chitosan biomaterials. In: M Yalpani (ed): Carbohydrates and carbohydrate polymers. ATL Press, Mt Prospect, 153 -164

ed. by P. Jolles and R. A.A. Muzzarelli
Immunological aspects of chitin and chitin

derivatives administered to animals
Seiichi Tokura 1, Hiroshi Tamura 1 and Ichiro Azuma 2
1
2
Faculty ofEngineering, Kansai University and HRC, Suita, Osaka 564-8680, Japan
Institute for the Immunological Sciences, Hokkaido University, Sapporo 060, Japan
Summary. Chitosan amino groups are recognized by the immune system. Therefore, every derivative of chitin should be assayed immunologically if biomedical applications are sought.
Macrophages are activated to various extents by chitin derivatives. Deacetylated chitin (30%
deacetylation) and chitin sulfate stimulate the production of circulating antibodies. Accumulation of carboxyrnethyl chitin takes place in granulocytes and macrophages. These polysaccharides activate complement in analogy to zyrnosan. Intraperitoneal injection ofN-acety1chitohexaose inhibits the growth of tumor cells and pathogens on a similar level as that of lentinan.
Introduction
Despite permanent aggression by bacteria and viruses, the enzymatic and

immunologie al defense systems protect the animal body from infection.
Lysozyme breaks down the bacterial cell wall, and the resulting hydro lysis
products become involved in normal metabolie cyc1es. The biodegradability and nontoxic properties of chitin stern from the capacity of the
physiological system of animals to protect from infection. However, pure
chitin isolated from other supporting materials such as protein might upset
the immune system due to the presence of a rigid crystalline structure
established by inter- and intraresidual hydrogen bonds between acetamido
groups and hydroxyl groups. Chitin derivatives, even though biodegradable, might stimulate the immune system when they carry unfamiliar
chemical groups.
Residual amino groups in chitosan also look unfamiliar to the immunological system despite the low pK a value for the amino group of chitosan
(pK a 6.3-6.4); an amino group with pK a higher than 9.0 in general tends
to stimulate the immune system. Imino derivatives of chitosan mayaiso stimulate the immune system because chemie als bond uncommon to animal
physiology.
Therefore, every derivative of chitin should be tested immunologically
before its application as a biomedical material. Chitosans and carboxymethyl chitin ofvarious degrees of deacetylation or substitution were used
to investigate the activation ofmouse peritoneal macrophages (Tabs. 1-3)
[1]. As can be seen in Table 1, mouse peritoneal macrophage activation by
chitosan is at a maximum at a degree of deacetylation of 70%. Chitin did
280
S. Tokura et al.
Table I. Effect of chitin and its deacetylated derivatives on macrophage activation in vivo [1]
Treatment"
Timing
(days)
Dose
(p.g)
% Iysis b
(mean s.e.)
% stasis b
(mean s.e.)
HzO z releasee
(nmolJmg protein)
Chitin
30% DA-chitin
-5
500
25.8 3.4
500
500
500
9.8 3.9
55.0 6.2
47.6 7.1"
34.5 5.2
98.0 0.2
-5
-10
-15
98.6 0.1"
97.5 0.5
98.0 0.1
37.0 1.2
16.9 0.8"
24.9 3.4
70% DA-chitin
-5
-10
-15
500
500
500
-5
-10
-15
500
500
500
98.5 0.1
97.5 0.3
95.7 0.3"
88.3 3.7
61.2 1.9 d
73.2 3.5 d
59.3 2.6
62.6 4.2
53.7 5.3
Chitosan
56.8 4.7
33.5 4.1
31.9 7.7 d
19.2 3.9
19.2 5.0 d
22.4 5.5 d
40.1 2.2"
24.6 2.2"
17.9 1.4"
-1.8 4.1
40.07.0
3.1 1.3
Control
Mice were injected intraperitoneally (i.p.) with each polysaccharide at indicated days before
harvest ofmacrophages.
b Each value is the standard error of six weHs in each group.
e Macrophages were stimulated with 20 nM ofPMA for 30 min. The results were expressed as
nanomoles ofHzOz per mg of ceH protein per 30 min.
d Significant difference from the control by Student's t-test (p < 0.05).
Significant difference from the control (p < 0.005).
a
Table 2. Effect of 6-0-substituted derivatives of chitin or chitosan on macrophage activation

[1]
Treatment"
Dose
(p.g)
% lysis
(mean s.e.)
% stasis
(mean s.e.)
HzOz release
(nmolJmg protein)
Chitin
CM-Chitin
P-Chitin
S-Chitin
Acetyl-chitin
HE-chitin
DHP-chitin
CM-chitosan
DHP-chitosan
70% DA-chitosan
Pyran copolymer
Contro!
500
500
500
500
500
500
500
500
500
500
500
2.5 3.0
53.04.0 e
3.44.2
2.8 6.6
0.3 2.6
22.2 4.8 b
15.3 3.2
3.9 3.7
18.6 7.2
55.94.3
66.4 3.0 c
1.0 5.6
58.2 1.3 e
58.3 2.0 c
55.6 2.9 b
43.1 4.1
N.D.
44.1 2.8
66.9 1.5 c
45.2 3.7
49.2 2.1 b
54.7 3.3 b
97.5 0.5 c
38.9 3.9
33.6 4.2 c
51.8 2.5"
33.0 5.3 b
44.1 2.4 c
N.D.
43.83.3 c
78.7 2.9 C
48.2 3.1 c
59.6 3.0 c
98.7 3.9 c
62.4 6.4 C
18.5 1.4
Mice were injected i. p. with each polysaccharide at 3 days before harvest of macrophages.
Significant difference from the control by Student's t-test (p < 0.05).
c Significant difference from the control (p < 0.005).
N.D., not done.
a
not activate mouse peritoneal macrophages. CM-chitin promoted macrophage activation to the same extent as 70% deacetylated chitin, and
hydroxyethyl-chitin was next, as seen in Table 2. DHP-chitin promoted
scarce macrophage activation despite the bigger substituting group.
P-chitin and S-chitin did not activate mouse peritoneal macrophages. The
Immunological aspects of chitin and chitin derivatives administered to animals
281
Table 3. Cytolytic and cytostatic activity ofmacrophages induced by various doses of70% DAchitin and CM-chitin [1]
Treatment a
Dose
(}lg)
Numberof
macrophages
(x 106/mouse)
% lysis
(mean s.e.)
% stasis
(mean s.e.)
70% DA-chitin
2500
500
100
20
2500
500
100
20
500
19.2
17.1
9.3
4.6
3.0
3.4
1.4
1.7
3.8
2.7
34.4 3.6'
39.3 3.1'
38.9 3.5'
29.5 2.8'
25.5 3.4'
30.8 3.2'
22.8 3.6 b
9.3 3.3
47.6 3.0'
2.1 3.4
84.2 1.8'
81.2 0.9'
81.7 1.9'
79.4 1.0'
87.00.5'
80.1 1.9'
87.0 1.0'
77.9 1.6'
81.2 1.6'
41.1 4.3
CM-chitin
Pyran copolymer
Control
Mice were injected i. p. with various doses of each polysaccharide at 3 days before harvest of
macrophages.
b Significant difference from the control by student's t-test (p < 0.05).
, Significant difference from the control (p < 0.001).
Table 4. Adjuvant activity of chitin derivatives on circulating-antibody formation to bacterial

a-amylase (BaA) in guinea-pigs [2]
Adjuvant
Anti-BaA titre a (U)

2wks
Water-insoluble derivatives
Chitin
Chitosan
DHP-chitin
DHP-chitosan
Water-soluble derivatives
DAC-30
DAC-70
CM-chitin
CM-chitosan
P-chitin
S-chitin
MDP
Control(BaA FIA)
4wks
48 11 (1.0)
32 7(0.7)
28 4(0.6)
52 8(1.1)
148
67
26
53
76
215
311
46
34(3.2)
15 (1.5)
3(0.6)
14(1.2)
15(1.7)
38(4.7)
114(6.8)
10(1.0)
462
483
459
490
11 (1.2)
32(1.2)
43(1.1)
12(1.2)
1393 119(3.5)
1216 135(3.0)
251 8(0.6)
835 41(2.1)
362 6(0.9)
1192 178(3.0)
1638 50(4.1)
401 16(1.0)
Hartley guinea pigs were immunized in each foot pad with 200 mg ofBaA in Freund's incomplete adjuvant with 100 mg of each adjuvant. The control group was immunized with 200 mg
ofBaA alone in Freund's incomplete adjuvant.
a One unit ofthe antibody titre was defined as dilution number of antiserum needed to neutralise 10 units of amylase activity. The data are expressed as mean units standard error. The
numbers in parentheses are stimulation indexes, as calculated with the contro!.
282
S. Tokura et al.
0.20,-----------------,
0.15
iii
~
0.10
CIl
.c
1l
0.05
10
100 x Reciprocal serum dilution
40
Figure 1. Titration of mouse anti-DNP serum collected three weeks after the injection of 20 Jlg
DNP-OVA in FIA(2). Three mice in each group were immunized with 50 Jlg OVA with or without 100 mg adjuvant in FIA two weeks before the injection ofDNP-OVA..; 70% deacety1ated
chitin, 0; BCG-CWS, .6.; saline.
free amino group seems to be the main factor in activating peritoneal

macrophages. Dose-dependent macrophage activation is shown in Table 3.
As shown in the table, 70% deacetylated chitin is a fairly strong effector
for the activation of mouse peritoneal macrophages compared with CMchitin. Although chitosan and CM-chitin were both found to accelerate
mouse peritoneal macrophage activity, only chitosan induced further production of circulating antibodies for bacterial a-amylase (BaA) (Tab. 4),
and dinitrophenyl-ovalbumine (DNP-OVA) induced anti-DNP serum in the
presence of 70 % deacetylated chitin as shown in Figure 1 [2]. As seen in
Table 4, 30% deacetylated chitin and S-chitin were the best effectors
among water-soluble chitin derivatives to stimulate the production of
circulating antibodies, whereas there was little difference among waterinsoluble chitin derivatives. Chitosans also induce delayed-type hypersensitivity to m-[4-(4' -Arsonophenyl-azo)-phenyl]-N-acetyl-L-tyrosine(ABAN-acetyl-tyrosine) in addition to the secretion of cytokines following
macrophage activation a shown in Table 5 [2]. The highest immunogenicity
was obtained by the administration of 70% deacetylated chitosan. Though
a negative mitogenic activity was found by the administration of chitosan,
a slightly positive activity was observed on normal spleen by CM-chitin in
spite of little serious value to cause allergy, as shown in Table 6. As the
mitogenic activity stimulates the induction of B cell formation, a hapten-
283
Table 5. Adjuvant activity of chitin derivatives on the induction of delayed-type hypersensitivity to azobenzenarsonate N-acetyltyrosine in guinea pigs [2]
Skin reaction with ABA-BSA at 24 h
Adjuvant
50 ]lg
100 ]lg
Chitin
DAC-30
DAC-70
P-chitin
S-chitin
(5.00.W
13.4 1.7
12.4 1.2
(6.5 0.2)
(2.3 0.9)
(7.8 0.6)
15.9 1.9
13.4 1.1
(7.3 0.2)
(5.0 0.0)
MDP
22.5 1.4
21.4 0.8
Control (ABA-Tyr + FIA)
(4.3 0.9)
(6.0 0.5)
Hartley guinea pigs were immunized with 50 mg of ABA-N-acetyltyrosine (ABA-Tyr) in FIA

with 100 mg of each adjuvant. The control group was immunized with ABA-N-acetyltyrosine
alone in FIA.
a After 2 weeks, a skin test was performed with 50 and 100 mg of ABA-BSA, and skin reaction
was measured 24 h after the intradermal injection of test antigen. The data are expressed as
mean standard error of the skin reaction for four guinea pigs.
b The numbers in parentheses indicate the size of faint erythaema.
304
(A)
256
(8)
E
::J
Qj
CIl
:;:::;
C
oe(
'5
c
0
:;:::;
..:!
128
i5
(C)
64
32
16
0
12345
Time course of Immunization (Month)
Figure 2. Time course of immunization for rabbits. Dilution of antiserum showed optimal
dilution to give an absorbance of 1.0 at 492 nm in the ELISA assay [3].
284
S. Tokura et al.
Table 6. Mitogenic activity of chitin derivatives on normal spleen cells ofBALB/c or C57BLl6
mice [2]
Mitogen
Dose
(Ilg mi-I)
[H] Thymidine incorporation (Counts per min)

BALB/C
Expt 1
CM-chitin
P-chitin
ConA
LPS
Control
Expt2
HE-chitin
5
10
3386
1855
2016
1665
5507
3522
2517
2376
250
50
10
2
250
50
10
2
DAC-70
250
50
10
2
5
10
ConA
LPS
Contro1
6065 108 (3.4)

3822 280 (2.2)
3478 296 (2.0)
2957 219 (1.7)
1432 102 (0.8)
2242 89 (1.3)
2801 288 (1.6)
2591 408 (1.5)
126058 2247 (71.2)
10466 190 (5.9)
1771 169 (1.0)
250
50
10
2
250
50
10
2
DHP-chitin
C57BLl6
158
165
101
54
(2.2)
(1.2)
(1.3)
(1.1)
393
266
133
223
(3.5)
(2.2)
(1.6)
(1.5)
123836 1818 (79.1)

8901 366 (5.7)
1566 177 (1.0)
5953 205 (1.6)

5953 205 (1.6)
3760 70 (1.0)
3575 162 (1.0)
6703 279 (1.8)
4219 237 (1.1)
3384 186 (0.9)
2869 87 (0.8)
3519 303 (0.9)
4150 96 (1.1)
3563 316 (1.0)
3325 210 (0.9)
88301 4633 (23.6)
15183 1113 (4.1)
3744 279 (1.0)
Normal spleen cells (5 x 10 5 ) of BALB/C or C57BLl6 mice were incubated with mitogen or
spleen cells alone for 72 h. ['H] Thymidine (0.5 }lCi) was added from 24 h until the end ofthe
culture. The numbers in parentheses are stimulation ratios.
CH3~
C~-CH2~ Hapten (33 mol/IOD residues)
H-N-(CH2)4- N
eH
o~~~~o~:'~o~
NHCOCH3
NHCOCH3
Scheme 1. Chemical structure ofhapten-bound CM-chitin.
NHCOCH3
285
Table 7. Cross-reaction ratios by metbamphetamine analogs [3]

Compound
Cross-reaction
(%)'
Methamphetamine
Amphetamine
Norephedrine
p-Hydroxymetbamphetamine
p- Hydroxyamphetamine
p-Hydroxynorephedrine
100
6.6
2
0.2
< 0.1
< 0.1
Ephedrine
Methylephedrine
N-Dimethylamphetamine
p-Hydroxyephedrine
Phentermine
Mephentermine
p- Phenetbylamine
Methoxyphenamine
(O-Methoxymethamphetamine)
p-Methoxymetbamphetamine
p- Methoxyamphetamine
13
20
266
< 0.1
6.1
100
0.9
1
1
0.4
Methamphetamine
O!J
~
yH3~
CH'-y-N-H
CH,
Phentermine
Mephentermine
Dlmelhylamphetamine
O!J
~
yH'yH3
CH,-CH-N-(CH,l,-NH-
MABA(Hapten)
Cross-reaction (%) was ca\culated from tbe equation, AlB X 100, where A = MA concentration to reduce absorbance at 492 nm to 50% and B = MA analog concentration to reduce
absorbance at 492 nm to 50%.
bound CM-chitin saline solution was administered subcutaneously to

rabbits every 2 weeks for 4 months (chemical structure of hapten-bound
CM-chitin in Scheme 1). There was little antibody production even after
4 months ofimmunization. The production ofhapten-specific antibody was
found first in the presence of a strong immunoadjuvant such as Freund's
complete adjuvant as shown in Figure 2. The specificity of produced antibody was studied, and the recognition of antibody was shown to be very
limited for the hapten analogues, as listed in Table 7; but quite low sensitivity of antibody was found either for the carrier CM-chitin or for CMchitin oligomers [3]. 14C-Iabelled CM-chitin was administered per os and
intravenously to mouse. The accumulation of 14C was observed mainly in
spleen on per os administration, and in bone marrow on intravenous injection, as shown in Figure 3. As the human organism has no -glycosidases
in the intestine to hydrolyse chitin and chitosan, the result of intravenous
injection of CM-chitin should be taken into consideration in the case of
human administration. The accumulation of radiolabelIed CM-chitin was
further studied to detect the distribution ofCM-chitin among bone marrow
cells. Fluorescent isothiocyanate (FITC)-labelled CM-chitin was incubated
in the presence ofbone marrow extracted from mouse bone (Scheme 2). The
fluorescence was first seen in the granulocytes after 8 h of incubation, and
macrophages accumulated fluorescence after 18 h. Little fluorescence was
observed in erythrocytes even after 98 h incubation, as shown in Figure 4
[4]. These observations would suggest that the accumulation ofCM-chitin
286
S. Tokura et al.
Kidney
Spleen
Lung
72h
1.V.
lnte tine
Bone Marrow
Brain
Liver
100 200
300 400 500 600

dpm I 100 mg of tissue
700 800
Kidney
Spleen
Lung
72h
p.O.
Stomach
lntestine
Bone Marrow
Brain
100 200 300 400 500 600 700 800

dpm I 100 mg of ti ue
Figure 3. Distribution of 14C-labeled CM-chitin on organs after intravenous and per os

administrations to mice [4].
is specific only for immune-related parts in the physiological pathway.

However, stimulation of the immune system seems to be almost the final
stage in producing antibody on the administration of CM-chitin, because
specific accumulations ofCM-chitin to granulocytes and macrophages was
observed among bone marrow components in addition to a slight mitogenic
activity.
In further studies of chitosan administration, secretion of cytokines was
observed upon mouse peritoneal administration of chitosan, and the degree
of deacetylation for maximum secretion of cytokines was 70% [5]. As
shown in Table 8, colony stimulating factor secretion was positive both in
vivo and in vitro, although the degree of secretion was not as much as that
by B cell growth cell wall skeleton (BCG-CWS), a strong immunoadjuvant.
But in the case of interferon and interleukin-l, secretions were positive
f.}:ry
~
V-/ thigh bone

murine
rinse with
PBS
~~o
o(
0,
~
V-n centrifugation
(1,200 rpm, 5 min)
37' C, 5% CO 2
t = 0, 4, 8, 18,24,
50,96hr.
I) centrifugation
2) washed with PBS
(twice)
V- FITC-CM-chitin
incubation with
Microscopic observation
Scheme 2. Scheme for the incubation ofbone marrow cells with FlTC-CM-chitin and microscopic observation.
Figure 4. Staining of mouse bone marrow cells at implantation stage with FITC probe. Left
side, bright field. Right side, fluorescent probe picture [4]. Top photographs were taken 18 h,
bottom photographs 98 h after incubation.
288
S. Tokura et al.
Table 8. Effect of DAC-70 on the induction of colony-stimulating factor (CSF) in vivo [5]
CSF activity
Adjuvant a
Dose
()1g)
Timing
(h)
[3H] Thymidine
incorporation b
(mean counts per
minute s.e.)
DAC-70
200
BCG-CWS
LPS (Salmonella)
Control
200
100
-3
-6
-12
-24
-6
-3
2558
6232
6885
3721
4383
48319
2460
262 (1.0)d
450 (2.5)
422 (2.8)
506 (1.59
374 (1.8)
2754 (19.6)
116 (1.0)
Colony formation C
(colonies/10 5 bone
marrow cells)
12.3 0.9 (1.4)d
81 4.7 (9.0)
69.7 10.3 (7.7)
3.7 1.7 (004)
78 3.5 (8.7)
69.5 1.5 (7.7)
9.0 1.5 (1.0)
Mice were injected intraperitoneally with each adjuvant at indicated times before collecting
ofserum.
b Incorporation of [3H] thymidine by bone marrow cells (1 x 105 cells) was assayed with CSF
sampies (5%). The results were expressed as mean standard eITor of quadruplicate wells in
eachgroup.
C Bone marrow cells (1 x 105 cells ml- ') were cultured with 5% CSF sampies for 7 days. The
results were expressed as mean colonies from 105 bone marrow cells standard eITor of
triplicate cultures in each group.
d Values in parentheses are stimulation ratios as calculated with control.
a
only in vivo (Table 9). Tumor necrosis factor (TNF) secretion was shown
both in vivo and in vitro, similar to lymphokines. Antiinfective activity was
also induced by the administration of chitosan against Sendai virus and
Escherichia coU as shown in Figure 5 [1]. These observations highlight the
immunoadjuvant properties of chitosan, which are almost similar to those
oflentinan, a polysaccharide from the mushroomLentinus edodes. Chitosan
microspheres were also applied to define the influence of the surface of
solid chitosan, and the level of immunoadjuvant activity was found to be
comparable to that of chitosan in liquid form [6].
Since chitosan flocculates upon adsorption of heavy metals or organic
materials in water, chitosan administered as a solution may precipitate onto
the surface of organs in vivo. Results of immunochemical studies on
chitosan and CM-chitin are summarized in Table 10 [25]. The proposed
immunological behaviour of chitosan is shown in Scheme 3.
Recent data indicate that chitin and chitosan induce effective complement activation and IL-8 production from fibroblasts when applied in a
wound. The alternative pathway complement activation by these polysaccharides seems to be similar to that promoted by zymosan. Interleukin
(lL-8) and C5a from the complement activation act on the polymorphonuclear cell migration to the wound site [26].
On the other hand, N-acetylchitohexaose, a water-soluble chitin oligomer,
was reported as an immunopotentiator by Suzuki et al. [32]. They found
inhibition of growth oftumour cells or pathogenic microbes in mice upon
289
Table 9. Comparison of cytokine production by DAC-70 and other adjuvants in mice [5]
Cytokine
Monokine
CSF (in vivo)
(in vitra)
INF (in vivo)
(in vitra)
IL-l (in vitra)
TNF (priming)
(eliciting)
Lymphokine
CSF (in vitra)
INF (in vitra)
IL-2 (in vitra)
MAF (in vitro)
DAC-70
LPS
MVE-2
BCG-CWS
MDP
+
+
+
+
+ (18, 25)
+
+ (26)
+ (11,12)
+ (33)"
- (33)
+
+ (7)
+ (10)
+ (12)
+(13)
+ (12)
+ (13)
+
+ (32)
+
+
" Nurnbers in parentheses are reference numbers.
Table 10. Biological activity terms ofDAC-70, CM-chitin, BCG-CWS and MDP [25]
Terms
DAC-70
CM-chitin
BCG-CWS
MDP
Adjuvant activities
(a) Macrophage activations
Cytolysis (in vivo)
Cytostasis (in vivo)
H2 0 2 release (in vivo)
(in vitra)
+
+
+
+
+
+
+
+
(b) Delayed type hypersensitivity

(ABA-N-acetyltyrosine)
(c) Antibody production

Bacterial amylase (in vivo)
Dinitrophenyl hapten (in vivo)
+
+
(d) Cytokine production

Interleukin-l (in vitro)
Interleukin-2 (in vitro)
Colony stimulating factor (in vivo)
Macrophage activating factor (in vitro)
Tumor necrosis factor (in vivo)
Interferon (in vivo)
(in vitro)
Mitogenic activity (in vitra)
Antitumor activity
Suppression ofMeth-A
Antiinfectious activity
E. eoli
Sendai virus
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
BCG-CWS, BCG cell wall skeleton; MDp, Muramyldipeptide (N-acetylmuramyl-L-alanyl-Disoglutamine ); ABA; azobenzenearsonate.
cn
::J
.~ 50
Cij
4.4X10 6 cfu
infection (s.c.)
246
Time after infection (days)
-,fr- _.. - 11 _111
..
6, \''ts.- -./: ;...
:".......
8
(i.p.)500jlQ
PS injection
- 5 day
!
~
.0
4.4X106 cfu
infection (s.c.)
E. coli
- 1 day
(s.c.) 100jlQ
MDP-Lys(LlB)
cn
:::J
.~ 50
ctl
;e
o
100
- - -0 - - -0 - . -0 - . 0
Time after infection (days)
b- .. 0
v---v---v
Figure 5. Prospective activities of chitin derivatives and MDP-Lys(L-18) in mice infected subcutaneously (s.c.) with E. coli [I]. Each mouse was treated intraperitoneally (i. p.) with chitin (6), 70% DA-chitin (A.), CM-chitin (.), and DHP-chitin (0) at a dose of 500 mg per mouse or PBS alone (0) five days before
or s.c. with MDP-Lys (LI8) (e) at a dose of 100 Jlg per mouse I day before infection with 3.7 x 10 6 cfu E. coli (a). Similarly, each mouse was treated i.p.
with 500 mg of30% DA-chitin (\7), 70% DA-chitin (A.), chitosan (T), CM-chitosan (.), DHP-chitosan (0) and PBS (0) at 5 days before or s. c. with 100 Jlg
ofMDP-Lys (LI8) (e) one day before infection with 4.4 x 10 6 . cfu E. coli (b). * Statistically different from contro! by X 2 method; p < 0.05.
(i.p. )500I-lQ
E. coli
- 1 day
(s.c.) 100jlQ
MDP-Lys(LlB)
PS injection
- 5 day
;g
o
100
t--J
\0
$P.
'"
Cl
291
Ag
Scheme 3. Proposed pathway of immunoresponse following administration of chitosan.
intraperitoneal injection ofN-acety1chitohexaose due to activation processes ofphagocytes and the immune system [27-31]. Growth inhibition of
tumour cells such as Sarcoma 180, MM 46, Meth A and 1ung carcinoma
solid tumours was observed. They also treated macrophage with N-acetylchitohexaose to produce IL-1 in vitro, but IL-2 secretion was not observed
from spleen T cells. They thus concluded that the main target ofN-acetylchitohexaose was macrophages when N-acety1chitohexaose was administered
intravenously and secretion of macrophage activating factor (MAF) was
accelerated from T cells to suppress microbial growth [32].
Upon intravenous or intraperitoneal injection of N-acety1chitohexaose,
acute influence is expected on the activation of the immune system due to
the fluidity of low molecular weight N-acetyl chitohexaose. However,
much lower concentations of oligomers are supposed to be supplied to the
immune system from chitosan or chitin than in the case ofN-acetylchitohexaose, due to the slow rate ofhydrolysis in animal bodies.
References
1 Nishimura K, Nishimura S-I, Nishi N, Saiki I, Tokura S, Azuma 1 (1984) Vaccine 2: 93-99
2 Nishimura K, Nishimura S-I, Nishi N, Murata F, Tone Y, Tokura S, Azuma 1 (1985) Vaccine
3: 379-384
3 Tokura S, Hasegawa 0, Nishimura S-I, Nishi N, Takatori T (1987) Analytical Biochem 161:
117-122
292
S. Tokura et al.
4 Tokura S, Matsubara N, Nishi N; inpreparation

5 Nishimura K, Ishihara C, Ukei S, Tokura S, Azuma I (1986) Vaccine 4: 151-156
6 Nishimura K, Nishimura S-I, Seo H, Nishi N, Tokura S, Azuma 1(1986) J Biomed Mater
Res 20: 1359-1372
7 HackmanRH(1958)AustJBioISci7: 168
8 Hayashi K, Imoto T, Funatsu M (1963) J Biochem 54: 381
9 Tokura S, Nishi N, TsutsumiA, Somorin 0 (1983) Polym J15: 485
10 Nishi N, Noguchi J, Tokura S, Shiota H (1979) Polym J 11: 27
11 Nishi N, Nishimura S-I, Ebina A, Tsutsumi A, Tokura S (1984) Int J Biol Macromol6: 53
12 Wolform ML, Shan TM, Summers CG (1953) J Am Chem Soc 54: 1519
13 Matumoto K, Otani T, Une T, Osaka Y, Ogawa H, Azuma I (1983) Infect Immun 39: 1029
14 Tabachnick M, Sobotka H (1959) J Biol Chem 238: 1726
15 Eisen HN, BeIman S, Carsten ME (1953) J Am Chem Soc 75: 4583
16 Schiffinan G, Kabat EA, Thompson W (1964) Biochemistry 3: 113
17 Azuma I, Yamawaki M, Yoshimoto T, Saiki I, Umemiya M, Tanio Y, Tokuzen R, Yasumoto
K, Yamamura Y (1975) Gann 70: 737
18 Saiki I, Kamisango K, Tonio y, Okumura H, Yamamura Y, Azuma 1(1982) Infect Immun 38: 58
19 Saiki I, Tokushima Y, Nishimura K, Yamamura Y, Azuma I (1983) Infect Immun 40: 622
20 Pick E, Mizel D (1981) J Immunol Methods 46: 211
21 Lowry OH, Rosebrough HJ, Farr AL, Randali RJ (1951) J Biol Chem 193: 265
22 Okada Y, Onoue K, Nakashima S, Yamamura Y (1963) J Biochem (Tokyo) 54: 477
23 Engval E (1980) Methods Enzymol70: 419
24 Brunner KT, Nauel J, RudolfH, Capuis B (1970) Immunology 18: 501
25 Tokura S, Nishi N, Azuma I (1987) In: MYalpani (ed): Industrial polysaccharides: genetic
engineering, structure/property relations and applications. Elsevier Science Publishers,
London and NewYork, 347
26 Okamoto Y, Minami S, Matsuhashi H, Fujinaga T (1994) Pro-Vet 70: 32
27 Suzuki S, Suzuki K, Tokoro A, Okawa Y, Suzuki M (1986) In: RAA Muzzarelli, C Jeuniaux,
GW Gooday (eds): Chitin in nature and technolgy. Plenum, NewYork and London, 485
28 Tsukada K, Tatewaki M, Aizawa K, Tokoro A, Naruse R, Suzuki S, Suzuki M (1990) Jpn J
Cancer Res 81: 259
29 Tokoro A, Tatewaki N, Suzuki K, Mikami T, Suzuki S, Suzuki M (1988) Chem Pharm Bult
36: 784
30 Tokoro A, Kobayashi M, Tatewaki N, Suzuki K, Okawa Y, Mikami T, Suzuki S, Suzuki M
(1990) Microbiol Immunol33: 357

31 Kobayashi M, Watanabe T, Suzuki S, Suzuki M (1990) Microbiol Immunol34: 413
32 Suzuki S, Watanabe T, Mikani T, Matsumoto T, Suzuki M (1991). In: CJ Brine, PA Sandfonl, JP Zikakis (eds): Advances in chitin and chitosan. Elsevier Applied Science, London
and New York, p. 96

ed. by ~ Jolles and R.A.A. Muzzarelli
Clinical and biochemical evaluation of chitosan

for hypercholesterolemia and overweight control
Center for Innovative Biomaterials, University ofAncona, Via Ranieri 67,
I-60JOOAncona, Italy
Summary. After providing basic information on enzymes involved in cholesterol homeostasis,
and on management of hypertriglyceremia and hypercholesterolemia, with the aid of
cholestyramine and fibric acid, this chapter examines the effects of the ingestion of chitosan.
Dietary chitosan is effective on serum cholesterol and in atherosclerosis in normal and diabetic
mice, and lends itself to the treatment of hypercholesterolemia in humans. It also exhibits
antiuicer, antiarthritic, antihypertension and antiuricemic properties. The published human
trials, analysed statistically, further indicate that chitosan is effective to control overweight when
associated to a diet. This chapter discusses several issues raised against the use of chitosan,
namely, depletion of zinc and liposoluble vitamins, as well as advantages such as enhanced
absorption of nutrients and competitive inhibition of lipases. It also directs attention to the
unexplored areas of fungal and algal chitosans, and the use of chitins instead of chitosans.
Introduction
Cholesterol, the most abundant sterol in the mammalian cell, is required for
normal cell growth and proper membrane structure and function. The brain
contains 10% of cholesterol (dry weight), most of which is incorporated
into mye1in. The liver is the key organ in the maintenance of cholesterol
homeostasis in the body. Adrenals, gonads and placenta transform cholesterol into hormones [1-2].
The most important elimination pathway for water-insoluble cholesterol
is the conversion to water-soluble bile acids. The flux of cholesterol and
bile acis regulates the activity of the following enzymes: cholesterol: acyl
coenzyme A transferase (ACAT); cholesterol 7a-hydroxylase (a monooxygenase located in the endoplasmic reticulum); 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R). Bile acids are efficient
suppressors ofthe HMG-CoA-R in vivo.
Unregulated accumulation of cholesterol is cytotoxic, and a failure to
maintain homeostasis ofthe sterol results in a number ofpathologies, such
as gallstone disease, atherosclerosis, corneal crystalline dystrophy and
tumour proliferation [3]. There is therefore much interest in keeping the
cholesterollevel under control and in limiting its ingestion. Of course, the
body reacts to interventions intended to lower the cholesterol concentration: the effect of cationic resins on cholesterol degradation is counteracted
by a compensatory increase in cholesterol synthesis. For instance, biliary
294
R.A.A. MuzzareJli
,,
Acetate
HMG-CoA
HMG-CoA ,.d",""
Mevalonate '.
+
+ ....."? ....:..
'. ?
Cholesterol'
Cholesterol
7a-hydr oxy 1 ase
7a-OH-cholesterol
+
+
Bile Acids
Figure 1. Regulation of cholesterol 7a-hydroxylase and HMG-CoA reductase at different
cholesterol precursors, cholesterol, and bile acids. (+) activation; (-) deactivation.
diversion or administration of bile acid-binding cationic resins leads to

severalfold stimulation of both bile acid biosynthesis and cholesterol 7 ahydroxylase activity (Fig. 1).
The hydrolysis of chitosan in the digestive tract appears to be of utmost
importance because (i) the current definition of dietary fiber indicates that
fibers should not be hydrolyzed by the digestive enzymes; (ii) the more or
less acetylated chitosans release N-acetylglucosamine and glucosamine,
which is recognized as a cytoprotective agent and a treatment for lower
gastrointestinal tract disorder [4]; (iii) the general behaviour of chitosan
might be partially reconducted to the behaviour ofits oligomers [5].
In this chapter, the effects of the ingestion of chitosan will be examined,
and several relevant topics will be discussed.
Management of hypertriglyceremia and hypercholesterolemia
Cholestyramine, cholestipol and other remedies

Frequently applied drugs are bile acid sequestrants and inhibitors ofHMGCoA-R, a rate-limiting enzyme catalyzing the synthesis of mevalonate, a
Clinical and biochemical evaluation of chitosan for hypercholesterolemia ...
295
precursor of all isoprenyl compounds inc1uding ubiquinones, heme A,

dolichols and isoprenylated proteins.
Cholestyramine and cholestipol are synthetic ion-exchange resins approved for hyperlipidemia treatment, but not indicated in the management
oflow HDL cholesterol. These bile acid sequestrants lower LDL cholesterol 15-30% but raise HDL cholesterol only 3-5% and may raise triglyceride levels. Cholestyramine sequesters bile salts in rats and alters
intestinal morphology in humans. Furthermore, there is a significant increase in rat intestinal tumour induction by various agents when
cholestyramine is added to the diet. DEAE-Dextran was also found effective in reducing intestinal fat absorption and speeding up bile turnover [6].
Nicotinic acid and derivatives reduce plasma levels of triglycerides and
cholesterol and increase the plasma concentration of HDL cholesterol.
Adverse effects are cutaneous flushing, nausea, vomiting and other gastrointestinal disturbances besides cholestatic jaundice. They are not approved
by the U. S. Food and Drug Administration (FDA).
The fibric acid derivatives reduce plasma cholesterol by 6-11 % and
triglycerides by 22-43% [3]. Their mode of action is complex and not fully established: they reduce cholesterol synthesis by suppressing HMGCoA-R activity and enhance LDL c1earance. Fibrates may be prescribed in
combination with nicotinic acid or bile acid sequestrants, or inhibitors of
HMG-CoA-R. They are generaIly weIl tolerated. HMG-CoA-R inhibitors
inc1ude lovastatin, fluvastatin, provastatin and simvastatin, and are the most
commonly prescribed c1ass oflipid-Iowering agents. They are competitive
inhibitors of the key regulatory enzyme of cholesterol biosynthesis. They
mayaiso reduce absorption of dietary cholesterol by reducing the activity
of acylcholesterol acyltransferase in erythrocytes. Reductase inhibitors are
effective in lowering LDL cholesterol [7].
Hypocholesterolemic action 0/ chitosan on animals

The ingested chitosan salts react with fatty acids and bind additional lipids
due to hydrophobic interactions (triglycrides, fatty and bile acids, cholesterol and other sterols), and a great portion of these bound lipids are excreted rather than absorbed. Bound triglycerides would escape hydro lysis
by lipase, promoting the excretion of fatty materials, inc1uding cholesterol,
sterols and triglycerides [8]. Studies done on various animal models have
been recently reviewed [9, 10].
Dietary chitosan is effective on serum cholesterol and atherosclerosis in
the hypercholesterolemic, apo lipoprotein E-deficient mouse, which deve10ps
high levels of blood cholesterol and atherosc1erosis without the need for
dietary intervention. Serum cholesterol was reduced by 52% in the
chitosan-fed animals foIlowing 20 weeks of treatment, and the area of
plaque in the aortic arch of treated animals was 50% less than that of the
296
R. A. A. Muzzarelli
controls. Interestingly, growth in the chitosan-fed animals was significantly enhanced, whereas in controls it was retarded. The overall weight gain of
the chitosan-fed mice was increased by 65% [11].
When chitin or chitosan in nylon net bags were administered orally to
dogs, the chitin did not undergo changes in weight and shape, but chitosan
was degraded in the stomach and large intestine. Chitosan was released
from the net into the gastrointestinal tract following the change to a gel in
the stomach.
When a chitosan bag was surgically placed in the large intestine for 24 h,
about 26% weight loss of the chitosan was observed in the presence of
feces. Bacterial flora seemed to influence the weight of chitosan. Total
cholesterol in the plasma significantly decreased when chitosan was administered orally for 2 weeks. The plasma total cholesterollevel decreased
to 77% on day 7 and subsequently to 54% on day 14, and then recovered at
the initial level on day 28. Chitin and cellulose had no effect [12].
The hyperglycemic and hypolipidemic effects of chitosan were studied
in normal and diabetic mice, the latter including obese ones with hyperinsulinemia (KK-Ay) and lean ones with hypoinsulinemia (neonatal
streptozocin-induced diabetic mice, NSZ) [13]. In no case was the body
weight altered after 4 weeks of diet at 5% chitosan.
Chitosan-treated normal mice had significantly decreased blood glucose,
cholesterol and triglycerides. In NSZ mice, significant hypoglycemic and
hypocholesterolemic effects were also observed after chitosan administration. No effect was observed in KK-Ay mice. Furthermore, chitosan improved lipid metabolism, indicating that it is useful for diabetic complications, because diabetic subjects have elevated blood cholesterol and
triglyceride levels caused by metabolic derangements. Chitosan was therefore proposed for treatment of non-insulin-dependent diabetes mellitus.
Management 0/ hypercholesterolemia in humans
There is general agreement on the fact that orally administered chitosan

lowers blood pressure and cholesterol in volunteers. In general, chitosan
administration is associated with a diet.
Chitosan exhibits anticholesterolemic, antiulcer, antiarthritic and antiuricemic properties. These properties are related to the capacity to bind fatty
acids, bile acids with consequent reduction of their enterohepatic recycling,
phospholipids and uric acid. In the presence of fatty acids, chitosan is able
to form the corresponding complex salts that bind triglycerides, fatty and
bile acids, cholesterol and other sterols, and a great portion of these bound
lipids are excreted [14-18]. The fecal loss of bile constituents demands
increased hepatic transformation of cholesterol into bile acids.
The existing in vitro data on the binding of fats to chitosan are of little
use for those interested in the reactions taking place in the stomach and
297
duodenum due to the biochemical complexity of the digestive tract.

Attempts at mimicking the in vivo situation were based on the following
approach: chitosan (0.5 g) was preincubated with 10 mM HCl (2 h, 37C);
then corn oil, taurocholic acid and lipase were added. Results were not
reproducible, and the hydrolytic action of lipase on chitosan was observed
in certain cases, thus no conclusion could be drawn [19]. Nevertheless, the
amount of corn oil bound to chitosan is currently taken by the producers as
evidence of the efficacy of the product, this parameter ranging from 2.81
to 10.8 ml/g (wheat flour control1.27-3.22).
Similarly, data have been produced on the absorption of cholesterol and
bile acids on various chitosan-based products [20-23]: again, these data
are hardly useful for understanding the mechanism of action due to methodologicallimitations (for instance, particle size and the identity ofthe other
ingredients were not specified). In general, it can be said that chitosan,
compared with cholestyramine, binds larger amounts of cholesterol and
smaller amounts of bile acids.
On the other hand, fat assimilation was reduced by 13-25% by chitosan
(glycerol tri[I-14C]0Ieate test), and serum cholesterol and triglycerides
were reduced in volunteers from 7.4 to 5.2 mmol/l and 1.6 to 1.3 mmol/l,
respectively, over a 5-week period. Weight reduction of 6.9 kg (2.5 kg in the
placebo group) and blood pressure reductions of 8.6 (vs. 3.9 placebo) and
19.6 (vs. 11.5 placebo) for diastolic and systolic values, respectively, were
also reported in a randomized double-blind study (0.96 g chitosan each
meal, 1000 kcal/day diet).
The most recent study has involved 1000 volunteers with body mass
index (kg' m-2) (BMI) over 25, for 12 weeks. In the course ofthis study, it
was realized that some subjects did not respond to the chitosan treatment,
but responders reduced their weight significantly [19-22].
The results on humans show a favourable effect with a very low dose
within a short period. Chitosan was administered to adult males in the form
ofbiscuit over a study period of 4 weeks [23]. When chitosan was given in
the diet (3-6 g/day), the total serum cholesterol level significantly decreased, whereas the serm HDL-cholesterol level significantly increased
when compared with the level for each of them before ingestion.
The chitosan intake (biscuits) by healthy volunteers was found to
produce a significant decrease ofthe fecal phenols,p-cresol and indole, in
analogy with other polysaccharides [24]. Chitosan inhibited the putrefactive activity of the intestinal microbiota, thus reducing the risk of
disease. The lecithinase negative clostridia were the only microbiota
significantly depressed by chitosan in humans after 2 weeks of chitosan
intake. The lipid-Iowering effect of chitosan in obese subjects was recently
assessed [25, 26].
Jing et al. [27] have shown the clinical importance of chitosan. Renal
failure patients undergoing hemodialysis were observed. After 12 weeks of
chitosan ingestion, the average total serum cholesterol level decreased
298
R.A.A. Muzzarelli
significantly to 5.822.19mM from 10.14 4.40 mM. A significant decrease in average serum lipoprotein level was also observed after 4 weeks
of chitosan ingestion. Practically no change was observed in the serum
HDL levels.
While the clinical trials with chitosan were directed to the abatement of
cholesterollevels, later on it appeared that chitosan could be used as a diet
integrator for the more general purpose of overweight control.
For obese patients, orally administered chitosan leads to overweight
reduction after a few weeks oftreatment. For example, according to Lassus
and Abelin [28], weight reduction is 4.4 kg better than that for placebo
groups. Similar data were obtained by various authors, among which
Veneroni et al. [29-30] and Ventura [31]. Hirano [32] has surveyed the
many authorized applications of chitosan in the food area in Japan. The
published human trials, when analyzed statistically, indicate that chitosan
groups showed greater weight loss than placebo groups.
The prescribed dose of chitosan is much lower than that used in animal
tests, and chitosan is recognized as a safe compound, being nontoxic and
deprived of activity on certain human enzymes involved in cholesterol
synthesis, as a point of difference from certain drugs. Immunopotentiating
and anticancerlantimetastatic actions have also been documented. Therefore, there is no risk of overdose, no side effects, no stimulant action:
chitosan is not a medicine, is easy for the overweight to use and is a nonaddictive substance.
Discussion
Chitosan is presently on the market as a food additive or dietary integrator
in several countries among which Japan, England, Italy and Portugal.
However, chitosan has met with difficulties and criticism by official organisms in several countries. In some cases the scarce correspondence between the characteristics of chitosan and the existing regulations has represented an obstacle; in other cases certain hypothetical problems have been
raised. This section is abrief survey of the topics debated.
Liposoluble vitamins
It has been remarked that chitosan could deprive the diet of liposoluble
vitamins [33] that might be incorporated in the chitosan-fat aggregates.
Nevertheless, some ofthe clinical reports cited above state that vitamin E
level was not depressed. Vitamins could be prescribed to be taken at different hours by patients. Furda [34] has indicated the value of the association of chitosan with vitamins for clinical purposes.
299
Trace metal ions

The chelating ability of chitosan could represent a worry in tenns of
depletion of iron. Jennings et al. showed that chitosan in rats does not reduce
serum iron or hemoglobin [35]. This subject was also addressed by other
authors with similar conclusions [36]. A more substantial rmding came from
research on mucoadhesive polymers that modulate the physiological barriers
enhancing peptide drug absorption by reducing the metabolie activity of
luminal and membrane-bound peptidase and proteases as weIl as by opening
the intestinal intracellular junctions [37]. Luessen et al. [38] evaluated the
potential of chitosan to inhibit the intestinal proteases trypsin and carboxypeptidase B, which degrade peptide drugs, and to improve the intestinal
transport of peptide drugs in a perfused intestinal model. Similar to trypsin,
carboxypeptidase B was only inhibited by polycarbophyl but not by chitosan
and a chitosan salt. Carboxypeptidase B is a Zn-containing metal enzyme,
and its inhibition by the poly(acrylates) is due to their chelating properties for
Zn and consequent depletion ofZn from the active site [38].
The substantial difIerence between the two biopolymers is that
poly(acrylate) acts as a protease inhibitor, whilst chitosan improves the
intestinal transport by increasing the paracellular penneability of the
intestinal epithelium. What is even more significant in these findings is that
the chitosan-chelating activity is not large enough to deplete the organism
of certain trace metal ions, which in any case occur as complexes, and
therefore chitosan administration per os should be regarded as safe from
this standpoint. Interestingly, the poly(acrylates) are FDA-approved, in
spite of their sequestering activity for Zn and metalloenzyme inactivation.
Moreover, most of the metal ions present in foods are complexed by
chelating agents more powerful than chitosan such as citric acid.
Administration of chitin or partially acid-degraded chitin in mice per os
resulted in anti-Candida albicans growth factor, which could be identified
to be a known factor, transferrin. In order to induce the same factor in large
amounts in the blood of mice, chitin was administered intraperitoneally to
young mice. It was confinned that the anti-Co albicans growth factor had
been induced in the blood of mice. Induction of apo-transferrin took place
in mouse blood, and this iron-binding protein displayed as a C. albicans
growth inhibitor. This result is indirect evidence that iron is not depleted as
a consequence of chitosan administration [39].
Lipases and other hydrolases

Pancreatic lipolytic activity originates from lipase and its cofactor colipase,
carboxyl ester lipase and phospholipase A2. Chitosan may act indirectly on
carboxyl ester lipase (a bile salt stimulated lipase) by depressing the available bile salts [40].
300
R. A. A. Muzzarelli
If chitosan is a substrate for human lipase in vivo (as it is in vitro), it

might be possible that the amount of chitosan introduced with the diet
would in part prevent lipases from hydrolyzing the lipids. It is well known
that the 2-monoglycerides and fatty acids are the partial hydro lysis products, yielded by pancreatic lipase, suitable for absorption. Studies on the
lipase-chitosan system inelude wheat germ lipase [41], porcine pancreatic
lipase and microbiallipase [42-43].
Le Houx and Grondin [44] studied the consequences of chitosan administration on 3-hydroxy-3-methylglutaryl CoA reductase in rats fed a
sterol diet. The enzyme levels remained elose to the normal value with a
7.5% chitosan formula, and plasma HDL cholesterol did not decrease. On
the other hand, the enzyme activity was overstimulated in the sterol +
cholestyramine group, where livers were smaller and yellowish. By this
means the advantages of chitosan over cholestyramine have been elearly
focused, but the nexus between chitosan and the enzyme has not been
elucidated.
Chitosan provides a sustained-release form of glucosamine. Oral ingestion (1 g/day) ofN-acetylglucosamine (NAG) or chitosan increase the
serum concentration of NAG, which is, however, eleared rapidly: in the
case of chitosan the serum NAG concentration, 48 h after ingestion, remains interestingly high [45]. Blood glucose levels are not altered. These
results confirm those about the efficacy of orally administered chitosan in
the treatment ofpatients with osteoarthritis [46].
Experimental evidence from various laboratories indicates that orally
administered chitosan is partially digested and absorbed. This is particularly due to the action of hydrochloric acid in the stomach and the
unspecific activities of enzymes present in saliva and gastric juice. In the
large intestine, other enzymes from microorganisms further digest chitosan.
Absorption 0/ nutrients
Because of the increasing interest in targeting peptides and protein drugs to

the colon, where proteolytic activities are lower than in other parts of the
digestive system, chitosan capsules were developed. For instance, a sealed
chitosan capsule containing insulin was coated with hydroxypropyl methyl
cellulose phthalate: this enteric coating prevents dissolution of chitosan in
the stomach, but dissolves in the small intestine, so that the chitosan capsule dissolves in the large intestine thus releasing its content. This mechanism was demonstrated with the release of insulin and the determination of
its pharmacological availability, with the detection of 6-carboxyfluorescein-vitamin B6 and salicylamide, and with a radiological survey of a
BaS04 fi1led capsule.
Chitosan enhances the absorption of macromolecules across the intestinal epithelia, an important property for the oral administration of
301
proteins and peptides. For instance, a pH-sensitive multicore microparticulate system, consisting of mucoadhesive chitosan microcores entrapped in
the enteric acrylic polymer Eudragit was developed [47]. The antiinflammatory drug sodium dic10fenac was tested after pre1iminary work with
fluorescein isothiocyanate-labeled bovine serum albumin. The micropartic1es escape dissolution in the stornach and reach the intestinal region,
where the acrylic coating dissolves. The naked chitosan microcores then
adhere to the intestinal mucosa, releasing the entrapped compound. The
release of the drug is completed as soon as chitosan is under the degradative action of colonic bacteria. These studies indicate that chitosan does not
depress absorption ofnutrients, consequent to its adhesion to the intestinal
surface.
Algal and fungal chitosans
The extracellular fibers of Cyclotella cryptica, Thalassiosira mentagrophytes and Thalassiosira fluviatilis are composed of pure chitin (15 % dry
weight) [48-49]. The cultivation ofthese algae has been proposed for the
production ofhigh-quality chitin [50]. The Poteriochramonas alga deposits
its chitin fibers on the cell surface [51-52].
The cell wall of certain fungi has been considered as an alternative to
crustacean shells. There were several advantages of using these fungi to
produce chitosan. The most important is that the cell wall of some of them
[53] contains a large quantity of chitosan whose physicochemical properties can be controlled by acting upon the fermentation parameters. The
extraction process is simple and produces litde waste [54].
The molecular weights of fungal chitosan show values in the range
100-450 kDa. A chitinous material produced from higher Basidiomycetes
is used orally as a sanitation and preventive remedy for c1inical treatment
of liver and kidney insufficiency, hepatic cirrhosis and cancer. Clinical
analysis indicated a significant fall of the intoxication level in the patients
[55]. Aged patients had noticeable improvement of general conditions and
working capacity, and lower rates of disease.
While algal chitins/chitosans have not been used c1inically so far, they
are important because botanists assimilate algae to plants [56]. It can then
be said that chitin is ofplant origin, and this would help in inc1uding chitin
in the definition of dietary fiber. On the other hand, fungal chitins/
chitosans provide the same advantages as animal chitinlchitosan.
Why not chitin?
Herrera and Mata-Segreda [57] have reported that chitin binds more
cholate than chitosan. Work in progress confirms these in vitra data. The
302
R.A.A. Muzzarelli
use of chitin would help in obviating criticism related to the chemical manipulation for chitosan production. Of course, chitin should be amorphous
for oral administration.
Conclusions
The biological significance of chitosan in the human body depends on the
actions that certain human hydrolases exert on it [58]. Research carried out
during the last few years has promoted chitins and chitosans to the forefront
of applied activities intended to offer genuinely valuable medical items in
the field of general medication, wound healing, plastic surgery, drug carriers, dietary supplements and immunostimulants.
In particular, chitosan appears to be a most effective and tolerable biopolymer suitable for the management of hypercholesterolemia and overweight.
Acknowledgements
The assistance of Maria Wecla in retrieving the bibliographic material and preparing the
typescript is gratefully acknowledged. This work was performed with the financial contribution
ofMURST.
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ed. by R Jolles and R.A.A. Muzzarelli
Microparticulate drug delivery systems

Ida Genta 1, Paola Perugini 1, Franca Pavanetto 1, Tiziana Modenal,
Bice Conti 1 and Riccardo A. A. Muzzarelli 2
1 Department
2
01 Pharmaceutical Chemistry, University 01 Pavia, Viale Taramelli 12,

1-271 00 Pavia, Italy
Center lor Innovative Biomaterials, University 01Ancona, Faculty 01 Medicine,
Via Ranieri 67, 1-60100 Ancona, Italy
Summary. Chitosan was proposed as a drug carrier for mucosal administration in ocular, buccal, nasal, gastroenteric and vaginal-uterine therapies based on its bioadhesive properties and
biodegradability in vivo under the action ofhydrolases. Examples are the delivery of acyclovir
via ocular administration, and the delivery of 5-aminosalicylic acid to the colon. Microparticles
may need to be cross-linked to retard their degradation in acidic media; yet cross-linking with
glutaraldehyde introduces cytotoxic characteristics and depresses bioadhesion. Alternative
cross-linking approaches are discussesd along with the suitability of chitosan for the oral
delivery of vaccines.
Introduction
In the pharmaceutical field the use of chitosan offers many advantages as

an excipient for increasing the dissolution rate of poorly soluble drugs, as
an auxiliary substance in direct tableting, as a binder and lubricant in wetgranulated tablets and as a stabilizing agent in emulsions [1- 3]. This biopolymer and its derivatives are employed in the preparation of modified
drug delivery systems such as implants, granules, pellets, xerogels and
micro- and nanoparticles [4].
Particulate carriers have important potential applications for the loading
and releasing of therapeutic molecules for oral mucosal and parenteral
administration. The term "microspheres", or "microparticles", is used to
describe particulate delivery systems, irrespective of size, chemical composition and manufacturing technique. Depending on process parameters
and physicochemical properties of the drug, either "true microcapsules"
are obtained, containing a drug core surrounded by a continuous coat of
polymer, or microspheres that contain the drug dispersed or dissolved in a
polymerie matrix. The distribution of the drug in the carrier system has
profound consequenees on the release and degradation properties of microparticles. The term "microcapsule" indicates reservoir type devices, whereas "microspheres" are monolithic or matrix-type microparticles [5].
The main drug classes successfully encapsulated into chitosan microparticles are antiinflammatory, chemotherapeutic, antibiotic and anticaneer
drugs [6]. Recently chitosan and its derivatives have been studied for their
306
I. Genta et al.
potential mucoadhesive properties; this characteristic is important for the

formulation of targeted particulate drug carriers and for the administration
of protein drugs.
There is increasing interest in the development of safe, efficient and
reliable mucosal delivery systems expecially for poorly absorbable drugs
such as peptides and proteins. Recent studies prove that chitosan has mueoadhesive properties and enhances the penetration of macromolecules
across the intestinal and nasal barrier [7 -1 0], and open new perspectives in
formulating bioadhesive dosage forms for mucosal administration (ocular,
nasal, buccal, gastroenteric and vaginal-uterine therapy).
Bioadhesive microspheres spread on a large area of mucosa and decrease
the rate of c1earanee ofthe drug from the mucosa, thereby allowing a longer
contact time with the absorptive epithelium. Chitosan's mucoadhesive
properties seem to be mediated by ionic interaetion between sialie acid
residues in mucus and the polymer amino groups [8]. Microspheres promote
a transient widening of the tight junction between Caco-2 cells, thereby
allowing larger moleeules to pass through the membrane [11]. Chitosan
microspheres applied to the nasal mucosal membranes promote nasal
peptide absorption into systemic circulation [12] and show no toxicity.
Genta et al. [13] prepared chitosan microspheres for the ophthalmie
administration of aeyc10vir for the treatment of localized or systemic
Herpes simplex infection. Results from "in vivo" ocular administration of
acyc1ovir-loaded chitosan microspheres to the rabbit eye showed prolonged
high coneentrations of acyc1ovir.
The nasal absorption of insulin in rat and sheep models with the aid of a
variety of chitosans has been performed according to a mechanism based
on bioadhesion and a transient widening of the tight junctions of the nasal
membranes; no membrane of cellular damage was reported. Mueociliary
transport rates in volunteers have been studied [12, 14].
Drug delivery to the colon

Colon-selective drug delivery systems have been the foeus of increasing
interest for the last decade. This is mainly due to the recently recognized
importance of this region of the gastrointestinal tract for systemic therapy.
At present, specific drug delivery to the colon is considered an important
alternative for the treatment of serious local diseases such as Crohn's
disease, ulcerative colitis, careinomas and infections. On the other hand,
specific systemic adsorption in the eolonie region offers interesting possibilities for the treatment of diseases susceptible to diurnal rhythm, such
as asthma, arthritis or inflammation [15]. Drug delivery to the various
regions of the gastrointestinal tract is based on the use of enteric polymers,
as protective drug coatings, able to release the drug at a particular pR. The
major drawback is that in contrast to what was believed in the past, the pR
307
of the proximal and transverse colon is more acidic than that in the small
intestine. Thus, the drug is rapidly released along the upper intestine before
reaching the colon. Despite this limitation, these enteric coating formulations are the only commercialized products for the treatment ofthe ulcerative colitis with 5-aminosalicylic acid (5-ASA). Timed release systems
have been also proposed for colonic drug delivery: they deliver drugs after
a particular time, which is the time normally required to reach the colon
(3-4 h). The only limitation associated with this approach is the enormous
variability in the gastric emptying of the dosage form depending on the
quantity and kind of food consumed.
A more realistic strategy for targeting drugs to the colon uses the ecosystem ofthe specific microflora in the large intestine. Bacterial hydrolases
are in sufficient quantity to be exploited in colonic drug targeting. Based
on this idea, chitosans have been evaluated for their susceptibility to c1eavage by these bacterial enzymes, expecially chitinases. Chitosans couple
their specific degradability in the colon with their good adhesiveness to the
gastrointestinal mucosa favouring the specific and persistent delivery of
the local drug. The only inconvenience of these polymers is their high
solubility in gastrointestinal fluids: this implies the need of cross-linking,
for instance with aldehydes, to preserve their integrity until they reach the
colon. Nevertheless, the toxicity of aldehydes enormously limits the
exploitation of these cross-linked microcapsules; furthermore, this crosslinking process is neither totally effective in preventing the early release of
the encapsulated drug nor does it maintain the mucoadhesive properties of
the polymer [7, 16, 17].
Lorenzo-Lamosa et al. [16] proposed a novel multiparticulate system based
on chitosan core microspheres coated with enteric polymers (Eudragit) to
overcome the problem due to the high solubility of chitosan in the gastric
cavity while avoiding chemical cross-linking with aldehydes. The probable
mechanism of drug release from these chitosan multicore microspheres at the
solubility pR ofthe coating polymer could be understood as follows (Fig.1):
Once the microspheres reach the small intestine, Eudragit slowly and continuously dissolves over time, thus leaving the chitosan microcores increasingly exposed to the release medium. The chitosan microcores, partially
cross-linked with Eudragit, swell upon contact with the basic release medium
and form a ge1 through which the drug diffuses. After 3 -4 h, the chitosan
microcores reach the colonic region where the chitosan undergoes a degradation process, thereby triggering the release of the entrapped drug. According
to this explanation, there are several factors which may affect the release ofthe
drug: (i) the pH-dependent solubility ofthe Eudragit coating; (ii) the size and
swelling behaviour of chitosan microcores; (iii) the core/coat ratio; (iv) the
microcore-coating interaction; (v) drug solubility and diffusion through the
chitosan gel; (vi) chitosan degradation in the colonic region [18]. Tozaki et al.
[19] studied the colon-specific delivery ofinsulin from chitosan capsules in
rats. They showed improvement of insulin absorption from the rat colon.
308
I. Genta et al.
1. Dissolution ofEudragit
2. Swelling of CS
Degradation ofCS
Gastrie
cavity
Small intestine

@@ @ @
@ @
@ @ @

@
Colon
Figure I. Scheme ofthe possible mechanism of drug release from the Eudragit microencapsulated chitosan (es) microcores.
In an in vitra study Genta et al. [17] showed that the bioadhesive characteristics of chitosan microspheres were depressed for glutaraldehyde crosslinked microspheres; scanning electron microscopy and transmission
electron microscopy observations ofmucin in contact with chitosan microspheres, uncross-linked or cross-linked with glutaraldehyde, have revealed
Figure 2. Scanning electron micrograph ofuncross-linked chitosan microspheres with mucin

in aqueous solution. In this picture it is possible to observe mucin adhering to microparticle
surface. Magnification 18000x.
310
I. Genta et al.
Bovine serum albumin (BSA) was loaded by passive absorption from

aqueous solution into preformed glutaraldehyde cross-linked chitosan
microspheres [23]. The study demonstrates the possibility of incorporating
biological macromolecules sensitive to organic solvents, pH, temperature
and ultrasound by a passive absorption technique into degradable biopolymer matrices by taking advantage of their swelling behaviour. It was also
shown that drugs passively adsorbed into such matrices are not necessarily
released completely in the initial burst, and a sustained release may be possible for macromolecules.
Remunan-Lopez et al. [24] used BSA as a model to prepare chitosan gel
micropartic1es for the controlled release of hydrophilie macromolecules.
Several formulation parameters (chitosan molecular weight, type of acid
and salt) were investigated with reference to encapsulation efficiency,
partic1e size, morphology and BSA-release.
Polk et al. [25] measured the release of BSA from chitosan-alginate
microcapsules and demonstrated that chitosan-alginate systems may be
used for the delayed release of a protein acid. Hari et al. [26] loaded insulin
into a chitosanlcalcium alginate system. Even though the load was low, the
results indicated the possibility of modifying the formulation to obtain the
desired controlled release of insulin for convenient oral administration.
Aydin and Akbuga [27] studied the release characteristics of salmon
calcitonin from chitosan beads. Chitosan presents favourable characteristics for gene delivery, such as the ability to condense DNA and form
small discrete particles in defined conditions [28, 29].
Hydrophilie chitosan nanoparticulate carriers have been proposed for the
delivery of a model protein drug [30]. The nanopartic1es incorporated polyethylene oxide in their structure for controlling the release of the loaded
protein.
Biodegradable particulate delivery systems may be useful in the field of
vaccine therapy. Currently used vaccines against viral infection consist of
parenterally administered inactivated virus, except the polio vaccine,
which is administered orally. Parenteral vaccinations need highly trained
personnel and sterilized equipment, which sometimes are limitations in
developing countries. Oral immunization is likely to offer advantages over
the parenteral route: no pain for the patient, no side effects - it would be
more acceptable to the majority of patients, and the logistics for mass
immunization would be simpler. In addition, oral immunization induces a
vigorous immune response in the mucosal surfaces of various organs, the
most common site of entry of infectious agents. Numerous experimental
systems have demonstrated the ability of oral immunization to induce antibody secretion into the mucous which bathes these surfaces [31]. These
mucosal antibodies are restricted almost entirely to the secretory form of
IgA (sIgA), an antibody type which is not effectively induced through conventional intramuscular or subcutaneous immunization. Oral administration of antigens can be effective in this respect because the small intestine
309
Figure 3. Scanning electron micrograph of cross-linked chitosan microspheres (5% glutaraldehyde) with mucin in aqueous solution: the smooth microsphere surface is deprived of any interaction with the protein. Magnification 5000 x.
that the protein was rejected from the microsphere surface (Fig. 2 and 3).
This indicates that glutaraldehyde, upon reaction with the amino groups
responsible for the chitosanlmucin interaction, reduces the affinity of
the polymer with mucin and depresses the mucoadhesive properties of
chitosan micropartic1es.
Protein delivery to other organs
A wide range of proteins such as vaccines, cytokines, enzymes, hormones

and growth factors are now commercially available in large quantity, but
several problems are associated with the therapeutic use of protein drugs.
Among them are the short in vivo half-lives and the side effects attributable
to the multiple and high-dose injections. An interesting approach to
maintain therapeutic levels is to deliver the proteins with the aid of biodegradable polymers [5, 20].
Over the past decade, various controlled release systems for peptide and
protein drugs have been extensively explored [21]. Among them, the most
promising delivery approach is the encapsulation of protein within injectable microspheres composed ofbiodegradable polymers [22]. For this aim
chitosans seem to be very useful because of their biomedical and hydrophilic properties, in addition to their biodegradability in vivo.
311
contains lymphoid aggregates, termed Peyer's patches, embedded along its

antimesenteric aspect. Peyer's patches are an organized lymphoid tissues
which contains functional T and B lymphocytes, the latter inc1uding a
significant fraction of cells committed to the synthesis of IgA c1ass antibodies. In addition, the Peyer's patches are separated from the lumen ofthe
gut by a layer of epithelial cells interspersed with a specialized type of
phagocytic cells that actively internalize sampies from the lumen contents,
and pass them to the underlying lymphoid cells. Here antigenic materials
trigger the c10nal expansion of specific B and T lymphocytes which then
extravasate through the lymphoid drainage. The IgA-committed B lymphocytes migrate through the mesenteric lymphonodes and into the blood
circulation so to lead to the immunization induction also in the glandular
tissues and the genitourinary and respiratory tracts [32].
Microencapsulation systems, in which antigens are incorporated into
liposomes or polymer microspheres, were tested as the most plausible
systems for oral vaccination. The micropartic1es were shown to be taken up
by Peyer's patches, where antigens were gradually released from the
particles to induce immune response [33].
Jameela et al. [23] loaded diphtheria toxoid by passive absorption into
cross-linked chitosan microspheres. Preliminary immunization studies carried out on rats using diphtheria toxoid-loaded chitosan spheres have
shown promise. Glutaraldehyde cross-linked chitosan spheres were well
tolerated by the living tissue and were not found to be degraded completely
in 6 months in vivo in rat musc1e.
Glutaraldehyde is the cross-linking agent most frequently used to
prepare chitosan microspheres. It is obvious, however, that because the
high reactivity of glutaraldehyde leads to chemical species of poorly understood formulae, the glutaraldehyde-re1ated toxicity of the products is a
serious limitation of the model. Recent observations confirm extreme
cytotoxicity of the glutaraldehyde cross-linking chitosan microspheres
[34]. With red blood cells, the glutaraldehyde cross-linked chitosan microspheres were considerably more lytic than dextran, and less membraneactive than soluble chitosan.
Improved microsphere biocompatibility can be achieved with other
reagents such as citric acid [35], alginates [20, 36, 37] and pectins [38].
Other cross-linking agents have been described [39-42], but no information is available on their biocompatibility and adequacy as drug carriers.
Acknowledgements
The present work was carried out with financial support from the Italian National Research
Council, "Progetto Finalizzato Materiali Speciali per Tecnologie Avanzate 11", Roma, Italy
(contract no. 98.00032.PF34).
312
1. Gcnta et al.
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19 Tozaki H, Fujita T, Yamamoto A, Muranishi S, Sugiyama T, Terare A, Matsumoto T, Suzuki
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24 Remunan-Lopez C, Lorenzo ML, Alonso MJ (1995) Deve10pment of new microencapsulated chitosan gel cores for the controlled release of hydrophilie macromolecules. Proc
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31 Mestecky J (1987) The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol7: 731-737
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microparticles. Arch Pharm Pharm Med Chem 331: 133-138

ed. by ~ Jolles and R... Muzzarelli

Raul G. Cuero
Summary. The objective of this chapter is to present fundamental factors (e. g. intrinsic and
extrinsic) influencing chitosan as antimicrobial agent, for effective practical application. The
antimicrobial activity of chitosan is well observed on a wide variety ofmicro-organisms inc1uding fungi, algae and some bacteria. However, the antimicrobial action is influenced by intrinsic
and extrinsic factors such as the type of chitosan (e. g. plain or derivative); degree of chitosan
polymerization; host natural nutrient constituency; substrate chemical andJor nutrient composition; and environmental conditions (e.g. substrate water activity (A w ) and/or moisture). Although both plain and derivative chitosans are effective as antimicrobial agents, there is a
differential effect between them. Their differential antimicrobial effect is mainly exhibited in
live host plants; thus the antifungal effect ofN-carboxymethyl chitosan (NCMC) is different in
vegetable as compared with graminea host. At the same time, pentamer and heptamer chitosan
units seem to have better antifungal action than larger units. Chitosan antimicrobial action is
more immediate on fungi and algae, followed by bacteria; the chitosan site of action is at the
microbial cell wall.
Introduction
Chitin and chitosan [42] show antimicrobial activity along with the ability to resist environmental conditions; they are also a source of nutrients.
Chitinlchitosan antimicrobial activity has been reported by several
authors [1, 10, 12-17,25]. Chitosan, has also been used to control growth
of algae [19], and to inhibit viral multiplication in plants and in vitro
[48,51].
Chitosan has been used as antimicrobial compound by its external application (exogenous) to the host, to the substrate or media, and to a physical surface containing microbial population [1, 12-17]. The chitosanase
and chitinase have been induced in plants as resistance mechanisms against
pathogens, especiaHy fungi [12, 15, 17, 28, 33]. Induction of chitinase
enzymes through genetic engineering as a contral alternative against
plant pathogens seems to be very promising. Broglie et al. [7], showed that
transgenic plants containing high constitutive levels ofbean endochitinase
are more resistant to infection by the soil-borne pathogen, Rhizoctonia
solani than are wild-type or control plants that lack the chimeric chitinase
gene. Chet et al. [10] tested three different chitinase genes from Serratia,
Aeromonas, and Trichoderma. The pure enzyme produced was tested
direct1y for its biocontrol activity, as weH as its properties as a lytic enzyme.
The cloned gene was then expressed in the nonrhizosphere bacteria
Escherichia coU for biocontrol experiments.
316
R.G. Cuero
Some mechanisms for antifungal activity of exogenous chitosans have

been suggested:
induction of phenylpropanoid and octadecanoid pathways
induction of chitosanase [16a, 21, 28, 33]
induction of chitosanase with many polypeptides [12-14, 16,20,28,37]
effect of chitosans on the plant enzymes in relation to plant resistance
against fungal pathogens [16, 18,26, 32a, 36, 37]
induction ofphenolic compounds and/or phytoalexins [15, 25, 37]
induction ofmorphological and/or physiological changes [12, 23].
In addition to the nature and/or physicochemical structure of chitosan,

many specific qualities, inherent in the host and/or substrate that can intluence the antimicrobial activity of the polymer include environmental
conditions (e. g. temperature, moisture), nutrient composition, pR and
water activity (a w )'
Some mechanisms have been suggested to understand this antibacterial
action [45]:
reaction with bacterial teichoic acids, polyelectrolyte complexes

chelation of metals present in metalloenzymes
alteration of the bacterial adhesion
inhibition of the enzymes that link glucans to chitin
prevention of nutrients permeation
Effect of the nature of chitinlchitosan on antimicrobial activity
The nature of chitinous material (e.g. plain or derivative), has a profound

effect on its antimicrobial efficacy. N-carboxymethylchitosan (NCMC) is
obtained by N-carboxymethylation of chitosan, which consists ofreacting
the free amino groups of chitosan with glyoxylic acid to produce a soluble
aldimine and then reducing the product with suitable reducing agent
[42, 43, 53]. The chemical structure differences between the plain and
derivative chitosan also has effects on their functional differences such as
antimicrobial activity, thus: NCMC, in vitra, even at lower concentration
exhibits more potency than plain chitosan, and its antimicrobial action in
vitra is more immediate or prompt than plain chitosan. Cuero and Lillehoj
[19] demonstrated that NCMC exhibits both algistatic and algicidal effects
by inhibiting total growth of the alga Anabaena sp. when applied to the
media before algal inoculation, or 48 h after algal inoculation, as compared
with native chitosan, which mainly showed an algistatic effect at the concentrations used (> 1 nM). NCMC's algicidal effect was shown in culture of
Anabaena sp. incubated at 22C under shaking conditions. All NCMCtreated algal cultures showed decreased biomass (40 mg/50 mllweek) as
compared with algal culture without NCMC or control, which showed
317
B
Figure I. Electron microscopic view showing algicidal effects of chitosan: A. Control/no
chitosan treatment: Normal algal filaments . B. Chitosan treated algal culture: PuncturedIHolel
dead algal filaments [19].
higher biomass (50 mg/50 ml/week). Although NCMC was effective in

inhibiting algal growth in liquid culture at higher concentrations (39 nM),
lower NCMC concentrations (12 and 24 nM) were also effective in inhibiting algal growth. The exogenous chitosan caused morphological changes
to the algal filaments. Big punctures and/or holes were observed under
electron microscopy in algal filaments treated with exogenous chitosan
(Fig. 1). Thus algal cells lyse through these holes and finally die. Both
the algistatic and algicidal effects of exogenous chitosan also depend on
the type of algae, and the age of the algal culture [19] (Tab. 1, Fig. 1). In
another study, Cuero (unpublished data) inhibited Fusarium species in agar
by using NCMC at concentrations between 0.05 % and 0.1 %, as compared
with native chitosan, which was effective at 0.1 % concentration only.
Cuero et al. [12, 13] also demonstrated a substantial antimicrobial effect of
NCMC on toxigenicAspergillusflavus. This in vitro antimicrobial efficacy
of the derivative chitosan, NCMC, could be due to its ability to chelate
transition metal salts such as copper, zinc, iron, and so on present in the
media, thus preventing nutrient availability to the microorganism. NCMC
has also shown antiviral effect in vitro by inhibiting viral adsorption to the
Table 1. NCMC algistatic effect on an Anabaena sp. in culture
Controls
Treatments
no NCMC
NCMC
Time(days)
Biomass (mg/50 ml)

Biomass (mg/50 ml)
Biomass (mg/50 ml)
21.7
25.5
25
29.0
30.0
42.6
54.3
48.7
51.0
Algal cells incubated at 22C under shaking conditions.

Source: [ 19].
12 nM
24 nM
39 nM
0
0
0
20
18
15.0
42.7
40.7
42.6
318
R.G. Cuero
CD4 receptor and reverse transcription of the viral genome. N-carboxymethylchitosan-N,O-sulfate (NCMCS), inhibited the propagation of the
human immunodeficiency virus type 1 (HIV-l) in human CD4 cells and
that ofRauscher murine leukemia virus (RLV) in murine fibroblasts [51].
NCMC functions at a wider pH range (3.5 to 9) than native chitosan. The
amphiphilic property ofNCMC is the reason for its pH versatility; thus it
is an ideal substance for in vitro studies. However, in vivo, especially in live
plants and in harvested grain and fruits, native chitosan exhibits a more
effective antimicrobial activity than NCMC. Other reports have also shown
marked antimicrobial activity of chitosan in growing plants as well as
in harvested grain and fruits [1, 13-17,22,25,26,48]. Cuero and Osuji
[16a] achieved complete inhibition of toxigenic A. jlavus in field growing com and peanuts treated with chitosan. The effect of chitosan on
growth inhibition of A. jlavus was correlated with reduction of aflatoxin
(Tab. 2). The chitosan effect on inhibiting the toxigenic A. jlavus also
depends on the management of the extrinsic and intrinsic factors, as
mentioned above. Growth of Botrytis species in field-grown eggplants was
similarly inhibited.
Plain chitosan seems to have a better molecular compatibility with the
plant host andlor grain and fruit substrates than derivative chitosans such
as NCMC, thus inducing resistance to microbial pathogens coming in
contact with the host. Several mechanisms have been suggested to explain
the chitosan induced microbial resistance in vivo (e.g. natural host): (1)
Exogenous chitosan from crustacean applied to a plant host seems to
activate genes in the host RNA; this will then induce the phenylpropanoid
pathway and some enzymes responsible for triggering resistance mechanisms against pathogens [1,28]; exogenous chitosan induces microbial and
plant chitosanase production. This enzyme has antifungal activity by
hydrolyzing the chitosan in the cell wall ofthe microorganism, thus causing
lysis of the fungal cells, and consequently growth inhibition andlor death
[1,12,14, R.G. Cuero, unpublished data] Cuero et al. [13], showed almost
Table 2. Mean Aspergillus flavus count in field grown corn, after single control treatments [12]
Treatments
Mean
ASFL
ASFLalone
water alone
chitosan alone
ASFL + chitosan
chitosan + ASFL
ASFL+BSUB
BSUB+ASFL
LSD
3.00
0.50
0.17
0.17
0.33
0.67
0.67
0.66
(STDE)
0.36
0.22
0.17
0.17
0.21
0.21
0.21
319
Figure 2. Morphological changes of Aspergillus jlavus hyphae after chitosan treatment. (R. G.
euero, 1988; unpub1ished data).
100% inhibition of A. jlavus growth in field-grown maize by treatment

with chitosan; consequently, the fungal toxin aflatoxin was also reduced to
almost nil. Cuero (unpublished data) observed morphological changes
such as weakening and swelling of A. jlavus hyphae, after treatment with
exogenous chitosan (Fig. 2). Plain chitosan seems to induce chitosanase
with more polypeptides than glycolchitosan. R.G. Cuero, 1988 (unpublished data) carried out a comparative electrophoretic study using both synthetic/derivative glycolchitosan or native chitosan as substrates to induce
chitosanase in peanut seeds. The results showed that native chitosan substrate induced chitosanase with a greater number of polypeptides than the
derivative/synthetic glycolchitosan (Fig. 3). The higher number of polypeptides seems to be advantageous because ofthe wider range of genes that
can be elicited for antimicrobial action. Also, it has been demonstrated that
exogenous chitosan elicits chitinase in melon plants and Japanese radish,
soybean, rice and black pine seeds during the germinating process [29, 49],
thus inducing antifungal activity in plants: (2) Exogenous chitosan from
crustacean has also shown elicitation of phytoalexins and/or their precursors (free phenolic, total phenols and unknown free phenolic compounds) after being applied to field peanut and maize plants, resulting in
control of pathogenic and/or toxigenic fungus A. jlavus, to no growth within 72 h, and consequently the fungal toxin (aflatoxin) was inhibited [15, 25,
26]. Cuero et al. [15], showed different levels ofphenolics (total phenols)
enhancement at different water activities as compared with control. There
320
R.G. Cuero
B nO.1
B nO.1
B nO. 2
Bno. 2
-1
- 1
- 2
-2
- 3
- 3
- 4
-5
- 6
- 7
- 4
GLYCOLCHITOSAN
AS SUBSTRATE
CHITOSAN
AS SUBST RATE
Figure 3. Different polypeptides horn two different Bacillus species. Induced by syntheticl
derivative and native chitosan substrates. Left: Glycochitosan substrate, induced 2- 4 polypeptides. Right: Chitosan substrate, induced 7 polypeptides. (R. G. Cuero, 1988; unpublished data)
were higher levels ofboth free phenolic compounds at lower A w (0.85) than
at higher A w (0.95), which increase with incubation with chitosan treatment. Marked increases of phenolics occurred after 48 h of incubation.
Levels of free phenolic acids were noticeably increased with incubation in
combined chitosan + A. flavus treatment at 0.85 A w . Cuero (unpublished
data) also corroborated the induction of higher concentrations of phenolic
compounds in tissue cultures ofpeanut (Fig. 4). Fajardo et al. [25] reported
significant enhanced elicitation of free ferulic and p-coumaric acids, and
bound p-coumaric acid by chitosan in peanut seeds betwen 9 and 72 h: (3)
The antimicrobial effect of exogenous chitosan also seems to be a result of
its ability to react with proteins and essential nutritional elements used by
microorganisms during growth, thus inhibiting availability of these nutrients to the microorganisms and causing slow growth and/or death of the
organisms. It has been demonstrated that chitosan chelates metals markedly [lla]. Chitosan also has the ability to immobilize enzymes relevant to
food processing such as proteases [35]. Chitosan is also believed to possess
a fungistatic property due to its ability to induce morphological changes in
the cell walls of Rhizopus stolonifer [13]. Thinning and lysis of the algal
321
...
.:i
At<
..
5
1IIjI.
aw,
ta
9'
Figure 4. Chitosanase induction in peanut seeds at 0.90 A w after treatment with: 1: water, 2: B.
subtilis # 1; 3: B. subtilis #2; 4: chitosan; 5: A. jlavus; 6: B. subtilis # 1 + chitosan. R. G. Cuero,
1995; unpublished data.
cells, as weIl as filamentous mal formation and flaccidity of the cell wall
along with perforations ofthe algal filaments was observed in Anabaena sp.
[19](Fig.1).
The ability of chitosan to enzymatically and/or mechanically inhibit
growth of fungi has effectively been used for practical applications such as
seed treatment, fruit and vegetable protection. When chitosan enters the
host plant cells, it triggers a sequence of reactions, thus inducing disease
resistance responses [28, 33].
Seed treatment, fruit and vegetable protection
Hadwiger et al. [28] reported seed and foliar treatments of field crops
with commercial chitosan. They applied seed treatments ranging from
60-1000 p.g of chitosan per gram of seed on winter and spring wheat, peas
and lentils during a 5-year trial. Plant yield increased 20-30%. Reduction
of dampoff, logging and other symptoms of fungal infection were observed. Similarly, induction of systemic resistance to Fusarium crown and
root rot in tomato plants was obtained after chitosan seed treatment [3].
Hirano et al. [30] observed a relationship between chitinase activities and
resistance of seedlings to pathogens in Japanese radish, soybean, rice,
hulled rice and black pine seed. R.G. Cuero and G. Osuji (unpublished
data) found inhibition of A. flavus growth in chitosan-coated com and
peanut seeds. However, chitosan was more effective in controlling the toxigenic fungi in peanut seeds. Cuero et al. [17] reported control of A. flavus
and concomitant aflatoxin production in postharvest com kemeis after
treatment with chitosan. Cuero et al. [l3] also reported control of A.flavus
and concomitant aflatoxin production in harvested com kerneIs treated
with chitosan in the field during plant development. The fungal population
was almost nil (1.7%) in kerneis from chitosan-treated plants as compared
to 30% in nontreated controls. Simultaneously, aflatoxin was reduced to
zero in kerneis from chitosan-treated plants as compared with 1104 p.glkg
in nontreated controls .
322
R.G. Cuero
The practical application of chitosan to protect fruits and vegetables

against pathogens such as fungi in postharvest, has been demonstrated by
EI Ghaouth et al. [23] and EI Ghaouth and Wilson [24]. They carried out in
vitro studies which showed that chitosan-coated strawberries were protected against decay-causing molds such as R. stolonifer. They also reported protection of vegetables (e. g. tomato, bell pepper, and cucumber)
against postharvest fungal pathogens). Complete protection offield grown
egg-plant against Botrytis spp. was achieved by treating the plants with chitosan; the fruits even increased in size as compared with untreated controls.
Seedlings of eggplants were inoculated with the fungus Botrytis spp. before
being transplanted into the field.
The agricultural applications reported by [29] mainly concern grain coating but a range of applications are being developed, such as the coating of
fruits [22] and vegetables [8]. Although the antibacterial activity of
chitosans has not been consistent and clear, other authors have reported
different antibacterial effects according the type of chitosan used. Muzzarelli [43] and Mattioli-Belmonte [40] found in vitro that N-carboxymethyl and N-carboxybutyl chitosans are more effective bacteriostatic
agents than other chitosans. Cationic or amphoteric polyaminosaccharides
interact with the bacterial cell wall, and alter its structure as a consequence
of polyelectrolyte complex formation and of disturbing action on the
equilibria involving metal ions. Autolysing enzymes, which normally control bacterial division, become lethaI substances when cationic compounds
are present; thus it is possible that a similar mechanism is promoted by the
strongly cationic chitosans.
Tanigawa et al. [54] examined the antibacterial effect of water-soluble
chitin derivatives, partially deacetylated chitins, N-trimethyl derivatives of
partially deacetylated chitins and chitosan oligomers in vitro. They found
that sulfuryl chitin, phosphoryl chitin and some chitosan oligomers preared by nitrous acid deamination of partially deacetylated chitins inhibited
bacterial growth. However, carboxymethyl chitin was less active, whereas
N-trimethyl derivatives ofpartially deacetylated chitins inhibited bacterial
growth more strongly than the partially deacetylated chitins. They suggest
that perhaps the inhibition ofbacterial growth is due to the cationic amino
groups in these chitosan oligomers which combine, by electrostatic interaction with anionic components, such as N-acetylmuramic acid, sialic acid
and muramic acid, on the cell wall surface, and may suppress bacterial
growth. However, D-glucosamine hydrochloride did not show inhibitory
activity. They also suggest that although a strict correlation between polysaccharide chain length and inhibition of bacterial growth was not found
in his study, the inhibition of growth might be caused by an optimum
chitosan oligomer chain length.
323
Effect 01 chitosans on plant enzymes in relation to resistance to fongal

pathogens
Chitosans trigger expression of plant enzymes other than chitinase/chitosanase. Kurosaki et al. [37] in addition to chitinase, induced phenylalanine
ammonia-Iyase (PAL) along with accumulation of phenolic acids in cultured carrot cells treated with chitinase or fungal mycelial walls. PAL is
a key enzyme in the phenylpropanoid pathway in plants which produces
phenolics inc1uding phytoalexin (antifungal compounds) [39, 60]. These
results corroborate earlier reports in pea-Fusarium pathogen interactions
regulated by chitosan [28]. The disease resistance response in peas was
correlated with increase in the activity of the fungal wall-hydrolyzing
enzymes (e.g. endo-B-glucanase and endo-chitinase) and with de novo
increases in the messenger RNA (mRNA) for and synthesis of phenylalanine ammonia lyase.
Bemasconi et al. [5] determined chitinase, lysozyme and a-mannosidase
activities in Rubus hispidus cultured in vitro. These enzymes were found in
the growth medium and in subcellular parts, although their activities varied
according the localization. Chitin oligosaccharides have been used as
elicitors of chitinase activity in melon plants [49]. They reported hexamer
and nonamer as the most efficient elicitors, thus suggesting chitinase elicitation by chitin oligosaccharides as an important element of molecular
communication in host-parasite interactions. These results also concur
with those ofother authors [28, L.A. Hadwiger, personal communication].
Walker-Simmons and Ryan [57], also demonstrated the effects of chitosan
oligomers and chemically modified chitosan and chitin in triggering
molecular signals, and receptors to activate plant defense responses in
tomato leaves.
The effect of chitosan on plant enzymes and/or defense response against
pathogenic and/or toxigenic fungi has been demonstrated in vivo in germinating peanut seeds, and also in tissue cultures [15]. Marked enhancement
of phenolic compounds (phytoalexin pecursors) and chitosanase was reported after treating the germinating seeds with chitosan. An increase in
phenolic compounds corresponds to an increase in some plant enzymes
such as PAL [39, 60].
Fajardo et al. [26] demonstrated changes in isozymes and protein molecular weights in mature peanut seeds treated with chitosan and/or the
fungus Aspergillus flavus. Enzymes involved with the synthesis of
phenolic compounds were analyzed. Cinnamyl alcohol dehydrogenase
(CAD), glutamate dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PDH) were resolved by native polyacrylamide gel
electrophresis (PAGE). Polyphenoloxidases (PPO), anodic perodixase
(PRX) and shikimate dehydrogenase (SKD) were also determined. After
48 h, chitosan and A. flavus treatments inconsistently enhanced G6PDH
activity, initially and near the end of the experiment. The combined pre-
324
R.G. Cuero
sence of chitosan + A. jlavus enhanced the activity of all GDH isozymes at

3 h in moderately susceptible cotyledonary tissues of peanut seed (Starr
variety). Seed tissues also responded to A. jlavus and chitosan + A. jlavus
invasion by inducing PRX enzymes. Fajardo et al. [26] also reported enhanced PPO and SKD activity after treatment with chitosan, A.jlavus, and
chitosan + A. jlavus treatments. These enzyme activities are linked to synthesis of polyphenolic compounds (e. g. tannin and quinone) and to the
shikimic acid pathway, respectively, which are involved in plant-pathogen
resistance mechanisms.
Kombrink et al. [36] also reported detection of N-acetyl--D-glucosaminidase and chitinase in parsley extracts, after treatment with chitinaceous elicitors. He suggested that despite the structural similarities of the
potential substrates, N-acetyl--D-glucosamidase(s) and chitinases are
separate enzymes in parsley with distinct substrate specificities.
The tetramers of glucosamine (GlcN)4 and of N-acetylglucosamine
(GlcNAC)4 were compared for their eliciting potency in Rubus protoplast
suspensions. The report indicated that xylosidase elicited the stronger
activation in presence of(GlcNAc)4 or (GlcN) 4, and laminarase increased
after glucosamine treatment only, while a-amylase was not elicited [38].
Type of chitosan: liquid or solid
Use ofliquid chitosan as an antimicrobial agent is more effective than solid

form. Liquid chitosan is readily or immediately uptaken by microbial and
plant cells as compared with slow uptake of the solid (usually powder
form). Rapid uptake (within 12 h) of NCMC by tomato plants has been
reported [12]. The uptake of chitosan by microbial cells has also been
determined through induction of chitosanase enzymes and phenolic compounds (within 8 h) in vitra and/or tissue culture after treatments with
exogenous chitosan, and by observing death of algal cells [19] and fungal
cells through staining with trypan and microscopy studies, and through
reduction ofthe fungal population [14, 15, 17]. Solid chitosan tends to act
slower and also to be less potent biologically. Cuero (unpublished data)
used powder and flaky chitosan to control toxigenic A. jlavus growing in
maize and peanut kemels. There was no immediate inhibition of fungal
growth, except a slight inhibition after 4 weeks when the experiment was
stopped. The effectiveness of powdered chitosan against bacterial strains
and fungi has been tested. Powdered chitinlchitosan or whole crab shell
showed no efficacy, as compared with solutions of chitosan in acetic acid
[34]. There are reports on the delayed action of solid chitinous material
against nematodes in soil under greenhouse conditions. The report also
showed the reduced potency of the solid chitinous material, since rates
equal or higher than 1% of the material were necessary to obtain adequate
control ofthe target nematode [50].
325
Also, the type of acid used in the preparation of chitosan solutions influences its antimicrobial activity. Chitosans prepared with acetic acid
exhibit more immediate antifungal effects as compared with chitosans
prepared with lactic acid [12, 17]. Chitosans prepared with acetic acid can
be kept longer than 3 years even at room temperature without loosing antimicrobial activity, as compared with chitosan prepared with lactic acid
which kept its full strength less than 3 years under room temperature.
Perhaps, this explains the higher efficacy of chitosan-acetate when applied
to substrates such as seeds with high fungal contamination, as compared
with lactate chitosan, which sometimes requires additional application,
depending upon the substrate and/or host where microorganisms are
growing.
Source of chitosan influencing its antimicrobial activity

Source of chitosan is mainly found in crustacea, bacteria, fungi, algae and
protozoa [32]. However, here I will mention only the most common sources
of chitosan used for antimicrobial activity, which includes crustacea,
bacteria and fungi. Many bacteria genera (e.g. Bacillus, Serratia) and
actinomycetes (e.g. Streptomycetes) produce chitosan in their cell walls
and exhibit antimicrobial activity [12, 47 a; R. G. Cuero and G. Osuji, unpublished data]. Fungal chitosan has also been reported as an antimicrobial
agent [12, 14, 16]. Crustacean chitosan seems to exhibit more diverse antimicrobial mechanisms, including induction of chitosanase, phenolic compounds and blocking nutrient availability of the microbial cells, as compared with microbial chitosan, whose main antimicrobial mechanism
seems to be induction of chitosanase only. However, bacteria, actinomycetes and fungi induced more chitosanase in germinating com and peanut
seeds than crustacean chitosan. Chitosanase production was clearly demonstrated in SDS-PAGE (Fig. 4). In agar plates, crustacean chitosan induced more chitosanase than the microbial chitosan, although some Bacillus species including a strain of B. subtilis, and also a strain of A. jlavus
produced chitosanase markedly [16, 17] (Fig. 5). This result is supported
by Tronsmo et al. [56] who found growth inhibition of Fusarium oxysporum by crustacean chitosan with high viscosity and deacetylation percentage in vitro. The production of chitosanase corresponded with antifungal activity [12, 14, 16]. Isolation of inducible chitosanase from Bacillus
spp. in agar media and B. circulans in liquid media have been reported [20,
58]. Fenton and Eveleigh [27] reported fungal chitosanase from Penicillium islandicum. Substantial antifungal activitity by bacterial and actinomycetes chitosanases induced by exogenous crustacean chitosan in germinating maize and peanut seeds was demonstrated. Maize and peanut plants
inoculated with A. jlavus under field or laboratory conditions were treated
with exogenous crustaceans chitosan alone or in duplex or tripie combina-
326
R.G. Cuero
tion with bacteria B. licheniformis, Pseudomonas aeruginosa, P jluorescens, Aeromonas hydrophila, and also in combination with actinomycetes
Streptomyces griseus. Although crustacean chitosan alone inhibited growth
of A. jlavus completely, antifungal activity was enhanced by microbial treatment in combination with the crustacean chitosan. Marked antifungal
activity corresponded with high production of chitosanase by the microorganisms. The best single antifungal treatment ws crustacean chitosan alone followed by Bacillus sp. However, Streptomyces showed the highest chitosanase production followed by Bacillus, and Aeromonas. The most
effective antimicrobial combined treatments were those carrying Bacillus
sp. The production of chitosanase was also markedly higher in mixtures of
Bacillus sp. plus crustacean chitosan. In SDS-PAGE studies ofthe enzyme,
the peptide profile of the microbial chitosanase was different among all the
different microorganisms used, and also in comparison with crustacean
chitosanase. However, the number of the enzyme polypeptides was influenced by the type of seed (e. g. monocotyledon or dicotyledon). Cuero
and Osuji [16] induced high production of A. jlavus chitosanase in maize
and peanuts after treatment with exogenous crustacean chitosan, resulting
in great growth dominance of A. jlavus over other microorganisms that corresponded with high chitosanase production by the toxigenic A. jlavus.
Plain chitosan induced more chitosanase polypeptides than synthetic
glycolchitosan in SDS-PAGE (Fig. 3) and that there is a synergistic effect
between exogenous native chitosan and microbial chitosan in inducing
chitosanase. However, there is an antagonistic effect between fungal and
bacterial chitosan in inducing chitosanase, thus resulting in reduced enzyme production. Mixtures of crustacean chitosan with fungus A. jlavus
yielded more chitosanase than mixtures ofthe crustacean chitosan with any
of the two bacterial strains of B. subtilis tested. However, the growth of
A.jlavus was completely inhibited, while inhibition ofbacterial growth was
too inconsistent to be considered as definite results [12, 16; R. G. Cuero
and G. Osuji, unpublished data]. The type of bacterial strain in mixtures
Figure 5. Chitosanase induction in corn seeds at 0.90 Aw after treatment with: 1: water; 2: B.
subtilis # 1; 3: B. subtilis #2; 4: Chitosan; 5: A. jlavus; 6: BS# 1 + chitosan. R. G. Cuero, 1995;
unpubJished data.
327
C.I-IITOSA
A.lAS ~
IN D II,"',()A!
BJlCIII"$ S~~~I
COHTIfOt. (WA+fR)
Figure 6. Induction of chitosanase by Bacillus species in chitosan agar R. G. Cuero et al., 1990;
unpublished data.
WATER
CHlr
Figure 7. Chitosanase induction by Aspergillus jlavus in chitosan-agar. Fungal inoculum was

placed in each disc (3). Left: Discs containing the fungus were placed on water-agar; there was
no chitosanase induction. Right: Discs containing the fungus were placed on chitosan-agar;
there was marked production of chitosanase.
with crustacean chitosan also influences the induction of chitosanase.

When two strains of B. subtilis (# 1 and #2) were independently mixed with
crustacean chitosan, the combination of one ofthe B. subtilis strains (#2)
with the crustacean chitosan yielded three times more chitosanase than the
combination of B. subtilis # 1 with the crustacean chitosan (Fig. 4).
328
R.G. Cuero
Degree of deacetylation and polymerization

The antimicrobial activity of chitosan has been correlated with its degree of
deacetylation and polymerization. Exogenous chitosan treatment with lower
deacetylation exhibited better inhibition of toxigenic A. flavus growth in
peanut and maize seeds as compared with chitosan with higher deacetylation. The degree of antimicrobial activity corresponded to the degree of
chitosanase production. Also, exogenous chitosan with lower deacetylation
induced better antifungal activity and chitosanase production in bacteria
and actynomycetes in peanut and maize seeds. Other workers also reported
chitosan with a low percentage of deacetylation and high viscosity exhibiting antifungal activity against plant pathogens [56]. Chitosan pentamer and
heptamer are effective in inhibiting some fungal germination and growth.
Both the antifungal and host-inducing resistance properties decreased
proportionally as the degree ofpolymerization decreased [33]. Yalpani et al.
[59] reported different effects of chitosan derivatives with different degrees
of polymerization. Chitooligosaccharides with varying degrees of polymerization displayed low antimicrobial activities against B. circulans, and
high activities against E. coli. Tokura et al. [55] conc1ude that chitosan
oligomers block nutrient permeation through the cell wall.
Types of hosts (plants) and their concentration of hexosamine:

Monocotyledons and dicotyledons
The antimicrobial activity of exogenous chitosan also depends on the type
of host where the microorganisms are growing. This antimicrobial activity
corresponds with the production of chitosanase and/or phenolic compounds in the host. The antifungal activity is more marked in peanut seed
host than in maize. The degree of antifungal activity in vegetables is
between peanut and maize seeds [16, 17]. Thus antifungal activity is more
marked in dicotyledons (e.g. peanuts, soybeans, vegetables etc.) than in
monocotyledons (e. g. maize). The best growth inhibition of A. flavus was
found in peanut seeds than in maize [16, 17]. Inhibition ofthe fungus Botrytis cinerea in tomato was also marked [16]. Peanut and vegetable plant
parts seem to uptake native chitosan faster than monocotyledons. Derivative NCMC us uptaken by the plant more slowly [16, 17]. Thus the type of
chitosan along with the their degree ofpolymerization can affect the antimicrobial activity of chitosan against microorganisms residing outside on
the surface of the plant. Since native chitosan is quickly and readily uptaken by the plant, its duration period outside on the surface of the plant
where some microorganisms reside is shorter than NCMC, which lasts
longer outside on the surface ofthe plant. This perhaps explains the sometimes short-lasting effet [12, 16, 17] of chitosan in host plants, and the need
to reapply it frequently. Cuero et al. [12] demonstrated chitosan uptake by
329
tomato plants within short time periods (12 h). Also, the concentration of
hexosamine in the fungus [11] and in the host influence the efficacy of exogenous chitosan as an antifungal agent in plants. Cuero (unpublished data)
found more hexosamine in peanut seeds than in maize; this also corresponded with the levels of chitosanase extracted and with antimicrobial
action.
Stage of plant development: germinating, flowering, fruiting and
senescence
Chitosan and N-carboxymethyl chitosan exhibit better antifungal effects in
germinating seeds, and in the flowering stage of the plant [12, 15-17].
Growth of A. flavus was marked1y inhibited in peanut and maize seeds
when chitosan was app1ied in germinating seeds, and also in field
flowering p1ants, as compared with fruiting and/or senescent p1ants.
Perhaps this is due to the highest hexosamine content of the plant during
germination and flowering, thus interacting with exogenous chitosan and
consequently inducing more endogenous chitosan to inhibit fungal growth.
Also, during earlier germination periods, seeds produce more phenolic
compounds after chitosan treatments, thus inhibiting funga1 growth, as
compared with senescent p1ants.
Extrinsic factors influencing chitosan antimicrobial activity
pH
It is weH established that pR is one of the factors influencing growth of
microorganisms in a substrate and in any host. NCMC functions at a wider

pR range, although it functions better at pRs between 3.5 and 5 as an antimicrobial agent, as compared with native chitosan, which functions better
at pR 5 or> 5; NCMC is more water-soluble than chitosan and has better
chelation properties [46] than chitosan [lla]. The function ofNCMC at a
wider range of pR is a result of its amphiphilic property. This enhances its
versatility as an antimicrobial agent, especially against fungi growing in
p1ants with different pRs. The fact that certain plant parts are acidic, whi1e
others are basic, affects the efficacy of the antimicrobia1 agent. Cuero and
Lillehoj [19] reported efficient algistatic and algicidal effects ofNCMC at
different pRs ranging from 7.1 to 9.1. In aseparate investigation, Cuero et
al. [12] demonstrated effective suppression of A.flavus growth by NCMC
in vitra under liquid conditions at pR changing from 5.5 (initial) to 3.5
(final).
330
R.G. Cuero
Water activitylwater content 0/ the host andlor substrate

Water activity or water content of the substrate influenced the efficacy of
chitosan on inhibiting fungal growth in monocotyledon and dicotyledon
plant seeds [17]. Although derivative chitosans such as NCMC tend to be
more hydrophilie than native chitosan, are still both equally influenced by
the water content of the host and/or substrate. Chitosans exhibited better
antifungal effects in maize and peanut seeds at 0.90 or 0.93 water activity
(A w) than at 0.95 [17]. This antifungal activity corresponded with higher
chitosanase production. Also, chitosan showed better inhibition of A. jlavus
growing in nonirrigated field peanut and maize plants than in irrigated
plants [12].
Concluding remarks
The use of chitosans as antimicrobial agents is a clear example of successful biological control. Undoubtely, chitosan is a versatile compound
endowed with antimicrobial activity affecting growth and physiology of
most microorganisms, including algae, fungi, bacteria, protozoa and
viruses. The degree of the effect and mode of action of chitosan varies
according to the microorganism targeted. The responses of the microorganisms to the chitosan as an antimicrobial agent also depend upon the
chemical makeup ofthe chitosan used, and the environmental conditions at
the time of the interactions. A clear understanding of the biological activity
of the different units andlor oligomers of the polymer chitosan, and the
fundamental baseline of the interactions between intrinsic and extrinsic
factors, is necessary for practical application of exogenous chitosan as an
effective antimicrobial agent.
3
4
5
1,2,3 = CORN
4,5,6 = PEANUT
Figure 8. Chitosanase from Bacillus sp. in corn and peanut at different water activities: 1 & 4
0.80 Aw. 2 & 5 = 0.85 Aw. R. G. Cuero, unpublished data.
331
Acknowledgements
This chapter is written in memory of my dear former professor, colleague and friend the late Dr.
John Lacey, who was a great microbiologist with incisive understanding of microbial control.
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335
Subject index
N-acetylchitohexaose 288
N-acetyl chito-oligosaccharide 112, 119
N-acetyldopamine 47
N-acetylglucosamine 114,237
-N-acetylgalactosaminidase 236
-N -acetylgalactosaminidase, concerted
action of 18
-l,4-linked N-acetylglucosamine
(GleNAc) 72
N-acetylglucosaminidase 114, 119, 148
N-acetyl-D-glucosaminidase 235,238,251
N-acetyl--D-glucosaminidase, urinary
excretion of 241
endo-N-acetylglucosaminidase 146
N-acetylhexosaminidase 114,235
N-acetylmuramic acid 114,322
ABA-N-acetyl-tyrosine 282
Acanthocheilonema viteae 224
acetylation, degree of 1
acromegaly 239
acyclovir, ophthalmie administration of 306
agriculture I 71
N--alanyldopamine 47
alginate 311
allergy 282
allosamidin 46, 113, 202, 230
allosamidin, competitive inhibition 204
allosamidin, cyst formation 231
allosamidin, mixed-type inhibition 205
allosamidin, non-competitive inhibition 205
Alteromonas 237
aminopterin 88
amoeba 50
a-amylase, bacterial 282
Anax inmaculifrons 46
angiogenesis 255
animal clinic 274
antibody, circulating 279
Aphrodite 44
apicectomy 259
Apis cerana indica 46
Arachnida 46
Arthropoda 46,48
Asburner's model 90
atherosclerosis 293
ATPase 44
avermeetin 88
Beeil formation 282
Beeil growth cell wall skeleton
(BCG-CWS) 285
bacteria, chitinolytic 160
bacteria, colonic 301
benzoylphenylurea 86
bile acid 293
bile salt 299
biocompatibility 252
biodegradability 251
biological control 172
biopesticide 172
biotechnology, agricultural 172
blackfly 225, 227
bone marrow 285
bone tissue, regeneration of 259
bovine serum albumin (BSA) 310
Brachiopoda 48
brefeldin 88
Brugia malayi 224
Brugia pahangi 224
buprofezin 88
C5a 288
calcium 48
Calcofluor 62, 97
Calcofluor white (CFW) 22, 24, 45
cancer 239
Candida albicans 299
carboxyl ester lipase 299
N,O-carboxymethy1chitosan 260
carboxypeptidase 299
carotenoid 48
CD44252
cell division 75
cell migration 80
cell wall 39,55
cell wall, bacterial 279
cellulose 44
cellulose binding protein 149
Chagas' disease 223,227
chitin 73, 119, 188,265
a chitin 44,97
chitin 44, 97
ychitin 44
chitin, alkali 3
chitin, clinical use of 251
chitin, colloidal 2, 119
chitin, fossil 4
chitin, gycol 119
chitin, in biosphere 4
chitin, molecular weight of 2
chitin, polymorphie form of 1
chitin, solubility of 2
chitin, supramolecular structure of 13, 39
chitin, tensile mechanical property of 45
chitin acetylase 45
chitin binding protein 149
336
chitin biosynthesis 39,86
chitin chain elongation 10
chitin chain tennination 12
chitin deacetylase 46
chitin fibril 12
chitin microfibril 39
chitin suture 252
chitin synthase 56, 66, 85
chitin synthase I (CSI) 55,57-59
chitin synthase 11 (CSII) 55, 56, 58-60, 62
chitin synthase III (CSIII) 55,56,58,61,
63-65
chitin synthase, activation of 14, 15
chitin synthase, amino acid sequence 26
chitin synthase, concerted action of 17, 18
chitin synthase, fungal 86
chitin synthase, 3D model of 27
chitin synthase, glycoconjugation of 30
chitin synthase, inhibition of 21
chitin synthase, latency of 16
chitin synthase, multiplicity of 24
chitin synthase, priming of 15
chitin synthase, purification of 15
chitin synthase, reaction components of 10
chitin synthase, regulation of 14
chitin synthase, structure of 25
chitin synthase allostery 14
chitin synthase co-operativity 14
chitin synthase (CHS) gene 55,61
chitin synthesis 18
chitin synthesis inhibitor 86
chitin synthetase 39
chitinase 45,57, 111, 188,307
chitinase, Acanthocheilonema viteae 229
chitinase, activator 113
chitinase, algae 111
chitinase, amino acid sequence of 137
chitinase, amphibian 111
chitinase, anomer fonnation 114
chitinase, arthropod 111
chitinase, bacteria 112
chitinase, baculovirus 148
chitinase, Brugia malayi 229
chitinase, c1ass 11 114
chitinase, c1ass III 119
chitinase, c1ass IV 119
chitinase, c1assification of 137, 154
chitinase, crustacean 111
chitinase, encystation 230
chitinase, endo-type 116
chitinase, Entamoeba 229
chitinase, exo-type 116
chitinase, family 18 113, 114, 126, 137,202
chitinase, family 19 114, 126, 148,202
chitinase, filarial 228
chitinase, fish 111, 165
chitinase, fungi 112
chitinase, glycoprotein 112
chitinase, human blood 165
Subject index
chitinase, inhibitor of 113, 230
chitinase, insect 111
chitinase, isoelectric point 112
chitinase, Killer toxin 158
chitinase, kinetics 119
chitinase, Leishmania 226, 230
chitinase, mammal 111
chitinase, mechanism of 146
chitinase, microorganism 111
chitinase, modular structure of 147
chitinase, molecular size of 111
chitinase, mollusk 111
chitinase, mycoparasitic fungi 159
chitinase, nematode 162
chitinase, octopus 162
chitinase, optimum pR 112
chitinase, parasite 161, 224
chitinase, plant 111, 163
chitinase, plasmid 158
chitinase, Plasmodium 162, 230
chitinase, reaction mechanism 113
chitinase, role in transmission ofparasite
226
chitinase, saliva 162
chitinase, seaweed 111
chitinase, splitting pattern 116
chitinase, stability of 112
chitinase, stage-specific 231
chitinase, transglycosylation reaction 119
chitinase, trypanosomatid 162
chitinase, venom 162
chitinase, vertebrate 111, 164
chitinase, virus 158
chitinase action, mechanism of 130
chitinase isofonn 230
chitinase like domain 212
chitinolysis 46
chitobiase 146
chitobiose 2,235,237
chitobiose, 4-methylumbelliferone- 230
chitodextrinase 146
chito-oligomer 44, 254
chitosan 2,46, 185,265
chitosan, algal 301
chitosan, N-carboxymethyl 3
chitosan, fungal 301
chitosan, methylpyrrolidinone 252
chitosan heptamer 328
chitosan microsphere 308
chitosan oligomer 191
chitosan pentamer 328
chitosan salt 3
chitosan/mucin interaction 309
chitosan-alginate 310
chitosan-inducible gene 185
chitosanase 47,114, 148
chitosanase, c1ass 46 127
chitosome 41
chitotriose 230
337
Subject index
cholesterol 293
cholesterol7a-hydroxylase 294
cholestipol 294
cholestyramine 294
chondrocyte 217
chondroitin sulphate 261
Choristoneura hormone receptor 3 (CHR3)
90,91
Choristoneura hormone receptor 75
(CHR75) 90, 91
chromosome 1 217
chs gene 44
Ciliata 48
CM-chitin 119
CM-chitin, 14C-Iabelled 285
CM-chitin, hapten-bound 282
colcemid 89
colon 307
colony stimulating factor 286
complement 279,288
composite 39
concanava1in B 146, 147
Congo red 2,45, 102
core structure 128
Crithidia Jasciculata 224
Crustacea 325
cuticle 47
cycloheximide 88
Cyclotella cryptica 301
cyromazine 88
cytokine 254, 286
cytosol 40
defense response 185
defense system 279
delivery, oral 260
depression 242
dermal substitute 258
detergent 48
development, embryonic 218
DG42 77,252
diabetes mellitus 239,241
diatom 44
dietary fiber 294
diflubenzuron 86, 88, 92, 93
digitonin 41
N,N-dimethy1acetamide 45
anti-dinitrophenyl serum 282
dinitropheny1-ova1bumine (DNP-OVA)
282
dipeptide cyclo(L-Arg-D-Pro) 205
diphtheria 311
disease resistance response 185
DNA 310
DNA degradation 190
drug delivery 305
dysentery, amebic 225
dystrophy, corneal crystalline 293
ecdysone receptor (EcR) 90,91

egg, hatching of 225,228
eggshell 223
electron-microscopic autoradiography 40
elephantiasis 224
elicitor 187
endop1asmic reticulum (ER) 42
Entamoeba 223,225
Entamoeba dispar 224
Entamoeba histolytica 225
Entamoeba invadens 50, 224, 229
Entamoeba, cyst of 44
entopathogen 160
environment 171
enzyme, exo-type chitinolytic 114
enzyme structure, comparison of 128
cpithelia, intestinal 300
erythrocyte 285
Escherichia coli 288
Eudragit 307
EuJolliculina uhligi 40
excystrnentprocess 225
exochitinasc 146
exoskeleton 39
exsheathment 225
extracellular matrix 254
fertilization 218
fibric acid 295
fibroblast 239,253,288
fibroblast growth factor, basic 253
fibronectin type III-like module 149
filariasis, cattle 225
filling agent 268
Flavobacterium meningosepticum 237
foot disease 272
fungi 40,55,60, 160
galactomannan 48
gallstone disease 293
Gaucher's disease 243
gene delivery 310
Giardia 223
glucan 46
-I,3 glucan 48
-I,6 glucan 48
-glucanase 188
glucosamine 260
a-glucosidase 241
-glucosidase 285
y-glutamyltransferase 241
glutaraldehyde 308, 311
glycohydrolase superfamily 129
glycol chitin 112
glycol chitosan 253
glycolipid 41
glycoprotein 85,211
338
glycoprotein, oviductal 211
glycoside hydrolase 137
Golgi apparatus 40, 41
Golgi cistemae 44
granulation 254
granulocyte 279
growth inhibition 291
HC gp-39 211
heavy metal 288
hemia, umbilical 271
hemia treatment 270
hevamine 113
hexosamine 329
histology 251
HMG-CoA reductase 294
host-parasite interaction 185
hyaluronan 77,261
hyaluronan synthase (has) 79
hyaluronan synthesis 252
3-hydroxy-3-methylglutaryl CoA reductase
300
20-hydroxyecdysone(20E) 89,91
hypercholesterolemia 293
hypersensitivity, delayed-type 282
hypertension 239,240
hypertriglyceremia 294
ICAM-l 252
immune system 279
immunoadjuvant 286
immunoassay, electron-microscopic 40
immunogenicity 282
infection 279
insect control 174
insect growth regulator 86
insect integument 47
insect vector 223
insulin 306
interferon 286
interleukin-l 254, 286, 291
interleukin-2 291
interleukin-8 288
intestine, large 307
invertebrate 40
isothiocyanate, fluorescent 285
isozyme 323
juvenile hormone (JH) 90
keratan sulphate 261
kissing bug 227
large animal c1inic 271
lectin 101
lectin-type protein 44
Subject index
leg ulcer 256
Leishmania 225
Leishmania braziliensis 224
Leishmania donavani 224
Leishmania in/antum 224
Leishmania major 224
leishmaniasis 223, 232
leishmaniasis, cutaneous 225
leishmaniasis, visceral 225
lentinan 288
Leptomonas seymouri 224
leukemia 239
lipase 251,299
lipase, microbial 300
lipase, porcine pancreatic 300
lipid, neutral 41
lipid, polar 41
lipochitin oligosaccharide (LCO) 71
lithium chloride 45
lithium thiocyanate 45
lithotripsy 242
Loligo 44
loricae 44
lymphedema 224
lysozyme 113, 148,251
macrophage 239, 279
macrophage, mouse peritoneal 279
macrophage activating factor (MAF) 291
macrophage activation 254
maize 325
malaria 223,226,232
mannoprotein 48
mechanism, inverting 130
mechanism, retaining 133
membrane, peritrophic 161
meniscus regeneration 25
metalloenzyme 316
MethA 291
mevalonate 294
Michael-type conjugate 47
Micrococcus lysodeikticus 113
microfibril 39
microfilaria, exsheathment of 228
microsphere 288
microsphere, bioadhesive 306
microvesicle 40
mineralization 48
MM46291
modulus 49
Mollusca 48
monensin 88
mosquito 224
mucin 215
mucoadhesive property 306
Mucor rouxii 40
mycoparasitism 176
Myriapoda 46
339
Subject index
nagstatin 237
narbonin 146,147
nematode, filarial 228
nephrology 241
Neptunes sanguinolentus 46
Neurospora crassa hyphae 40
nicotinic acid 295
nikkomycin 88
nitrite (HN02 ) 47
nitrogen content 2
nitrogen oxide 254
nodB gene 46
NodC 73
nodulation 73
NodZ 74
nonhost resistance 186
nuc1eoside-peptide 21
Oecophila longinoda 46
oligomer 288, 322
Onchocerca gibsoni 224
Onchocerca gibsoni, eggshell of 225
Onchocerca volvulus 224
ophthalmology 260
organ, masticatory 48
osteoinductive property 259
osteoporosis 259
overweight control 293
oviduct 211
oviductin 211
oxazolinium intermediate 133
oxocarbonium intermediate 133
6-oxychitin 4
parasite, development of 231

parasite, emergence of 225
parasite, intestinal protozoan 225
parasite, metazoan 223
parasite, protozoan 223
parasite, transmission of 231
pathogen, human 224
pathogen, intestinal 223
pathogenesis-related gene 185
peanut 325
pectin 311
pentachloronitrobenzene 22
peptidoglycan deacetylase 46
peritrophic matrix (PM) 226
phagocyte 291
phenylpropanoid 316
phosphatidylcholine 41
phosphatidylserine 41
phospholipase A2 299
Phycomyces blakesleeanus 46
phytoalexin 316
plant, pathogenesis-related protein 163
plasmalemma 40
Plasmodium 226
Plasmodiumfalciparum 228
Plasmodium gallinaceum 224, 228
pogonophore 44
polio vaccine 310
poly(acrylate) 299
polyaminosaccharide 322
polyelectrolyte 316
polyene macro1ide 23
polyoxin-D 88
polypeptide 316
polysaccharide 47
Poteriochromonas 301
pregnancy 239
primulin 102
protein, chitin-binding 97
protein, chitinase-like 211
prothoracicotrophic hormone (PTTH) 89
protoplast, fungal 49
protozoa 40
Ptinus 44
puromycin 88
pyothorax 270
o-quinone 47
p-quinonemethide 47
renal functionality 260
RHAMM 252
Rhizobiaceae 71
Rhizopoda 48
Rickettsia-like organism (RLO) 227
river blindness 224
route, exocytic 40
Saccharomyces cerevisiae 40,55-57,63
Sacculina rotundata 46
Sagitta 1
Sandhoff disease 243
Sanfilippo's syndrome 243
Sarcoma 180 291
scar formation 270
Schiff's base 47
Schizosaccharomyces pombe 46
sclerotin 48
sclerozation 47
sedimentation, isopycnic 40
Sendai virus 288
Serratia marcescens 235,237
sheath 225
sialic acid 306, 322
signal 187
silica 48
site-directed mutagenesis 130
skin, artificial 269
skin, regeneration of 270
340
skin substitute 258
spleen 282
spleen T cell 291
sporangiophore 46
spore germination 46
sterol 41
Stigmatella aurantiaca 237
strategy, antiparasitic 231
styloguanidin 205
substrate binding c1eft 130
16S subunit 41
synovial cell 217
tandem repeat 215
Tay-Sachs disease 243
tebufenozide (RH-5992) 89,92
termite, physogastric queen of 47
Thalassiosira jluviatilis 301
thyroiditis 239
trace metal ion 299
transcription factor, ecdysone-induced 91
transgene,plant 178
transglycosylation 39
transmission-blocking vaccine 224, 231, 232
treatment, antimicrobial 172
Triatoma infestans 227
Tribolium castaneum 41
Trichoderma 176
Trypanosoma 227
Trypanosoma brucei 224
Trypanosoma cruzi 227
Trypanosoma lewisi 224
trypanosomiasis 232
trypsin 299
tryptophan 97, 104
tumor ce1l 288
tumor necrosis factor (TNF) 288
tumor necrosis factor-a 254
tumor proliferation 293
tunicamycin 88
turgor pressure 46
Subject index
Ustilago maydis 48
ultraspirac1e (USP) 90,91
uranyl acetate 42
uridine diphosphate N-acetylglucosamine
39
vaccine candidate 228

vaccine therapy 310
vascularisation 255
vesic1e, apical 40
vesic1e, cortical 40, 44
veterinary practice 265
Vibrio harveyi 237
vinblastine 89
vitamin, liposoluble 298
wall integrity 55
water activity 319
waxe 48
wheat germ agglutinin 102
wheat germ lectin 47
wheat germ lipase 300
worm, filarial 224
worm, microfilarial 223
wound dressing 253, 268
wound healing 251,253,260
Wuchereria bancrofti 224
X-ray diffraction 45
YKL-40 213
zebrafish 76
zona pellucida 218
zoo animal c1inic 274
zygomycete 46
zymosan 279

(EXS 87) Riccardo A. A. Muzzarelli (Auth.), Prof. Dr. P. Jolles, Prof. R. A. A. Muzzarelli (Eds.) - Chitin and Chitinases-Birkhauser Basel (1999)

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(EXS 87) Riccardo A. A. Muzzarelli (Auth.), Prof. Dr. P. Jolles, Prof. R. A. A. Muzzarelli (Eds.) - Chitin and Chitinases-Birkhauser Basel (1999)

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EXS87

Chitin and Chitinases

Prof. R.A.A. Muzzarelli

Library of Congress Cataloging-in-Publication Data

Jose Ruiz-Herrera andAlfredo D. Martinez-Espinoza

M Henar Valdivieso, Angel Duran and Cesar Roncero

Jeroen Bakkers, Jan W. Kijne and Herman P. Spaink

Subba Reddy Palli and Arthur Retnakaran

Jon D. Robertus and Arthur E Monzingo

Alfredo Herrera-Estrella and Ilan Chet

Klaus-Dieter Spindler and Margarethe Spindler-Barth

Gilles Bleau, FrMeric Massicotte, Yannick Merlen and

Ida Genta, Paola Perugini, Franca Pavanetto, Tiziana Modena,

Graham W Gooday, Department of Molecular and Cell Biology,

Masahiro Matsumiya, Laboratory ofMarine Products Uzilization,

F. Pavanerto, Department of Pharmaceutical Chemistry,

Paola Perugini, Department of Pharmaceutical Chemistry,

Herman P. Spaink, Leiden University, Institute of Molecular Plant

Pierre Jolles and

Chitin and Chitinases

Native, industrial and fossil chitins

Chitosan indicates a family of deacetylated chitins. In general, chitosans

Native, industrial and fossil chitins

granules submitted to heterogeneous deacetylation. The acetyl groups in

In the past, chitin has been often considered as an intractable biopolymer

Chitin is amply distributed in the biosphere where it constitutes the

Native, industrial and fossil chitins

tissues such as the flying apparatus of insects, and tendons of crustaceans.

22 Nishimura SI, Kai H, Shinada K, Yoshida T, Tokura S, Kurita K, Nakashima H, Yamamoto

Chitin and Chitinases

Biochemistry of chitin synthase

chitin synthase (eS)

Components of the CS reaction

Biochemistry of chitin synthase

Fig. 1, R j = priming polypeptide). In any case, the experimental evidence

To reiterate what is known of the chemical structure of chitin lies outside

R.A. Merz et a1.

Biochemistry of chitin synthase

Homotropic-heterotropic regulation with GlcNAc acting as an allosteric

Biochemistry of chitin synthase

be detected by the filtration assay) as the cause ofthis phenomenon can be

Non-allosteric activation or priming o/es

erating a glucosyl-tyrosine linkage [Glc(I-O)TyrI94; see [60] for amino

Biochemistry of chitin synthase

vide for a tight regulation of chitin synthesis in vivo, at the transcriptional

The topology ofthe site ofaction ofthe chitin synthesizing system at

Biochemistry of chitin synthase

"'~ .' "..t/~

o ,GlcNAc; l>-O, UDPGlcNAc; 00, chitobiose; the sign

Figure 3. Legend see p. 18

and two types of chitinases (A, B) differing in pH optimum, affinity

The -l,4-linked partial structures present in individual chitin chains

Biochemistry of chitin synthase

of the polymer, stretches generated de novo by es sensu stricto, and

R.A. Merz et a1.

Biochemistry of chitin synthase

at 100 J.1M), 16 S-CS ex chitosomes was slightly stimulated (some 30%

RA. Merz et al.

Biochemistry of chitin synthase

extracts obtained from the membrane-bound species simply by solubilization

Despite the wealth of articles on CS published during the last decade,

-210 . ---220 -- ---

-. ----- - -110. ----- - - -200. --- - -