JBC Hist Persp Glycobiology

HISTORICAL PERSPECTIVES
Glycobiology &
Carbohydrates
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The Journal of Biological Chemistry

TABLE OF CONTENTS
2010
HISTORICAL PERSPECTIVES ON GLYCOBIOLOGY
AND CARBOHYDRATES
PROLOGUE
H34 Proteoglycans and Orchids: the Work of Vincent Hascall
H1 JBC Historical Perspectives: Glycobiology and Carbohydrates.
H37 Lactose Synthesis in the Mammary Gland: Lactose Synthase
Nicole Kresge, Robert D. Simoni, and Robert L. Hill
and the Work of Robert L. Hill

H40 Lysosomal Storage Disease Factors: the Work of Elizabeth F.
CLASSICS
H3 Benedicts Solution, a Reagent for Measuring Reducing Sugars:
the Clinical Chemistry Work of Stanley R. Benedict

H5 The Discovery of Hyaluronan by Karl Meyer
H7 Otto Fritz Meyerhof and the Elucidation of the Glycolytic
Pathway
H10 Luis F. Leloir and the Biosynthesis of Saccharides
H13 Bernard L. Horeckers Contributions to Elucidating the Pentose
Phosphate Pathway
H15 The Entner-Doudoroff Pathway for Glucose Degradation: the
Work of Michael Doudoroff
Neufeld
H43 The Biosynthesis of Membrane Glycoproteins: the Work of
William J. Lennarz
H45 Hepatic Carbohydrate Binding Proteins and Glycoprotein
Catabolism: the Work of Gilbert G. Ashwell

H47 The Pathway of Complex Oligosaccharide Biosynthesis: the
Work of Stuart A. Kornfeld

H49 The Isolation and Localization of Laminin by Rupert Timpl
H51 The Formation of N-Glycosidic Linkages: the Work of Phillips
W. Robbins
H53 The Transient Glucosylation of Glycoproteins: the Work of
Armando J. Parodi
H17 Albert Dorfman and the Biosynthesis of Hyaluronic Acid

H20 Hexosamine Metabolism, Sialic Acids, and the
Phosphotransferase System: Saul Rosemans Contributions to

Glycobiology
H23 The Regulation of Glucose Uptake in Muscle: the Work of
Charles R. Park
H26 Plant Carbohydrates and the Biosynthesis of Lactose: the Work
of William Zev Hassid

H29 The Control of Gluconeogenesis: the Work of John Exton
H31 Mycobacterial Glycophosphoinositides: the Work of Clinton E.
Ballou
REFLECTIONS
H56 Reflections on Glycobiology. Saul Roseman
H72 The Pentose Phosphate Pathway. Bernard L. Horecker
H79 My Brief Encounter with the Phosphoinositides and IP3. Clinton
E. Ballou
H87 Lectins: Carbohydrate-specific Reagents and Biological
Recognition Molecules. Nathan Sharon

H99 In Search of the Message. John H. Exton
H108 From the -Glutamyl Cycle to the Glycan Cycle: A Road with
Many Turns and Pleasant Surprises. Naoyuki Taniguchi
JOURNAL OF BIOLOGICAL CHEMISTRY
PROLOGUE
This paper is available online at www.jbc.org

2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in the U.S.A.
JBC Historical Perspectives: Glycobiology and Carbohydrates*

Defined in the broadest sense, glycobiology is the study of

the roles of carbohydrates in cellular life. Carbohydrates are
the primary products of plant photosynthesis and the metabolic precursors of all other organic compounds. Often they
are covalently bound to proteins (glycoproteins) and lipids
(glycolipids) to form glycoconjugates. Many glycoconjugates
have structural roles. However, the carbohydrate groups of
glycoconjugates also can be involved in cellular processes
including adhesion, transformation, growth, endocytosis,
and fertilization.
The Classics and Reflections included in this collection trace
many of the discoveries that have led to our current knowledge
of these important molecules.
The earliest Classic paper in this collection, published just 3
years after the founding of the Journal in 1908, is an article by
Stanley R. Benedict reporting an analytical method for determining the reducing sugar content of biological fluids such as
urine. As explained in the Classic, the resulting Benedicts Solution, and the many variants that evolved from it, was used as the
reagent of choice for measuring sugar content for more than 50
years. Benedict also served as managing editor of the Journal of
Biological Chemistry (JBC) from 1926 until his death in 1936.
Carbohydrate Metabolism
Several of the articles in this collection explain the events
surrounding milestones in carbohydrate metabolism. For
example, Nobel laureate Otto Fritz Meyerhof published
three papers in the JBC in the mid-1940s detailing several
steps in the glycolytic pathway, the process whereby glucose
is converted into pyruvate and ATP. A few years later, in
1952, Michael Doudoroff published a JBC paper containing
experiments that eventually led to the formulation of the
Entner-Doudoroff pathway, a series of reactions that catabolize glucose to pyruvic acid using a set of enzymes different
from those used in either glycolysis or the pentose phosphate
pathway. Finally, in the 1960s, JBC Associate Editor John
Exton published two papers on the control of gluconeogenesis, the metabolic pathway that results in the generation of
glucose from non-carbohydrate carbon substrates such as
lactate, glycerol, and glucogenic amino acids. Exton used
isolated, perfused rat liver, which allowed them to study the
process and its regulation without the interference of other
changes in the body.
Carbohydrate Biosynthesis
Carbohydrate biosynthesis is a topic that also figures prominently in the collection. In the early 1950s, Luis F. Leloir published three papers in the JBC detailing the discovery of the
* To cite articles in this collection, use the citation information that appears in
the upper right-hand corner on the first page of the article.
nucleotide sugars uridine diphosphate glucose (UDPG),

UDP-N-acetylglucosamine (UDPAG), and guanosine diphosphate mannose (GDPM). The importance of this and
Leloirs other work on sugar nucleotides was recognized
when he received the Nobel Prize in Chemistry in 1970 for
his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.
Another seminal carbohydrate biosynthesis paper published
in the early 1950s was Bernard L. Horeckers article describing
the oxidation of 6-phosphogluconate and the metabolic intermediates involved in the formation of the pentose phosphate
pathway. To obtain these results, Horecker had to purify
6-phosphogluconate dehydrogenase from brewers yeast and
show that the first products in a TPN-dependent reaction were
carbon dioxide and ribulose 5-phosphate. The ribulose 5-phosphate was then converted to ribose 5-phosphate.
In 1960, Saul Roseman published a paper in the JBC on the
structures of one of the sialic acids, N-acetylneuraminic acid,
and the N-acetylneuraminic acid aldolase from Clostridium
perfringens. He also described the enzymatic synthesis of CMPsialic acid, the donor substrate in the synthesis of sialic acidcontaining oligosaccharides, and the requirements for the
acceptor substrate of CMP-sialic acids. About 10 years later,
Roseman published another JBC paper describing a novel series
of phosphotransferase reactions that transport sugars across
the bacterial membrane. Central in this work was Robert D.
Simoni, Deputy Editor of the JBC, who has made many JBC
innovations, including the JBC Classics.
A little more than 15 years after Horeckers paper was published, Clinton E. Ballou explained the biosynthesis of glycophosphoinositides in mycobacteria in his Classic paper. His coauthor Patrick Brennan, now at Colorado State University, is a
member of the JBC editorial board and continues to work on
the biochemistry and genetics of glycophosphoinositides in
these organisms.
Many advances in our knowledge about carbohydrate biosynthesis were made in the 1970s, including William J. Lennarzs description of the enzymatic synthesis of mannolipids in
Micrococcus lysodeikticus. As reported in his Classic, Lennarz
found that mannosyl phosphoryl undecaprenol participates in
this process. Subsequently, he elucidated the enzymatic transfer of mannose from GDP-mannose to endogenous acceptors
in bovine thyroid and hen oviduct that are associated with
membrane components of the cell. Stuart A. Kornfeld also published a Classic paper in 1978. His experiments described the
glycosylation of glycoproteins containing asparagine-linked
oligosaccharides. Kornfeld noted that a lipid-linked oligosaccharide is first formed and then transferred en bloc to an
asparagine-linked oligosaccharide, which was then partially
degraded before excess mannose residues were removed and
outer sugars were added.
H1
PROLOGUE: Glycobiology and Carbohydrates

Rounding out the biosynthesis collection is a 1982 paper by
Phillips W. Robbins in which he reports on the pathway of
N-linked oligosaccharide glycosylation in mammals and also
characterizes temperature-sensitive yeast mutants that had a
deficiency of the different enzymes involved in this process. A
year later, Armando J. Parodi explained the dolichol-phosphatedependent pathway of protein N-glycosylation in his Classic
JBC paper. Several protein-linked glycans were identified and
initially synthesized as dolichol-P-P-glycans.
Carbohydrates in Biology
Carbohydrates also play a prominent role in physiology and
disease, as exemplified by several other papers in the collection.
Charles R. Park was very interested in the hormonal regulation
of carbohydrate metabolism and performed a classic series of
experiments studying the regulation of glucose uptake in muscle by insulin and other hormones. He used isolated perfused
rat heart, and his work resulted in the publication of a series of
six Classic papers in the JBC in 1961. He determined that the
limiting step for glucose uptake was the transport of the sugar
across the cell membrane, which was accelerated by insulin and
anoxia.
In the mid-1960s, William Zev Hassid published two Classic
JBC papers reporting the first studies of lactose biosynthesis in
mammary glands. He recognized that lactose was synthesized
from UDP-galactose and glucose and that partial purification
and some of the properties of the galactosyltransferase were
responsible for synthesis of lactose. In the early 1970s, JBC
Associate Editor Robert L. Hill published several papers in
which he reported the complete amino acid sequence and the
location of four disulfide bonds in the milk protein -lactalbumin, one of two proteins that are required for lactose synthesis.
Hill noted that the covalent structure of -lactalbumin was very
similar to egg white lysozyme and proposed that the two proteins arose from a common ancestral gene. He also purified the
second protein required for lactose synthesis, galactosyltransferase, which normally catalyzes synthesis of N-acetylgalactosamine but, in the presence of -lactalbumin, synthesizes
lactose.
The 1961 Gilbert G. Ashwell Classic describes membrane
proteins (lectins) that remove senescent glycoproteins from
blood. Ashwell identified two types, one from rabbit liver that
binds terminal galactose and the other from chicken liver that
binds proteins with terminal N-acetylglucosamine. His work on
hepatic binding proteins has served as a stimulus for the iden-

H2
tification of a host of carbohydrate-specific receptors on various cell surfaces and has inaugurated the current concept of a
cellular lectin.
The inability to remove carbohydrates from the body can
cause disease. For example, a group of lysosomal storage diseases called mucopolysaccharidoses (MPS) and related disorders result from the failure to properly store or metabolize
mucopolysaccharides. Elizabeth F. Neufeld studied three of
these diseases, Hurler syndrome, Sanfilippo syndrome, and
Hunter syndrome, and their corrective factors. She discovered
that Hurler syndrome was corrected by -L-iduronidase, Sanfilippo syndrome by heparan sulfate sulfatase, and Hunter syndrome by iduronate sulfatase and published these results
between 1971 and 1972 in three JBC papers.
Hyaluronan (also called hyaluronic acid or hyaluronate) is an
anionic, non-sulfated glycosaminoglycan distributed widely
throughout connective, epithelial, and neural tissues. Many of
the experiments leading to our current understanding of this
molecule were published in the JBC. In his 1934 paper, Karl
Meyer described the repeating disaccharides that are the basic
unit of the hyaluronan polymer, namely glucuronate--1,3-Nacetylglucosamine-1,4. Nineteen years later, Albert Dorfman
detailed the synthesis of hyaluronic acid from UDP-glucuronic
acid and UDP-N-acetylglucosamine in a cell-free system from
streptococci. Sixteen years after Dorfmans paper was published, JBC Associate Editor Vincent Hascall showed how the
proteoglycan aggregates from cartilage were formed by noncovalent binding of glycosaminoglycans to hyaluronic acid with
the aid of a small molecular weight link protein in three JBC
papers. Finally, in 1979, Rupert Timpl described the characterization of extracellular matrix glycoproteins in basement membranes in his JBC paper. A high molecular weight non-collagenous glycoprotein that was a major constituent of tumors was
identified and named laminin.
Reflections
The collection also contains six JBC Reflections articles written by several of the above scientists, including Clinton E. Ballou, John H. Exton, Bernard L. Horecker, and Saul Roseman,
who explain many of the seminal discoveries in carbohydrate
biochemistry in their own words. Also included are Reflections
by Nathan Sharon and Naoyuki Taniguchi.
This is just a brief overview of the many papers that we have
assembled in this collection. We hope you take the time to read
them, and that you find them both enjoyable and educational.
THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 277, No. 16, Issue of April 19, p. e5, 2002

Printed in U.S.A.
Classics
A PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
19052005
100 Years of Biochemistry and Molecular Biology
Benedicts Solution, a Reagent for Measuring Reducing

Sugars: the Clinical Chemistry of Stanley R. Benedict
A Reagent For the Detection of Reducing Sugars
(Benedict, S. R. (1908) J. Biol. Chem. 5, 485 487)
Stanley Rossiter Benedict was born in Cincinnati in 1884. While a student at the University
of Cincinnati he worked with J. F. Snell, and together they published nine papers describing
new analytical methods in inorganic chemistry. This research experience as a college student
provided the intellectual foundation for his career. After a mistaken year in medical school at
Cincinnati, he went to Yale, to the Department of Physiological Chemistry, to study with
Russell Chittenden and Lafayette Mendel where he received training in metabolism and
physiology. He received his Ph.D. in 1908, 2 years after entering graduate school. (Current
students take note.) In 1910, he became Professor of Chemistry at Cornell University Medical
College, the position he held until his death in 1936 at the age of 52 (1).
In a biographical review of Benedicts career, E. V. McCollum wrote, It is not possible to give
an accurate account of the scientific work of Stanley Benedict without at the same time
discussing the parallel researches of Otto Folin . . . they succeeded, through many years of
intensive investigations, in devising and refining analytical procedures for determination of
minute amounts of the principal non-protein constituents of blood and urine so that, for the
first time, chemical analysis became a highly useful technic (sic) for the discovery of the
chemical processes in the normal functioning of the body (1).
Of Benedicts relationship with Folin, Shaffer wrote, Both excelled in designing very clever
analytical methods of the widest usefulness, and in using these tools with rare success for the
discovery of new facts about metabolism. In spite of seventeen years difference in their age
(Folin was the older), of the rivalry and controversy sometimes evident in their papers, there
early developed between them a warm friendship which reveals the fine character of both.
They were kindred spirits (2). (We will present a classic paper by Otto Folin in a subsequent
installment of JBC Classics, stay tuned.)
As McCollum and Shaffer described, Benedicts major contributions to biochemistry were in
devising analytical methods. Although he published many papers in the Journal of Biological
Chemistry (JBC), the paper reprinted here seemed appropriate to characterize a distinguished
career. It had been known for many years that the common sugars had carbonyl groups and
were therefore, reducing sugars. That is, they were oxidized by a variety of metal ions, Ag,
Fe3, and Cu2. Treatment with hot alkali fragments the sugars, and the resulting products
reduce Cu2 to Cu with the formation of a precipitate of Cu2O. As noted in the paper,
Benedicts goal was to improve this general method to make the reagent less corrosive and
more stable. He accomplished this by substituting carbonate for hydroxide as the alkali
component, to reduce the corrosiveness, and by substituting citrate for tartrate as the agent to
chelate the Cu2, to make the reagent more stable.
Benedicts Solution, or one of the many variants that evolved over the years, was used as the
reagent of choice for measuring sugar content for more than 50 years. It was the most common
test for diabetes and was the standard procedure for virtually all clinical laboratories. Saul
Roseman remembers that all inductees into the army during World War II had their urine
H3
This paper is available on line at http://www.jbc.org
Classics
Stanley R. Benedict. Photo courtesy of the National Library of Medicine.
tested for sugar with Benedicts Solution.1 Although Benedicts assay was the method of choice
for more than 50 years, it suffered from lack of sugar specificity and was eventually supplanted
by the use of enzymatic methods such as glucose oxidase.
Benedicts work on analytical methods was particularly important for clinical applications.
There was, for many years, a very close relationship between basic biochemistry research and
biochemical clinical applications. Many biochemists were employed as clinical chemists because academic jobs as biochemists were difficult to find. Many of the methods that have been
taken for granted for many years find their origins with biochemists working in clinical
laboratories.
Benedict was active in the Society and the JBC. He served as Secretary of the Society and,
in 1919, served as President. It was during his tenure as President that the JBC was
transferred to the Society for management. (Officially the financial relationship between the
Society and the Journal Corporation was not finalized until 1942.) Benedict became a member
of the JBC Editorial Board and in 1926 became Managing Editor, a position he held until his
death in 1936. An interesting characterization of Benedicts service as JBC Managing Editor
is made in the obituary of Benedict written by Philip A. Shaffer (2), With many other
contributor (sic), the present writer has occasionally smarted under sometimes sharp criticism
of the editor; but rarely if ever were the criticisms unjustified. His standards were high, he
expected clarity, logic, and brevity in exposition, his opinions were definite and outspoken his
judgements were based on essential facts and were impartial as to individuals. (During this
period of the Journal, and later as well, all communication between authors and the Journal
was conducted personally by the Managing Editor.)
Robert D. Simoni, Robert L. Hill, and Martha Vaughan
REFERENCES
1. McCollum, E. V. (1974) Memoir of Stanley Rossiter Benedict, Vol. 27, National Academy of Sciences, Washington,
D. C.
2. Shaffer, P. A. (1937) Obituary for Stanley Rossiter Benedict. J. Biol. Chem. 117, 428
1
Much of the background information for sugar chemistry and clinical usage of Benedicts Reagent was kindly
provided by Professor Saul Roseman, Professor of Biology, Johns Hopkins University. Professor Roseman knew
Stanley R. Benedict personally.
H4

Vol. 277, No. 39, Issue of September 27, p. e27, 2002

Printed in U.S.A.
Classics
JBC Centennial
19052005
The Discovery of Hyaluronan by Karl Meyer

The Polysaccharide of the Vitreous Humor
(Meyer, K., and Palmer, J. W. (1934) J. Biol. Chem. 107, 629 634)
Karl Meyer (1899 1990) was born in the village of Karpen, Germany, near Cologne. In 1917,
he was drafted into the German army and served in the last year of World War I. After the war,
he entered medical school at the University of Cologne and received the M.D. degree in 1924.
He then went to Berlin for a year of study in medical chemistry and met several promising
young biochemists including Fritz Lippman, Hans Krebs, and Ernst Chain. To gain more
training in chemistry, he enrolled as a graduate student with Otto Meyerhof. For his Ph.D.
thesis work, he investigated lactic acid formation in yeast and muscle demonstrating the
requirement for a co-enzyme later identified as ADP. His research career was launched.
After three years as a Rockefeller Fellow in Zurich studying heme-catalyzed oxidation of
unsaturated compounds with Professor Kuhn, he was offered a position as Assistant Professor
at the University of California at Berkeley with Herbert Evans.
In 1932, while at Berkeley, he attended a conference in Europe. During the conference, he
received notice that Evans had terminated his position with the suggestion that he stay in
Germany. He decided, no doubt in part because of the rising anti-Semitism in Europe and the
increasing probability of war, to go back to the United States. His return was facilitated by
Hans T. Clarke at Columbia University who provided interim fellowship support until 1933
when he received a position as Assistant Professor in the Department of Ophthalmology at the
College of Physicians and Surgeons. In part because of the mission of his department, Meyer
began to study the lysozyme present in tears and undertook to identify a physiological
substrate for the enzyme. Examination of the viscous vitreous humor of the eye as a plausible
source of substrate quickly led to the discovery of hyaluronan, which is reported in this
Journal of Biological Chemistry (JBC) Classic.
Meyer and his assistant John Palmer isolated a novel, high molecular weight polysaccharide
and reported that it was composed of a uronic acid, an amino sugar, and possibly a pentose.
(The last is incorrect.) They proposed for convenience, the name hyaluronic acid, from hyaloid
(vitreous) uronic acid. Nearly 25 years of work were required to establish the structure of
the repeating disaccharide that is the basic unit of the hyaluranan polymer, namely
glucuronate--1,3-N-acetylglucosamine-1,4-.
Hyaluronan is one member of a family of glycosaminoglycans that includes chondroitin/
dermatan sulfate, keratan sulfate, and heparin/heparan sulfate, each with a characteristic
disaccharide-repeat structure of an amino sugar, either glucosamine or galactosamine, plus a
negatively charged sugar, a carboxylate and/or a sulfate. The polymers are found as cell
surface molecules and in the extracellular matrix. Glycosaminoglycans, with the exception of
hyaluronan, are covalently bound to proteins to form proteoglycans. These ubiquitous and
structurally diverse macromolecules are found as cell surface molecules and in the extracellular matrix. The multiplicity of their functions that is now recognized was not always
appreciated. In a symposium at the 1958 annual meeting of the American Society of Biological
Chemists entitled Acid Mucopolysaccharides of Animal Origin, which was chaired by Meyer,
he states in an opening remark, It is my opinion that the mucopolysaccharides will never be
a highly popular field in biochemistry, but they will probably not be relegated again to the
insignificance and disregard in which they were held not so long ago.
H5
Classics
Karl Meyer. Photo courtesy of the National Library of Medicine.
Meyers scientific contributions were not limited to the discovery of hyaluronan. He is

considered the father of glycosaminoglycan chemistry and received many honors including
election to the National Academy of Sciences in 1967. On the occasion of his induction he
commented as follows: Looking back on my scientific career, I have often wondered whether
it was worthwhile to stick so tenaciously to a technically difficult and conceptually apparently
unexciting field, while my colleagues and friends shifted over to more fashionable and rewarding areas. The reasons for my persistence are manifold, among them a distaste for jumping in
on ground broken by others. Besides, I felt committed to problems such as the biological
functions of the mucopolysaccharides of connective tissues to their role in differentiation, in
cell membranes and in inherited diseases.
His persistence was biochemistrys gain.1
Robert D. Simoni, Robert L. Hill, Martha Vaughan, and Vincent Hascall
REFERENCES
1. McDonald, J., and Hascall, V. C. (2002) J. Biol. Chem. 277, 4575 4579
1
A recent overview of glycosaminoglycan biochemistry and additional information about Karl Meyer are included
in the JBC Hyaluronan Minireview Series edited by John McDonald and Vincent Hascall (1).
H6

Vol. 280, No. 4, Issue of January 28, p. e3, 2005

Printed in U.S.A.
Classics
JBC Centennial
19052005
Otto Fritz Meyerhof and the Elucidation of the Glycolytic

Pathway
The Equilibria of Isomerase and Aldolase, and the Problem of the Phosphorylation
of Glyceraldehyde Phosphate
(Meyerhof, O., and Junowicz-Kocholaty, R. (1943) J. Biol. Chem. 149, 7192)
The Origin of the Reaction of Harden and Young in Cell-free Alcoholic Fermentation
(Meyerhof, O. (1945) J. Biol. Chem. 157, 105120)
The Mechanism of the Oxidative Reaction in Fermentation
(Meyerhof, O. and Oesper, P. (1947) J. Biol. Chem. 170, 122)
The elucidation of the glycolytic pathway, the process whereby glucose is converted into
pyruvate and ATP, began in 1860 when Louis Pasteur observed that microorganisms were
responsible for fermentation. Several years later, in 1897, Eduard Buchner made the significant discovery that cell-free extracts could carry out fermentation. The next important
contribution was from Arthur Harden and William Young in 1905. They realized that inorganic phosphate was necessary for glycolysis and that fermentation requires the presence of
both a heat-labile component they called zymase and a low molecular weight, heat-stable
fraction called cozymase. (It was later shown that zymase contains a number of enzymes
whereas cozymase consists of metal ions, ATP, ADP, and coenzymes such as NAD.) Building
on these initial observations, the complete glycolytic pathway was elucidated by 1940 by the
combined efforts of several scientists including Otto Fritz Meyerhof (1884 1951).
Meyerhof was born in Hanover, Germany and grew up in Berlin. In 1909, he graduated as
a doctor of medicine from the University of Heidelberg. Around this time, Ludolf von Krehl was
building a small research program on metabolism at the University of Heidelberg Medical
Clinic, and he offered Meyerhof a position in his laboratory. There, Meyerhof met Otto
Warburg whose innovative ideas and confident approach inspired him to focus his career on
physiological chemistry.1
In 1912, Meyerhof took a position at the University of Kiel. A year later, he delivered a
lecture on the energetics of living cells, one of the very first adaptations of the physical laws
of thermodynamics to physiological chemistry. Meyerhof had recognized that after energy is
input as food it is transformed through a series of intermediate steps and finally dissipated as
heat. He soon began using muscle to look at energy transformations and chemical changes
during cellular function. Meyerhof was also interested in analogies between oxygen respiration in muscle and alcoholic fermentation in yeast and proved, in 1918, that the coenzymes
involved in lactic acid production were the same as the yeast coenzymes discovered by Harden
and Young, revealing an underlying unity in biochemistry.
Soon after World War I, Meyerhof began collaborating with Archibald Vivian Hill who was
investigating heat production in muscle. The pair worked to decipher metabolism in terms of
heat development, mechanical work, and cellular chemical reactions. Meyerhof determined
that glycogen is converted to lactic acid in the absence of oxygen and showed that in the
presence of oxygen only a small portion of lactic acid is oxidized and the rest is converted back
to glycogen. This discovery of the lactic acid cycle provided the first evidence of the cyclical
1
All biographical information on Otto Fritz Meyerhof was taken from Ref. 6.
H7
Classics
Otto F. Meyerhof. Photo courtesy of the National Library of Medicine.
nature of energy transformation in cells. These results also confirmed and extended Louis
Pasteurs theory (now called the Pasteur-Meyerhof effect) that less glycogen is consumed in
muscle metabolism in the presence of oxygen than in its absence. Meyerhof and Hill won the
Nobel Prize in Physiology or Medicine in 1922 for their analysis of the lactic acid cycle and its
relation to respiration.
Two years after wining the Nobel Prize, Meyerhof joined the Kaiser Wilhelm Institutes in
Berlin-Dahlem. Then, in 1929, he took charge of the newly founded Kaiser Wilhelm Institute
for Medical Research at Heidelberg.
By this time, it was clear that glycolysis was far more complicated than anyone had
imagined. The sheer number of components and their short lived nature made the task of
sorting out the pathway daunting. However, during his time at Heidelberg, Meyerhofs group
was extremely successful at breaking down glycolysis into its many separate components. In
1932, Meyerhof made the first associations between the uptake of phosphate during the
breakdown of carbohydrates to lactic acid and the splitting of ATP. By 1934, Kurt Lohmann in
Meyerhofs laboratory provided direct evidence that ATP synthesis was the byproduct of
utilization of glucose. Lohmann also established that creatine phosphate is an energy source
for ATP phosphorylation, which led Meyerh of to the conclusion that the energy release from
ATP hydrolysis was the primary event leading to muscle contraction.
By the 1930s Meyerhof had managed to isolate and purify the co-enzymes involved in the
conversion of glycogen to lactic acid and had reconstructed the main steps of this set of
reactions in cell-free solution. All in all, Meyerhofs group discovered more than one-third of
the enzymes involved in glycolysis. In 1932, Gustav Embden constructed a detailed proposal
for reaction sequences for almost the entire glycolytic pathway. Over the next 5 years,
Meyerhof, along with Warburg, Jacob Parnas, Carl Neuberg, Gerti and Karl Cori, and Hans
von Euler worked out the details of glycolysis, which is often referred to as the EmbdenMeyerhof pathway.
With Adolf Hitlers rise to power, Meyerhof left Germany in 1938 and became director of the
Institut de Biologie Physiochimique in Paris. In 1940, when the Nazis invaded France,
Meyerhof fled to the United States where the post of Research Professor of Physiological
Chemistry was created for him by the University of Pennsylvania and the Rockefeller Foundation. He remained at Pennsylvania where he continued to study metabolism until his death.
The three Journal of Biological Chemistry (JBC) Classics reprinted here are from Meyerhofs
time at Pennsylvania.
H8
Classics
The first paper deals with one of the intermediate reactions that occurs in glycolysis: the
splitting of hexose diphosphate (now known as fructose 1,6-bisphosphate) into two triose
phosphate isomers, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, by zymohexase (fructose-1,6-bisphosphate aldolase). Triose-phosphate isomerase then converts dihydroxyacetone phosphate into glyceraldehyde 3-phosphate. In the next step of glycolysis,
glyceraldehyde 3-phosphate is oxidized and phosphorylated to become 1,3-diphosphoglyceric
acid. Warburg and Christian (1, 2) and Negelein and Bromel (3, 4) proposed that this step
occurs through the intermediate 1,3-diphosphoglyceraldehyde with the aid of an oxidizing
enzyme and cozymase. If this were true, then inorganic phosphate could be used to remove
glyceraldehyde 3-phosphate from the hexose diphosphate reaction.
To investigate this matter further, Meyerhof and Renate Junowicz-Kocholaty redetermined
the equilibrium constant for the isomerase and aldolase reactions in the presence and absence
of inorganic phosphate, cozymase, and Warburgs oxidizing enzyme. They found that their
values agreed with those previously determined and that equilibrium is not influenced by the
presence of inorganic phosphate, cozymase, or Warburgs enzyme. They were also unable to
detect the formation of any substance that would break down into glyceraldehyde phosphate
and phosphate, prompting them to write that Warburgs claims of a diphosphoglyceraldehyde
intermediate may have been premature.
The second Classic deals with the next two steps of glycolysis shown as Reactions 1 and 2.
Glyceraldehyde 3-phosphate phosphate cozymase ^
1,3-diphosphoglyceric acid dihydrocozymase
REACTION 1
1,3-Diphosphoglyceric acid ADP ^ 3-phosphoglyceric acid ATP
REACTION 2
Harden and Young stated that during fermentation, one sugar molecule is fermented to CO2
and alcohol while a second is esterfied to hexose diphosphate (5). In a cell-free system, this
reaction can be divided into two phases, a rapid phosphate period and a slower phase that
depends on the rate of hexose diphosphate fermentation. Meyerhof proposed that the rate of
the hexose diphosphate reaction was much slower in cell-free systems than in live yeast
because the majority of the enzyme needed to split ATP, adenylpyrophosphatase (apyrase),
was lost during the extraction process. He backed up his claim by studying the distribution of
apyrase in the yeast cell and showing that it remains mainly with solid elements that are not
used in cell-free systems. Meyerhof also purified apyrase from potatoes and added it to cell-free
preparations to prove that it raises the rate of hexose diphosphate fermentation.
The final JBC Classic revisits the phosphorylation of glyceraldehyde 3-phosphate and its
subsequent oxidation. In this paper, Meyerhof and Peter Oesper use a Beckman spectrophotometer to follow the reaction and provide further proof that a diphosphoglyceric aldehyde
intermediate does not exist. They also alter the equation for this step of glycolysis to reflect the
fact that the reduction of cozymase is accompanied by the formation of an H ion.
REFERENCES
1.
2.
3.
4.
5.
6.
Warburg, O., and Christian, W. (1939) Biochem. Z. 301, 201

Warburg, O., and Christian, W. (1939) Biochem. Z. 303, 40
Negelein, E., and Bromel, H. (1939) Biochem. Z. 301, 135
Negelein, E., and Bromel, H. (1939) Biochem. Z. 303, 132
Harden A., and Young, W. J. (1908) Proc. R. Soc. Lond. Ser. B Biol. Sci. 80, 299
States, D. M. Otto Meyerhof and the Physiology Department: the Birth of Modern Biochemistry. A History of the
Max Planck Institute for Medical Research (http://sun0.mpimf-heidelberg.mpg.de/History/Meyerhof.html)
H9

Vol. 280, No. 19, Issue of May 13, p. e16, 2005

Printed in U.S.A.
Classics
JBC Centennial
19052005
Luis F. Leloir and the Biosynthesis of Saccharides

Isolation of the Coenzyme of the Galactose Phosphate-Glucose Phosphate
Transformation
(Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184,
333350)
Uridine Diphosphate Acetylglucosamine
(Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 10551070)
Guanosine Diphosphate Mannose
(Cabib, E., and Leloir, L. F. (1954) J. Biol. Chem. 206, 779 790)
Luis Federico Leloir (1906 1987) was born in Paris but moved to Buenos Aires with his
Argentine parents when he was 2 years old. He attended the University of Buenos Aires and
graduated with an M.D. in 1932. Leloir got a job at the University hospital but left the bedside
for the bench 2 years later. As he recalled, When I practiced medicine, except for surgery,
digitalis, and a few other active remedies, we could do little for our patients. Antibiotics,
psychoactive drugs, and all the new therapeutic agents were unknown. It was therefore not
strange in 1932 that a young doctor such as I should try to join efforts with those who were
trying to advance medical knowledge (1).1
The most active research laboratory in town was run by Bernardo A. Houssay, who would
later be awarded the Nobel Prize with Carl and Gerty Cori for their work on the role of the
pituitary gland in carbohydrate metabolism. Leloir joined Houssays laboratory as a graduate
student and studied the role of the adrenals in carbohydrate metabolism.
After Leloir finished his thesis work, Houssay advised him to study abroad. So, in 1936
Leloir moved to England to work at the Biochemical Laboratory of Cambridge University.
There, he collaborated with Malcolm Dixon on the effect of cyanide and pyrophosphate on
succinic acid dehydrogenase, Norman L. Edson on ketogenesis using liver slices, and David E.
Green on the purification and properties of -hydroxybutyrate dehydrogenase.
Leloir returned to Buenos Aires after his time at Cambridge and started investigating the
oxidation of fatty acids in the liver with J. M. Munoz. They managed to produce an active
cell-free system, which was an accomplishment since at that time it was thought that oxidation
could only occur in intact cells. Leloir also worked with E. Braun Menendez, Juan Carlos
Fasciolo, and A. C. Taquini on the mechanism of renal hypertension and the formation of
angiotensin.
In 1944, Leloir left Buenos Aires again. This time he went to Washington University in St.
Louis to work with Carl and Gerty Cori, who were featured in a previous Journal of Biological
Chemistry (JBC) Classic (2). While in the States, Leloir reunited with Green and spent some
time at the College of Physicians and Surgeons at Columbia University working on the
purification of aminotransferases.
After his stay in the United States, Leloir returned to the Institute of Physiology in Buenos
Aires. He worked there for a time and then left for a private institution recently created, the
Instituto de Investigaciones Bioquimicas Fundacion Campomar (now Fundacion Instituto
Leloir), where he remained until his death. In collaboration with Ranwel Caputto, Carlos E.
1
All biographical information on Luis F. Leloir was taken from Refs. 1 and 8. We thank Armando J. Parodi, Ph.D.,
of the Fundacion Instituto Leloir, for helpful comments in the preparation of this JBC Classic Introduction.
H10
Classics
Luis F. Leloir. Photo courtesy of the National Library of Medicine.
Cardini, Raul Trucco, and Alejandro C. Paladini, Leloir started to work on the metabolism of
galactose. The project was initiated when Caputto presented some preliminary results that
indicated that mammary gland homogenates could produce lactose when incubated with
glycogen. The group performed many experiments with mammary gland extracts but generally got ambiguous results, mainly due to their lack of a reliable method for lactose detection.
Discouraged, they decided to focus on the breakdown of lactose by Saccharomyces fragilis,
hoping that this would give them information on the mechanism of lactose synthesis.
Leloir and his colleagues isolated lactase from the yeast and determined that galactose was
phosphorylated to produce galactose 1-phosphate. They synthesized glucose 1-phosphate and
galactose 1-phosphate and observed that the esters were used when incubated with enzymes
from galactose-adapted yeast. At first they thought that only one factor was required for this
reaction, but soon realized that two factors were involved: one for the conversion of galactose
1-phosphate into glucose 1-phosphate and another for the formation of glucose 6-phosphate, as
shown in the following reaction.
Galactose 1-phosphate 3 glucose 1-phosphate 3 glucose 6-phosphate
Factor 1
Factor 2
REACTION 1
The group first concentrated on finding Factor 2 and eventually determined that it was
glucose 1,6-diphosphate (3). Next, they turned their attention to identifying Factor 1. The
purified factor absorbed light at 260 nm and had a spectrum similar to that of adenosine, with
some differences. They were stumped on the identity of this factor for quite some time until
Caputto came in one morning with an issue of the JBC that showed a spectrum identical to
theirs. The spectrum was that of uridine. The group published their results in a preliminary
communication in Nature (4) and then in the JBC, which is the first classic reprinted here. In
addition to uridine, the co-factor was found to contain glucose and two phosphates and hence
was named uridine diphosphate glucose (UDPG). The presence of uridine in a co-factor was
rather novel as, until then, all known factors (ATP, NAD, FAD) only contained the nucleotide
adenosine. The occurrence of a sugar derivative combined with a nucleoside was also novel.
Eventually, Leloir determined that UDPG acts as a glucose donor in the synthesis of trehalose
(5), sucrose (6), and glycogen (7).
Another result of the discovery of UDPG was the isolation and characterization of the sugar
nucleotides UDP-N-acetylglucosamine (UDPAG) and guanosine diphosphate mannose
(GDPM), which are the subjects of the remaining two JBC Classics reprinted here. UDPAG
was originally detected as an impurity in UDPG concentrates and was called UDP-X until
Leloir was able to identify the sugar moiety as N-acetylglucosamine. Similarly, GDPM was
first detected by paper chromatography of UDPG preparations that were purified by anion
exchange. UDPAG and GDPM are now known to be involved in the biosynthesis of numerous
glycoconjugates.
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Classics
Leloirs extensive work on sugar nucleotides and his contributions to biochemistry received
the recognition they deserved when he was awarded the Nobel Prize in Chemistry in 1970, for
his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.
REFERENCES
1. Leloir, L. F. (1983) Far away and long ago. Annu. Rev. Biochem. 52, 115
2. JBC Classics: Cori, C. F., and Cori, G. T. (1928) J. Biol. Chem. 79, 321341; Cori, G. T., Colowick, S. P., and Cori,
C. F. (1938) J. Biol. Chem. 124, 543555; Cori, G. T., Colowick, S. P., and Cori, C. F. (1939) J. Biol. Chem. 127,
771782; Green, A. A., and Cori, G. T. (1943) J. Biol. Chem. 151, 2129; Cori, G. T., and Green, A. A. (1943)
J. Biol. Chem. 151, 3138 (http://www.jbc.org/cgi/content/full/277/29/e18)
3. Cardini, C. E., Paladini, A. C., Caputto, R., Leloir, L. F., and Trucco, R. E. (1949) Arch. Biochem. 22, 87
4. Cardini, C. E., Paladini, A. C., Caputto, R., and Leloir, L. F. (1950) Uridine diphosphate glucose: the coenzyme of
the galactose-glucose phosphate isomerization. Nature 165, 191193
5. Leloir, L. F., and Cabib, E. (1953) The enzymic synthesis of trehalose phosphate. J. Am. Chem. Soc. 75, 54455446
6. Cardini, C. E., Leloir, L. F., and Chiriboga, J. (1955) The biosynthesis of sucrose. J. Biol. Chem. 214, 149 155
7. Leloir, L. F., and Cardini, C. E. (1957) Biosynthesis of glycogen from uridine diphosphate glucose. J. Am. Chem.
Soc. 79, 6340
8. Leloir, L. F. (1971) Two decades of research on the biosynthesis of saccharides. Science 172, 1299 1302
H12

Vol. 280, No. 29, Issue of July 22, p. e26, 2005

Printed in U.S.A.
Classics
JBC Centennial
19052005
Bernard L. Horeckers Contributions to Elucidating the

Pentose Phosphate Pathway
The Enzymatic Conversion of 6-Phosphogluconate to Ribulose-5-Phosphate and
Ribose-5-Phosphate
(Horecker, B. L., Smyrniotis, P. Z., and Seegmiller, J. E. (1951) J. Biol. Chem. 193,
383396)
Bernard Leonard Horecker (1914) began his training in enzymology in 1936 as a graduate
student at the University of Chicago in the laboratory of T. R. Hogness. His initial project
involved studying succinic dehydrogenase from beef heart using the Warburg manometric
apparatus. However, when Erwin Hass arrived from Otto Warburgs laboratory he asked
Horecker to join him in the search for an enzyme that would catalyze the reduction of
cytochrome c by reduced NADP. This marked the beginning of Horeckers lifelong involvement
with the pentose phosphate pathway.
During World War II, Horecker left Chicago and got a job at the National Institutes of
Health (NIH) in Frederick S. Bracketts laboratory in the Division of Industrial Hygiene. As
part of the wartime effort, Horecker was assigned the task of developing a method to determine the carbon monoxide hemoglobin content of the blood of Navy pilots returning from
combat missions. When the war ended, Horecker returned to research in enzymology and
began studying the reduction of cytochrome c by the succinic dehydrogenase system.
Shortly after he began these investigation changes, Horecker was approached by future
Nobel laureate Arthur Kornberg, who was convinced that enzymes were the key to understanding intracellular biochemical processes. Kornberg suggested they collaborate, and the
two began to study the effect of cyanide on the succinic dehydrogenase system. Cyanide had
previously been found to inhibit enzymes containing a heme group, with the exception of
cytochrome c. However, Horecker and Kornberg found that cyanide did in fact react with
cytochrome c and concluded that previous groups had failed to perceive this interaction
because the shift in the absorption maximum was too small to be detected by visual
examination.
Two years later, Kornberg invited Horecker and Leon Heppel to join him in setting up a new
Section on Enzymes in the Laboratory of Physiology at the NIH. Their Section on Enzymes
eventually became part of the new Experimental Biology and Medicine Institute and was later
renamed the National Institute of Arthritis and Metabolic Diseases.
Horecker and Kornberg continued to collaborate, this time on the isolation of DPN and TPN.
By 1948 they had amassed a huge supply of the coenzymes and were able to present Otto
Warburg, the discoverer of TPN, with a gift of 25 mg of the enzyme when he came to visit.
Horecker also collaborated with Heppel on the isolation of cytochrome c reductase from yeast
and eventually accomplished the first isolation of the flavoprotein from mammalian liver.
Along with his lab technician Pauline Smyrniotis, Horecker began to study the enzymes
involved in the oxidation of 6-phosphogluconate and the metabolic intermediates formed in the
pentose phosphate pathway. Joined by Horeckers first postdoctoral student, J. E. Seegmiller,
they worked out a new method for the preparation of glucose 6-phosphate and 6-phosphogluconate, both of which were not yet commercially available. As reported in the Journal of
Biological Chemistry (JBC) Classic reprinted here, they purified 6-phosphogluconate dehydrogenase from brewers yeast (1), and by coupling the reduction of TPN to its reoxidation by
H13
Classics
FIG. 1
pyruvate in the presence of lactic dehydrogenase, they were able to show that the first product
of 6-phosphogluconate oxidation, in addition to carbon dioxide, was ribulose 5-phosphte. This
pentose ester was then converted to ribose 5-phosphate by a pentose-phosphate isomerase.
They were able to separate ribulose 5-phosphate from ribose 5-phosphate and demonstrate
their interconversion using a recently developed nucleotide separation technique called ionexchange chromatography. Horecker and Seegmiller later showed that 6-phosphogluconate
metabolism by enzymes from mammalian tissues also produced the same products.
Over the next several years, Horecker played a key role in elucidating the remaining steps
of the pentose phosphate pathway. His total contributions included the discovery of three new
sugar phosphate esters, ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose
4-phosphate, and three new enzymes, transketolase, transaldolase, and pentose-phosphate
3-epimerase. The outline of the complete pentose phosphate cycle was published in 1955 (2).
Horeckers personal account of his work on the pentose phosphate pathway can be found in his
JBC Reflection (3).1
Horeckers contributions to science were recognized with many awards and honors including
the Washington Academy of Sciences Award for Scientific Achievement in Biological Sciences
(1954) and his election to the National Academy of Sciences in 1961. Horecker also served as
president of the American Society of Biological Chemists (now the American Society for
Biochemistry and Molecular Biology) in 1968.
REFERENCES
1. Horecker, B. L., and Smyrniotis, P. Z. (1951) Phosphogluconic acid dehydrogenase from yeast. J. Biol. Chem. 193,
371381
2. Gunsalus, I. C., Horecker, B. L., and Wood, W. A. (1955) Pathways of carbohydrate metabolism in microorganisms.
Bacteriol. Rev. 19, 79 128
3. Horecker, B. L. (2002) The pentose phosphate pathway. J. Biol. Chem. 277, 47965 47971
All biographical information on Bernard L. Horecker was taken from Ref. 3.
H14


Printed in U.S.A.
Classics
JBC Centennial
19052005
The Entner-Doudoroff Pathway for Glucose Degradation:

the Work of Michael Doudoroff
Glucose and Gluconic Acid Oxidation of Pseudomonas saccharophila
(Entner, N., and Doudoroff, M. (1952) J. Biol. Chem. 196, 853 862)
Michael Doudoroff (19111975) was born in St. Petersburg, Russia but moved to San
Francisco when he was 12 years old. He entered Stanford University in 1929, planning to
major in biology and specialize in entomology. However, as his exposure to different types of
science broadened, he became fascinated with bacteriology and protozoology. As a result, he
studied the survival of Paramecium at elevated temperatures for his Masters thesis with A. C.
Giese and the adaptation of E. coli to elevated salt concentrations for his Ph.D. thesis with
C. B. van Niel at the Hopkins Marine Station.
While assisting van Niel in a course in general microbiology at the Marine Station, Doudoroff was introduced to the physiological and biochemical diversity of the microbial world. He
subsequently started to study luminous bacteria, resulting in his discovery that riboflavin is
directly involved in bacterial luminescence. Doudoroff also isolated a new species of H2oxidizing bacteria, Pseudomonas saccharophila, which could oxidize a number of mono-, di-,
and polysaccharides. This was a surprising discovery because most bacteria only oxidize diand polysaccharides after first hydrolyzing them to monosaccharides. P. saccharophila, in fact,
oxidized sucrose much more rapidly than its constituent monosaccharides, glucose and fructose. This anomaly was later shown to be caused by the lack of permeases in P. saccharophila
for monosaccharides.
In 1940, Doudoroff joined the bacteriology department at the University of California,
Berkeley. There he discovered that P. saccharophila extracts catalyze the reversible formation
of glucose 1-phosphate and fructose from sucrose and inorganic phosphate. He used the
reverse reaction to synthesize sucrose, a compound that had not yet been made by chemical or
enzymatic methods. Doudoroff also used the synthetic reaction to make novel analogues of
sucrose by replacing fructose with D-ketoxylose and L-sorbose.
He subsequently purified sucrose phosphorylase from P. saccharophila and studied its mode
of action using radioactive inorganic phosphate. He learned that the enzyme is a transglucosidase that transfers the glucosyl residue from a suitable donor such as sucrose or glucose
1-phosphate to an appropriate acceptor such as fructose or orthophosphate. This was some of
the first evidence for the formation of a substrate-enzyme complex as an intermediate in an
enzymatic reaction. Doudoroff also discovered a second type of phosphorolytic enzyme, maltose
phosphorylase, in Neisseria meningitides.
Doudoroff and his associates soon began to study the oxidative degradation of other sugars
by P. saccharophila. He and Nathan Entner examined the enzymatic oxidation of glucose
labeled with C14, as reported in the Journal of Biological Chemistry (JBC) Classic reprinted
here. They determined that glucose is phosphorylated to glucose 6-phosphate, which is oxidized to 6-phosphogluconic acid. This compound is then split to give rise to pyruvic acid and
glyceraldehyde phosphate. 2-Keto-3-deoxy-6-phosphogluconate was thought to be an intermediate in this reaction, and this was subsequently confirmed by Doudoroff and Joseph MacGee
who isolated and characterized the compound in 1954 (1). The enzyme that cleaved the keto
acid, ketodeoxyphosphogluconate aldolase, was later purified and crystallized in 1967 (2).
H15
Classics
These experiments eventually led to the formulation of the Entner-Doudoroff pathway, a
series of reactions that catabolize glucose to pyruvic acid using a different set of enzymes from
those used in either glycolysis or the pentose phosphate pathway. The novel feature of this
pathway is the cleavage of 6-phosphogluconate to yield pyruvate and glyceraldehyde 3-phosphate. Other sugars were shown to be metabolized by similar, but divergent, pathways.
Later in his career, Doudoroff studied assimilatory processes in aerobic and photosynthetic
bacteria and showed that poly--hydroxybutyric acid is an important energy reserve that is
utilized by both intracellular and extracellular enzymes. He was also involved in an extensive
clarification of taxonomic and phylogenetic relationships in Pseudomonas and other aerobic
bacteria. The first publication resulting from this collaboration was a massive survey of 169
phenotypic characters of 267 strains of Pseudomonas (3).
Doudoroff also had a profound influence on how bacteriology was taught at Berkeley. When
he joined the faculty in 1940, the bacteriology courses emphasized the medical and paramedical aspects of the subject. Doudoroff reorganized the curriculum to present bacteria and other
microorganisms as creatures whose structures, behaviors, and metabolic activities were worthy of study independent of their roles in agriculture, industry, or disease. He eventually,
along with Roger Y. Stanier and Edward A. Adelberg, wrote the popular textbook, The
Microbial World, based on the Berkeley courses.
In recognition of Doudoroffs contributions to microbiology and biochemistry he received the
first Sugar Research Award from the National Academy of Sciences in 1945 with Horace A.
Barker and William Z. Hassid (both of whom will be featured in future JBC Classics). He also
became a J. S. Guggenheim Foundation fellow in 1949 and was elected to membership in the
National Academy of Sciences in 1962.1
REFERENCES
1. MacGee, J., and Doudoroff, M. (1954) A new phosphorylated intermediate in glucose oxidation. J. Biol. Chem. 210,
617 626
2. Shuster C. W., and Doudoroff, M. (1967) Purification of 2-keto-3-deoxy-6-phosphohexonate aldolases of Pseudomonas saccharophila. Arch. Mikrobiol. 59, 279 286
3. Stanier, R. Y., Palleroni, N. J., and Doudoroff, M. (1966) The aerobic pseudomonads: a taxonomic study. J. Gen.
Microbiol. 43, 159 271
4. Barker, H. A. (1993) Biographical Memoir of Michael Doudoroff, Vol. 62, pp. 118 141, National Academy of
Sciences, Washington D. C.
All biographical information on Michael Doudoroff was taken from Ref. 4.
H16

Vol. 280, No. 31, Issue of August 5, p. e28, 2005

Printed in U.S.A.
Classics
JBC Centennial
19052005
Albert Dorfman and the Biosynthesis of Hyaluronic Acid

The Biosynthesis of Hyaluronic Acid by Group A Streptococcus. I. Utilization of
1-C14-Glucose
(Roseman, S., Moses, F. E., Ludowieg, J., and Dorfman, A. (1953) J. Biol. Chem. 203,
213225)
Albert Dorfman (1916 1982) was born and raised in Chicago. While in high school, he
became interested in science because of his older brother who was studying chemistry at the
University of Illinois. After graduating, Dorfman obtained a scholarship to the University of
Chicago where he enrolled as a chemistry major; however, during his senior year he switched
to biochemistry and entered the University of Chicago School of Medicine. He eventually found
biochemistry so appealing that he dropped out of medical school after 2 years to pursue
graduate work. His thesis research was on the identification of nicotinamide as a growth
requirement for Shigella dysenteriae and the synthesis of various nicotinic acid derivatives to
correlate structure with biological activity.
Dorfman received his Ph.D. from the University of Chicago in 1939. He remained at the
University as a research associate and started to study the role of bacterial growth factors in
metabolism. These studies led to Dorfmans development of the technique of growing deficient
cells to determine the role of growth factors in metabolism. He also elucidated the roles of
pantothenic acid in pyruvate metabolism and of biotin in aspartic acid biosynthesis.
With the arrival of World War II and lack of an academic position, Dorfman returned to
medical school and graduated in 1944. This experience rekindled his interest in medicine,
particularly pediatrics. An encounter with a child with rheumatic fever also sparked an
interest in the mechanism of action of aspirin and would profoundly affect Dorfmans subsequent career.
When Dorfman finished medical school he got an internship in internal medicine at Beth
Israel Hospital and then became a resident in pediatrics at the University of Chicago. After
completing his residency, he served 2 years in the U. S. Army where he was assigned to the
Army Medical School and was able to resume research in biochemistry. Around this time a
study emerged claiming that aspirin exerted its antirheumatic effect by inhibiting hyaluronidase. Dorfman promptly initiated studies on connective tissue polysaccharides, an area of
research he would pursue for the next 30 years. He started by studying the biosynthesis of
hyaluronic acid in group A streptococci, which led to the development of quantitative methods
for assays of hyaluronidase, the discovery that chondroitin sulfate is a substrate for testicular
hyaluronidase, and the recognition that hyaluronidase is unusually stable to heat and acid pH.
After his 2 years in the army, Dorfman returned to the University of Chicago as an assistant
professor of pediatrics and continued to study the biosynthesis of hyaluronic acid. His goal was
to determine the origins of the 14 unique carbon atoms of the polysaccharide using specifically
labeled precursors. Dorfman embarked on this project with his postdoc, Saul Roseman, who
was an author in a previous Journal of Biological Chemistry (JBC) Classic on Karl Paul Link
(1) and will be featured in his own Classic in the future. Dorfman and Roseman, along with
Julio Ludowieg and Frances Moses, synthesized [1-14C]glucose and incorporated it into medium upon which they could grow streptococcus. They devised a method for isolating the
radioactive hyaluronic acid from the streptococcus filtrate and then analyzed its components.
It became immediately evident that glucose was the major carbon precursor of hyaluronic acid.
The glucose was then converted to the glucosamine and glucuronic acid portions of the
H17
Classics
Albert Dorfman. Photo courtesy of the Office of NIH History, National Institutes of Health.
molecule without cleavage of the carbon chain. These results are presented in the JBC Classic
reprinted here, which is the first in a series of JBC papers Dorfman published on the
biosynthesis of hyaluronic acid by group A streptococcus.
Dorfman and his colleagues subsequently synthesized [6-14C]glucose and [1-14C]acetic acid
and used those compounds to establish that acetate is a precursor of the acetyl group of
N-acetylglucosamine and that glucosamine but not N-acetylglucosamine serves as a precursor
of the N-acetylglucosamine residue in hyaluronic acid. These results were also published in the
JBC series (2, 3).
The discovery of uridine nucleotide sugars by Luis Leloir, as reported in a previous JBC
Classic (4), suggested to Dorfman that these compounds might be intermediates in polysaccharide synthesis. Together with J. A. Cifonelli, Dorfman established that streptococci contain
two uridine nucleotide sugars, UDP-N-acetylglucosamine and UDP-glucuronic acid, which are
requisite for the biosynthesis of hyaluronic acid (5). Using labeled nucleotides, they were able
to demonstrate the synthesis of hyaluronic acid in a cell-free preparation of streptococci. This
was published as the final paper in Dorfmans hyaluronic acid series in the JBC (6).
In addition to his work on hyaluronic acid, Dorfman also contributed significantly to
understanding the biosynthesis of other glycosaminoglycans. As well, he discovered the cause
of Hurlers syndrome, a genetic disease that affects bones and cartilage and results in mental
retardation. He deduced that the condition results from elevated levels of dermatan sulfate
and heparin sulfate due to a defect in -L-iduronidase, an enzyme needed for the normal
catabolism of the two glycosaminoglycans.
In 1967 Dorfman became the Richard T. Crane Distinguished Service Professor of Pediatrics
and Biochemistry and acted as Chairman of the Department of Pediatrics from 1962 to 1972.
He also served as Director of the La Rabida University of Chicago Institute (19571972) and
Director of the Joseph P. Kennedy, Jr. Mental Retardation Research Center (19671982). In
addition to his research activities, Dorfman was President of the Society for Glycobiology
(1975) and President of the Pediatric Society (1979). His contributions to science were recognized with his election to the National Academy of Sciences in 1973.1
REFERENCES
1. JBC Classics: Campbell, H. A., and Link, K. P. (1941) J. Biol. Chem. 138, 2133; Stahmann, M. A., Huebner, C. F.,
and Link, K. P. (1941) J. Biol. Chem. 138, 513527; Overman, R. S., Stahmann, M. A., Huebner, C. F., Sullivan,
1
All biographical information on Albert Dorfman was taken from Ref. 7.
H18
Classics
2.
3.
4.
5.
6.
7.
W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153,
524 (http://www.jbc.org/cgi/content/full/280/8/e5)
Roseman, S., Ludowieg, J., Moses, F. E., and Dorfman, A. (1954) The biosynthesis of hyaluronic acid by group A
Streptococcus. II. Origin of the glucuronic acid. J. Biol. Chem. 206, 665 669
Dorfman, A., Roseman, S., Moses, F. E., Ludowieg, J., and Mayeda, M. (1955) The biosynthesis of hyaluronic acid
by group A Streptococcus. III. Origin of the N-acetylglucosamine moiety. J. Biol. Chem. 212, 583592
JBC Classics: Caputto, R., Leloir, L. F, Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333350;
Cabib, E., Leloir, L. F., and Cardini, C. E. (1953) J. Biol. Chem. 203, 10551070; Cabib, E., and Leloir, L. F.
(1954) J. Biol. Chem. 206, 779 790 (http://www.jbc.org/cgi/content/full/280/19/e16)
Cifonelli, J. A., and Dorfman, A. (1957) The biosynthesis of hyaluronic acid by group A Streptococcus. V. The
uridine nucleotides of group A Streptococcus. J. Biol. Chem. 228, 547557
Markovitz, A., Cifonelli, J. A., and Dorfman, A. (1959) The biosynthesis of hyaluronic acid by group A Streptococcus. VI. Biosynthesis from uridine nucleotides in cell-free extracts. J. Biol. Chem. 234, 23432350
Schwartz, N. B., and Roden, L. (1997) Biographical Memoir of Albert Dorfman, Vol. 72, pp. 70 87, National
Academy of Sciences, Washington, D. C.
H19

Vol. 281, No. 1, Issue of January 6, p. e1, 2006

Printed in U.S.A.
Classics
JBC Centennial
19052005
Hexosamine Metabolism, Sialic Acids, and the

Phosphotransferase System: Saul Rosemans
Contributions to Glycobiology
The Sialic Acids. I. The Structure and Enzymatic Synthesis of N-Acetylneuraminic
Acid
(Comb, D. G., and Roseman, S. (1960) J. Biol. Chem. 235, 2529 2537)
Sugar Transport. I. Isolation of a Phosphotransferase System from Escherichia coli
(Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 13931406)
Saul Roseman was born in Brooklyn, New York, in 1921. He received his Bachelor of Science
in Chemistry from the City College of New York in 1941 and began his graduate studies in the
Biochemistry Department at the University of Wisconsin, earning his masters degree in 1944.
He then served for 2 years as an infantryman in Europe in World War II. Upon his return, he
completed his Ph.D. in 1947, studying biochemistry with Karl Paul Link and organic chemistry with Homer Atkins. Rosemans graduate work focused on the synthesis and metabolism
of coumarin derivatives (e.g. Dicumarol), which Link had discovered and which was the subject
of a previous Journal of Biological Chemistry (JBC) Classic (1). It was during his graduate
studies that Rosemans life-long interest in carbohydrates began, when he started working on
the metabolism of 4-hydroxycoumarin, which is secreted into the urine of dogs as the glucuronide or glucuronic acid derivative. A growing interest in complex carbohydrates led Roseman
to do his postdoctoral studies with Albert Dorfman at the University of Chicago School of
Medicine, who was also featured in a previous JBC Classic (2). With Dorfman, Roseman
developed new methods of glycan radioisotopic labeling to study hyaluronic acid and chondroitin sulfate biosynthesis and showed that the carbon chain of glucose was converted in vivo
directly, with no cleavage, to that of glucosamine. He remained at Chicago until 1953 and was
promoted to Assistant Professor.
Roseman then became Assistant Professor of Biological Chemistry at the University of
Michigan Medical School and Chemist of the Rackham Arthritis Research Unit. In the next
few years, he rose in academic rank to Professor. While at Michigan, Roseman started to work
with enzymes and cell-free extracts, which took him to glucosamine metabolism, which, in
turn, led to the sialic acids. His work on the identification of the structure and metabolism of
sialic acids is the subject of the first JBC Classic reprinted here.
In the Classic, Roseman and one of his first postdoctoral fellows, Donald G. Comb (founder
and President of New England Biolabs), report on their studies on the structure of one sialic
acid in particular, N-acetylneuraminic acid, and the properties of the bacterial enzyme,
N-acetylneuraminic acid aldolase, that cleaves the sialic acid into N-acetyl-D-mannosamine
(which was not previously known to occur naturally) and pyruvate. Using N-acetylneuraminic
acid purified from both human blood and Escherichia coli and N-acetylneuraminic acid
aldolase from Clostridium perfringens, Roseman and Comb established the products of the
cleavage reaction, showed that it was reversible (yielding NAN), and characterized the enzyme. They also used 1-[14C]- and 6-[14C]glucose to follow the biosynthesis of N-acetylneuraminic acid and concluded that it is the product of a 3-carbon and 6-carbon condensation.
At that time, and over a period of 3 decades, over 11 possible structures had been suggested
for N-acetylneuraminic acid. Roseman and Combs work was critical in establishing the correct
H20
Classics
Saul Roseman
structure. Subsequently, Roseman and Comb isolated, characterized, and enzymatically synthesized a unique sugar nucleotide, CMP-sialic acid. This led to a series of novel studies on
glycosyltransferases. Roseman and colleagues showed that families of glycosyltransferases
existed, in which each family transferred a specific sugar, and that each member of the family
had specific requirements for the acceptor and/or the linkage of the newly synthesized glycosidic bond. The sum of these studies was to establish the individual steps in the metabolic
pathways between fructose-6-P and complex carbohydrates such as the gangliosides (with
Basu), the oligosaccharide chains in the mucins (with Schachter), and the carbohydrate
termini in the blood glycoproteins (with Jourdian, Carlson, and others).
In 1965 Roseman was recruited to the McCollum-Pratt Institute and Department of Biology
at the Johns Hopkins University, where he later served as Director and Chairman for two
terms (1969 1973 and 1988 1990). Just before leaving Michigan for Baltimore, Roseman,
while working on N-acetylmannosamine metabolism, discovered what turned out to be a novel
sugar transport system in bacteria, the PTS (phosphotransferase system). The system involves
a series of sequential phosphotransfer reactions between proteins and simultaneously phosphorylates and translocates its sugar substrates across the membrane. Surprisingly, the
source of the phosphoryl group is not ATP or another nucleoside triphosphate but rather
phosphoenolpyruvate.
Rosemans isolation of a PTS from E. coli is the subject of the second JBC Classic reprinted
here. In the paper, Roseman and Werner Kundig purify the two enzymes (Enzyme I and II)
and one protein (HPr) that comprise the system and determine that Enzyme I and HPr are
soluble while Enzyme II is part of the cell membrane. Using 32P and 14C to assay the activity
of the enzymes, they conclude that Enzyme I catalyzes the transfer of phosphate from
phosphoenolpyruvate to HPr and Enzyme II catalyzes the transfer of phosphate from phosphoHPr to the carbohydrate. The phosphate is linked to HPr via an imidazole nitrogen atom on a
histidine residue. In two subsequent JBC papers (3, 4), Roseman and Kundig further characterized the enzymes that constitute the PTS, and somewhat later, with Simoni, established
that it was, in fact, a sugar transport system, not just a novel kinase.
Still at Johns Hopkins, Roseman retains his position of Professor in the Department of
Biology. His laboratory continues to make key contributions to the molecular understanding of
complex carbohydrate glycosyltransferases, bacterial sugar transport, and intercellular adhesion. Most recently, he has made novel forays into the complex metabolism of chitin, the second
most abundant organic compound in nature. More information about the history of glycobiology can be found in Rosemans JBC Reflections (5).
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Classics
In recognition of his contributions to glycobiology, Roseman has been the recipient of many
national and international awards and honors, among which was his election, in 1972, to the
National Academy of Sciences, and the degree of Doctor of Medicine Honoris causa from the
University of Lund. He has also received the Sesquicentennial award from the University of
Michigan (1967), the T. Duckett Jones Memorial award from the Helen Hay Whitney Foundation (1973), the Rosenstiehl award from Brandeis University (1974), the International
award from the Gairdner Foundation (1981), and the Karl Meyer award from the Society of
Glycobiology (1993). Roseman is also a former member of the JBC editorial board and has
published 136 papers in the journal over a 60-year period.
REFERENCES
W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153,
524 (http://www.jbc.org/cgi/content/full/280/8/e5)
2. JBC Classics: Roseman, S., Moses, F. E., Ludowieg, J., and Dorfman, A. (1953) J. Biol. Chem. 203, 213225
(http://www.jbc.org/cgi/content/full/280/31/e28)
3. Kundig, W., and Roseman, S. (1971) Sugar transport. II. Characterization of constitutive membrane-bound
Enzymes II of the Escherichia coli phosphotransferase system. J. Biol. Chem. 246, 14071418
4. Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) Sugar transport. III. Purification and properties of
a phosphocarrier protein (HPr) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli. J. Biol. Chem. 246, 70237033
5. Roseman, S. (2001) Reflections on glycobiology. J. Biol. Chem. 276, 41527 41542
H22


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Classics
JBC Centennial
19052005
The Regulation of Glucose Uptake in Muscle: the Work of

Charles R. Park
Regulation of Glucose Uptake in Muscle. I. The Effects of Insulin and Anoxia on
Glucose Transport and Phosphorylation in the Isolated, Perfused Heart of Normal
Rats
(Morgan, H. E., Henderson, M. J., Regen, D. M., and Park, C. R. (1961) J. Biol. Chem.
236, 253261)
Charles Rawlinson Rollo Park was born in Baltimore in 1916. He attended Harvard
College and began medical studies at Johns Hopkins School of Medicine in 1941. After
interning at Johns Hopkins, he was appointed Assistant Resident and Chief Resident at the
Peter Bent Brigham Hospital in Boston. In 1944, Park served in the U.S. Army Medical Corps
at Fort Knox, Kentucky, where he studied thermoregulation during the acclimatization of
military personnel exposed to desert-like conditions. Park jocularly refers to this as his front
line experience during the war.
In 1947, Park joined the laboratory of Carl and Gerty Cori at Washington University in St.
Louis. The Coris, who were featured in a previous Journal of Biological Chemistry (JBC)
Classic (1), won the Nobel Prize for Physiology or Medicine that year, and many Nobel
laureates emerged from their laboratory. Parks work in St. Louis focused on the effects of
growth hormone, hypophysectomy, and adrenalectomy on glucose uptake by isolated rat
diaphragms. He relates his consternation when Carl Cori looked at some of his early results
and commented in his German-accented English, I do not like these data! Despite this
encouraging comment, Park enjoyed interacting with the stellar group of scientists working
in the laboratory at that time.
In 1952, at a relatively young age, Park was appointed to the Chair of Physiology at
Vanderbilt University Medical School. At that time, the department consisted of three physiologists, a technician, a graduate student, and a secretary, and the rooms were full of
abandoned, dusty, obsolete equipment. However from these humble beginnings, Park built one
of the most distinguished physiology departments in the country with a stellar reputation for
research.
Park developed a vigorous research program in the hormonal regulation of carbohydrate
metabolism and recruited faculty with a strong research background. The most notable of
these was his wife, Jane Harting Park, who also came from the Cori department. Another
recruit was Howard Morgan, who was an obstetrician working at the Fort Campbell Army
Base in Clarksville, Tennessee. In an effort to relieve the tedium of deliveries, he applied to
Park who, in an enlightened choice, took him on as a researcher. Morgan turned out to be a
very gifted investigator who later became Chair of Physiology at Pennsylvania State Medical
School, President of the American Physiological Society, and President of the American Heart
Association. In an interesting turn of events, Parks son Edwards A. Park later obtained his
Ph.D. under Morgan.
Together with Morgan, Margaret J. Henderson, Robert Post, and David M. Regen, Park
began a classic series of experiments studying the regulation of glucose uptake in muscle by
insulin and other hormones. The experimental system was the isolated perfused rat heart, and
the work resulted in the publication of a series of six classic papers in the JBC in 1961 (2 6),
the first of which is reprinted here as a JBC Classic. The papers showed that the limiting step
H23
Classics
Charles Rawlinson Rollo Park
for glucose uptake was the transport of the sugar across the cell membrane and that this was
accelerated by insulin and anoxia. Diabetes was shown to decrease this transport and also
reduce its sensitivity to insulin. Hypophysectomy reduced basal glucose transport but made it
more sensitive to insulin, whereas growth hormone treatment in vivo had the opposite effect.
Diabetes was also shown to decrease glucose phosphorylation, which was relieved by hypophysectomy or adrenalectomy and restored by treatment with growth hormone or cortisol. All
these findings were of fundamental relevance to human diabetes, and Park later won the
Banting Award of the American Diabetes Association.
In 1963, Park recruited Earl Sutherland from Western Reserve University. Sutherland had
initiated seminal studies of the hormonal regulation of glycogen phosphorylase in the Cori
laboratory, which led to the discovery of cyclic AMP and the award of a Nobel Prize in 1971.
This was the subject of a previous JBC Classic (7). Also in 1963, a postdoc named John Exton
arrived from New Zealand and began a new area of research with Park: the hormonal
regulation of hepatic gluconeogenesis. The research utilized the isolated perfused rat liver and
again resulted in a series of Classic papers published in the JBC (8).
Parks scientific career didnt stop there but extended to studies of the regulation of cyclic
AMP-dependent protein kinase, cyclic AMP phosphodiesterase, fatty acid transport, and, in
collaboration with Jane Harting Park, studies of muscular dystrophy and muscle metabolism.
In addition to the Banting Award, Park has won many distinctions and awards at Vanderbilt
University and was elected to the National Academy of Sciences in 1980. He was listed among
the 10 most frequently cited authors in Physiology during 19651978.
The Parks have a long history with the JBC and the American Society for Biochemistry and
Molecular Biology (ASBMB). Jane Harting Park was Treasurer of the American Society of
Biological Chemists (now the ASBMB) from 1979 to 1982, and Edwards A. Park served on the
JBC editorial board from 1998 to 2003 and started another 5-year term in 2006. Rollo and Jane
were also both JBC editorial board members in the 1960s and 1970s.
H24
Classics
Rollo Park has a relaxed, aristocratic demeanor and can properly be called a gentleman and
a scholar. He is notable for treating the lowliest members of society the same as the accomplished. His prowess as a fisherman, runner, and kayaker is legendary, and he has terrified
many famous scientists as he fearlessly guided his kayak through the rapids. His attitude to
publishing and deadlines was relaxed, and priority was not a concern. When an anguished
postdoctoral fellow would protest when a manuscript lay on his desk for many months or Park
spent almost a day studying a single result, he would say, Dont worry, the best paper wins
in the end! Parks record proves he was right.
John H. Exton, Nicole Kresge, Robert D. Simoni, and Robert L. Hill
REFERENCES
1. JBC Classics: Cori, C. F., and Cori, G. T. (1928) J. Biol. Chem. 79, 321341; Cori, G. T., Colowick, S. P., and Cori,
C. F. (1938) J. Biol. Chem. 124, 543555; Cori, G. T., Colowick, S. P., and Cori, C. F. (1939) J. Biol. Chem. 127,
771782; Green, A. A., and Cori, G. T. (1943) J. Biol. Chem. 151, 2129; Cori, G. T., and Green, A. A. (1943)
J. Biol. Chem. 151, 3138 (http://www.jbc.org/cgi/content/full/277/29/e18)
2. Morgan, H. E., Cadenas, E., Regen, D. M., and Park, C. R. (1961) Regulation of glucose uptake in muscle. II.
Rate-limiting steps and effects of insulin and anoxia in heart muscle from diabetic rats. J. Biol. Chem. 236,
262268
3. Post, R. L., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. III. The interaction of
membrane transport and phosphorylation in the control of glucose uptake. J. Biol. Chem. 236, 269 272
4. Henderson, M. J., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. IV. The effect of
hypophysectomy on glucose transport, phosphorylation, and insulin sensitivity in the isolated, perfused heart.
J. Biol. Chem. 236, 273277
5. Henderson, M. J., Morgan, H. E., and Park, C. R. (1961) Regulation of glucose uptake in muscle. V. The effect of
growth hormone on glucose transport in the isolated, perfused rat heart. J. Biol. Chem. 236, 21572161
6. Morgan, H. E., Regen, D. M., Henderson, M. J., Sawyer, T. K., and Park, C. R. (1961) Regulation of glucose uptake
in muscle. VI. Effects of hypophysectomy, adrenalectomy, growth hormone, hydrocortisone, and insulin on
glucose transport and phosphorylation in the perfused rat heart. J. Biol. Chem. 236, 21622168
7. JBC Classics: Rall, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 10651076; Sutherland, E. W., and
Rall, T. W. (1958) J. Biol. Chem. 232, 10771092 (http://www.jbc.org/cgi/content/full/280/42/e39)
8. JBC Classics: Exton, J. H., and Park, C. R. (1967) J. Biol. Chem. 242, 26222636; Exton, J. H., and Park, C. R.
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Classics
JBC Centennial
19052005
Plant Carbohydrates and the Biosynthesis of Lactose: the

Work of William Zev Hassid
A Soluble Lactose-synthesizing Enzyme from Bovine Milk
(Babad, H., and Hassid, W. Z. (1964) J. Biol. Chem. 239, 946 948)
Soluble Uridine Diphosphate D-Galactose:D-Glucose -4-D-Galactosyltransferase
from Bovine Milk
(Babad, H., and Hassid, W. Z. (1966) J. Biol. Chem. 241, 26722678)
Zev Hassid (1899 1974) was born in Jaffa, Palestine. He added William to his name after
he came to the United States in 1920. Hassid was educated in Palestine at a Hebrew language
school and then at an Agricultural High School, from which he graduated in 1916. He then
worked as a farm laborer until 1918 when he joined the British army to help liberate Palestine
from the Turks. While in the army, Hassid was never involved in combat; instead he guarded
prisoners and supplies in transit, which allowed him to travel to places like Alexandria in
Egypt. It was in Alexandria that Hassid first heard about the University of California from a
fellow soldier who had studied there.
After leaving the army, Hassid decided to use his savings to go to California to study
agronomy at the University, intending to return to Palestine to assist in the development of
scientific agriculture. He arrived in Berkeley in 1920 and registered at the University of
California. However, his knowledge of English was so limited that he could not follow the
lectures or read the textbooks. After a week of frustration he took a leave of absence from the
University and moved to Fresno where he attended Fresno State Teachers College, majoring
in Letters and Science with an emphasis on Chemistry, French Language, and Mathematics.
In August 1924, he returned to UC Berkeley to major in Chemistry, but he changed his major
to general literature and obtained a Bachelor of Arts degree in 1925. He then enrolled in
graduate studies at the School of Education and graduated in 1926, at the same time earning
a General Secondary School Credential from the State Board of Education. However, instead
of teaching, he worked as a chemical analyst for a year.
In 1927, Hassid was offered a position as a research assistant with D. R. Hoagland in the
Division of Plant Nutrition at Berkeleys Agricultural Experiment Station. Working with
Hoagland, Hassid analyzed plant materials and soils for a variety of inorganic constituents.
This renewed his interest in plant research, and he enrolled at UC Berkeley as a graduate
student in Plant Nutrition. He earned his Ph.D. in 1934, investigating the structure of
polysaccharides in marine algae for his thesis. After graduating Hassid joined the staff of the
Division of Plant Nutrition as a junior chemist and rose to the rank of Professor of Plant
Biochemistry in 1947. In 1959 he transferred to the Biochemistry Department and in 1965 he
became Emeritus.
Hassids independent scientific research started with his investigation of the structure of a
galactan that was a major component of the fleshy marine alga, Iridea laminarioides. In
elucidating the structure he applied methylation methods that had recently been developed by
the English chemist and Nobel laureate, Walter N. Haworth. Later he used the same methods
to establish the primary structures of several other types of starch and glycogen, including
canna starch, dog liver glycogen, the dextran formed from sucrose by Betacoccus arabinosaceus, an insoluble polysaccharide derived from Saccharomyces cerevisiae, and glycogen and
H26
Classics
William Zev Hassid, professor of biochemistry at the University of California Berkeley. Credit: Bob Lackenbach,
University of California Berkeley (1951 or earlier).
starch derived from sweet corn. These initial investigations led to an interest in the biochemistry of carbohydrates that remained with Hassid throughout his career.
Hassid started collaborating with Samuel Ruben and Martin D. Kamen in 1939 on the first
application of 11C to the study of photosynthesis. When 14C became available in 1946, Hassid
and his students pioneered in the development of biological methods for the preparation of
uniformly 14C-labeled carbohydrates from plant tissue, including D-glucose, D-fructose, Dgalactose, sucrose, and starch. He generously supplied the radioactive sugars to many other
investigators before they became commercially available.
In 1943, Hassid initiated a collaboration with Michael Doudoroff and Horace A. Barker, both
authors of previous Journal of Biological Chemistry (JBC) Classics (1, 2), as well as Nathan O.
Kaplan. They investigated the biosynthesis of sucrose by sucrose phosphorylase, an enzyme
from Pseudomonas saccharophila. Their demonstration of the first enzymatic synthesis of
sucrose caught the attention of officials at the Coca-Cola Company who were having trouble
obtaining sucrose because of wartime rationing. The company sent a representative to Berkeley to offer them $500,000 for research on sucrose phosphorylase if a commercial process for
sucrose synthesis seemed feasible. Unfortunately, Hassid and his associates were away at the
time, and the representative could only discuss the problem with Hoagland who was pessimistic about the method. Due to Hoaglands lack of optimism, Coca-Cola did not end up
providing funding for sucrose phosphorylase research.
Subsequent efforts to show the presence of a similar enzyme in plants were unsuccessful
until Luis Leloir and his associates discovered uridine diphosphate D-glucose (UDPG) and
demonstrated the synthesis of sucrose from UDPG and fructose, as reported in a previous JBC
Classic (3). This prompted Hassid and his colleagues to undertake a systematic investigation
of the occurrence of nucleoside diphosphate sugars in plants. They isolated nucleoside diphosphate derivatives of D-xylose, L-arabinose, D-galactose, D-galacturonic acid, D-mannuronic acid,
and 2-acetamido-2-deoxy-D-glucose and established the roles of several of these compounds in
sugar interconversions and polysaccharide formation.
Hassids reputation attracted scientists from around the world to work in his laboratory.
One of these scientists was Winifred N. Watkins, who embarked on a study of the biosynthesis
of lactose in mammary tissue with Hassid. Using guinea pig and bovine mammary glands,
they established that lactose was synthesized according to the following reaction.
H27
Classics
UDP-D-galactose D-glucose 3 lactose UDP
REACTION 1
They also discovered that mammary tissue contained an enzyme activity that transfers
D-galactose to N-acetyl-D-glucosamine.
This led to Hassids isolation of lactose synthetase with Helene Babad, which is the subject
of the two JBC Classics reprinted here. The first Classic is a communication that describes
how Hassid and Babad used centrifugation and ammonium sulfate fractionation to obtain a
soluble enzyme preparation from bovine milk capable of catalyzing synthesis of lactose from
UDP-D-galactose and D-glucose. They confirmed that their preparation contained lactose
synthetase activity using [1-14C]UDP-D-galactose and -D-[14C]glucose 1-phosphate. The second Classic describes the partial purification and some of the properties of the galactosyltransferase responsible for the synthesis of lactose. It was later discovered that lactose
synthetase is composed of two proteins: galactosyltransferase and -lactalbumin, which increases the affinity of galactosyltransferase.
Hassids numerous contributions to understanding plant carbohydrates were recognized by
several awards and honors. He was given the first Sugar Research Award (1945) of the
National Academy of Sciences (jointly with Doudoroff and Barker), the Charles Reid Barnes
Honorary Life Membership Award of the American Society of Plant Physiologists (1964), and
the C. S. Hudson Award of the American Chemical Society (1967). In 1972 he was honored at
the Sixth International Symposium on Carbohydrate Chemistry as one of three outstanding
senior American carbohydrate chemists. He was a member of the National Academy of
Sciences and the American Academy of Arts and Sciences, Chairman of the Division of
Carbohydrate Chemistry of the American Chemical Society (1949 1950), and a member of
numerous editorial boards including that of the JBC.1
REFERENCES
1. JBC Classic: Entner, N., and Doudoroff, M. (1952) J. Biol. Chem. 196, 853 862 (http://www.jbc.org/cgi/
content/full/280/27/e24)
2. JBC Classic: Brady, R. O., Castanera, E. G., and Barker, H. A. (1962) J. Biol. Chem. 237, 23252332
3. JBC Classics: Caputto, R., Leloir, L. F, Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333350;
4. Ballou, C. and Barker, H. A. (1979) Biographical Memoir of William Zev Hassid, Vol. 50, pp.196 231, National
Academy of Sciences, Washington, D. C.
All biographical information on William Zev Hassid was taken from Ref. 4.
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Vol. 282, No. 10, Issue of March 9, p. e7, 2007

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Classics
JBC Centennial
19052005
The Control of Gluconeogenesis: the Work of John Exton

Control of Gluconeogenesis in Liver. I. General Features of Gluconeogenesis in the
Perfused Livers of Rats
(Exton, J. H., and Park, C. R. (1967) J. Biol. Chem. 242, 26222636)
Control of Gluconeogenesis in Liver. II. Effects of Glucagon, Catecholamines, and
Adenosine 3,5-Monophosphate on Gluconeogenesis in the Perfused Rat Liver
(Exton, J. H., and Park, C. R. (1968) J. Biol. Chem. 243, 4189 4196)
John H. Exton was born in Auckland, New Zealand in 1933. He received his medical degree
from the University of New Zealand in 1958 and his Ph.D. in biochemistry from the University
of Otago, New Zealand in 1963. Exton then left New Zealand for Nashville, Tennessee to do
postdoctoral research in the Department of Physiology at the Vanderbilt University School of
Medicine with Charles R. Park and Journal of Biological Chemistry (JBC) Classic author Earl
Sutherland (1).
Along with Park, who will be featured in an upcoming JBC Classic, Exton worked on
elucidating the pathways of gluconeogenesis, the formation of glucose from non-sugar carbon
substrates like pyruvate, lactate, glycerol, and certain amino acids. Gluconeogenesis occurs
principally in the liver and is essential for survival in starvation; it also plays a major role in
the disposal of lactate and maintenance of glucose during exercise. The process is controlled
directly or indirectly by many hormones, including insulin, glucagon, catecholamines, and
glucocorticoids. Exton and Park published a series of papers in the JBC on the control of
gluconeogenesis using the isolated, perfused rat liver. This allowed them to study the process
and its regulation without the interference of other changes in the body. Two of those papers
are reprinted here as JBC Classics.
The first paper studied the basic process of gluconeogenesis (i.e. the conversion of lactate,
pyruvate, glycerol, alanine, and a mixture of amino acids to glucose) and demonstrated that
physiological increases in these substrates alone led to increased glucose production by the
liver, indicating the importance of substrate supply in the regulation of gluconeogenesis.
Comparison of the rates of gluconeogenesis from lactate, pyruvate, fructose, and dihydroxyacetone suggested that the rate-limiting step for lactate gluconeogenesis was located between
pyruvate and triose phosphate in the gluconeogenic pathway. Measurements of the conversion
of [14C]pyruvate to glucose and CO2 were used in a mathematical analysis to determine the
flow of isotope from this substrate into the gluconeogenic pathway and the Krebs cycle.
In the second paper, the stimulatory effects of glucagon and catecholamines on lactate
gluconeogenesis were analyzed. Physiological concentrations of glucagon were effective in
stimulating gluconeogenesis. However, blood levels of epinephrine were not, leading to the
proposal that the stimulatory effects of the sympathetic nervous system were due to the
release of norepinephrine from adrenergic nerve endings in the liver. Exogenous cyclic AMP
stimulated gluconeogenesis, consistent with this being the mediator of the effect of glucagon.
Studies of gluconeogenesis from fructose indicated that glucagon/cyclic AMP stimulated the
pathway somewhere between pyruvate and phosphoenolpyruvate in the gluconeogenic
pathway.
In 1966, Exton became an Assistant Professor of Physiology at Vanderbilt and was promoted
to Associate Professor in 1968. Later, in 1970, he became Professor of Molecular Physiology
H29
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John Exton
and Biophysics and in 1989 he became a Professor of Pharmacology. Exton remains at

Vanderbilt where he studies signal transduction.
Exton has received many honors for his research including the Lilly Award from the
American Diabetes Association, a Doctor Honoris Causa from the Autonomous University of
Barcelona, the Drummond Award from the University of Calgary, and the Sutherland Award,
Vanderbilts highest research award. He is also a University National Scholar, New Zealand,
and a Commonwealth Scholar, United Kingdom, as well as a fellow of the American Association for the Advancement of Science and a member of the National Academy of Science. He
was named an investigator of the Howard Hughes Medical Institute in 1976 and has served as
an Associate Editor for the Journal of Biological Chemistry since 1988.
REFERENCES
1. JBC Classics: Rall, T. W., and Sutherland, E. W. (1958) J. Biol. Chem. 232, 10651076; Sutherland, E. W., and
Rall, T. W. (1958) J. Biol. Chem. 232, 10771092 (http://www.jbc.org/cgi/content/full/280/42/e39)
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JBC Centennial
19052005
Mycobacterial Glycophosphoinositides: the Work of

Clinton E. Ballou
Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family of
Dimannophosphoinositides
(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046 3056)
Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acylation of the Dimannophosphoinositides
(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 29752984)
Clinton Edward Edgerton Ballou was born in
King Hill, Idaho in 1923. After graduating from
high school, he enrolled in a premed program at
Boise Junior College, but his interests quickly
turned to chemistry after he dissected a poorly
embalmed cat in a comparative anatomy course.
After 2 years at Boise, Ballou pursued a degree in
chemistry at Oregon State College in Corvallis
where he became involved in two research
projects: the first during his junior year synthesizing new antimalarial drugs with Bert Christensen, and the second during his senior year
studying the guinea pig antistiffness factor with
Willem van Wagtendonk.
The military draft was in effect as Ballou entered his last year of college so he decided to join
the U. S. Navy after graduating in 1944. He was
discharged 2 years later and decided to apply for
graduate study in biochemistry with Karl Paul
Link at the University of Wisconsin-Madison. As
detailed in a previous Journal of Biological
Chemistry (JBC) Classic (1), Links research centered on blood anticoagulants, and when Ballou
Clinton E. Ballou
arrived in his laboratory in 1946, the primary
focus was the structure-function relationship of coumarin anticoagulants. Ballou was immediately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarol
because the acetylated intermediate was degraded in the alkali conditions used for deacetylation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Ballou
decided to study a variety of synthetic compounds to try to understand the structural basis for
alkali sensitivity. This research formed the core of his doctoral dissertation, and his exposure
to carbohydrate chemistry influenced the direction of his career.
After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L.
Hirst in the Department of Chemistry at the University of Edinburgh. There he studied the
structure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to work
with Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at the
University of California, Berkeley. Ballou explained his choice: I was attracted to Fischer in
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Classics
part because of his research on phosphorylated sugars but also because during graduate school
I had drawn heavily on the published works of his father, Emil Fischer. I guess the idea of
being associated with the son of Emil Fischer just seemed real cool to me (2).
The 1950s was a time of active research on biosynthetic pathways involving short chain
phosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou undertook the syntheses of several such metabolic intermediates, including D-glyceric acid 2-phosphate, D-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid
3-phosphate, and D-erythrose 4-phosphate. He also became interested in inositol chemistry as
a result of studies on the cyclitols in sugar pine heartwood.
In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about setting
up an independent research program. He decided to work on inositol-containing phospholipids
and was able to synthesize and characterize D-myoinositol 1-phosphate. He also spent several
years isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide.
This culminated in his discovery of D-myoinositol l,4,5-trisphosphate or IP3. More information on
Ballous studies of these phosphoinositides can be found in his JBC Reflections (2).
Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at the
National Center for Scientific Research (CNRS) in France studying the glycophosphoinositides
of mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experiments
to establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositol
ring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structures
of the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4 6). Ballou and
his postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intact
dimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBC
Classics reprinted here.
In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyze
the biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reported
that the major products are a family of three dimannophosphoinositides (A, B, and C) that
differ in the number of fatty acyl groups they contain (four, three, and two, respectively). On
the basis of their results, they proposed that guanosine diphosphate mannose acts as the sugar
donor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside
(dimannophosphoinositide C), which is then acylated in a two-step process to first yield
dimannophosphoinositide B and then dimannophosphoinositide A.
In the second JBC Classic, Ballou and Brennan provide further evidence for their proposed
biosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fatty
acids into the dimannophosphoinositides. They showed that label from [14C]palmityl-CoA is
incorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After a
short incubation period, this molecule is converted to dimannophosphoinositide A, but with
longer incubation periods the product is deacylated to isomeric forms of dimannophosphoinositides B and C.
Brennan continued to work on these glycophosphoinositides after completing his postdoctoral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) and
lipoarabinomannan (LAM)) were multiglycosylated extensions of Ballous phosphatidylinositol
mannosides and are very important in the pathogenesis of tuberculosis and leprosy. More
recent research has defined the biochemistry and genetics of synthesis of these molecules.
Brennan is currently University Distinguished Professor in the Department of Microbiology,
Immunology, and Pathology at Colorado State University. He served on the editorial board of
the Journal of Biological Chemistry for several years and was also named Colorado State
University Researcher of the Year in 1992.
In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California,
Berkeley, although he continued research and teaching for a few years. In recognition of his
contributions to science, Ballou has received many awards and honors including election to the
National Academy of Sciences (1975), the American Chemical Societys Claude Hudson Award
in Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the University
of Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), a
National Science Foundation Senior Fellowship (1961), and a University of California Berkeley Citation (1992). Ballou also served as an editorial board member for the Journal of
Biological Chemistry.
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REFERENCES
W. R., Spero, L., Doherty, D. G., Ikawa, M., Graf, L., Roseman, S., and Link, K. P. (1944) J. Biol. Chem. 153, 524
2. Ballou, C. E. (2004) My brief encounter with the phosphoinositides and IP3. J. Biol. Chem. 279, 5497554982
3. Ballou, C. E., Vilkas, E., and Lederer, E. (1963) Structural studies on the myo-inositol phospholipids of Mycobacterium tuberculosis (var. bovis, strain BCG). J. Biol. Chem. 238, 69 76
4. Lee, Y. C., and Ballou, C. E. (1964) Structural studies on the myo-inositol mannosides from the glycolipids of
Mycobacterium tuberculosis and Mycobacterium phlei. J. Biol. Chem. 239, 1316 1327
5. Ballou, C. E., and Lee, Y. C. (1964) The structure of myoinositol mannoside from Mycobacterium tuberculosis
glycolipid. Biochemistry 3, 682 685
6. Lee, Y. C., and Ballou, C. E. (1965) Complete structures of the glycophospholipids of mycobacteria. Biochemistry 4,
13951404
H33

Vol. 282, No. 52, Issue of December 28, p. e35, 2007

Printed in U.S.A.
Classics
JBC Centennial
19052005
Proteoglycans and Orchids: the Work of Vincent Hascall

Proteinpolysaccharide Complex from Bovine Nasal Cartilage. A Comparison of Low
and High Shear Extraction Procedures
(Sajdera, S. W., and Hascall, V. C. (1969) J. Biol. Chem. 244, 77 87)
Proteinpolysaccharide Complex from Bovine Nasal Cartilage. The Function of
Glycoprotein in the Formation of Aggregates
(Hascall, V. C., and Sajdera, S. W. (1969) J. Biol. Chem. 244, 2384 2396)
Aggregation of Cartilage Proteoglycans. III. Characteristics of the Proteins Isolated
from Trypsin Digests of Aggregates
(Heinegrd, D., and Hascall, V. C. (1974) J. Biol. Chem. 249, 4250 4256)
Vincent C. Hascall, Jr. was born
in Burwell, Nebraska in 1940. He
attended the California Institute of
Technology and earned a B.S. in
1962. After graduating he enrolled
at Rockefeller University, where he
earned his Ph.D. in 1969. While at
Rockefeller, Hascall met Alan Kapuler who had won a Westinghouse
Prize in high school for using a new
method for meristem cultures on an
orchid species. As a result Kapuler
was taken to Bogota, Colombia to
learn how specimens were collected
and preserved.
Alan suggested that we take a
summer and go collecting orchids in
Central America and Colombia, recalls Hascall. Rockefeller University agreed to let us do it, and Alans
parents gave us an old Renault Dauphin 2-cylinder car, and we drove
from Manhattan down through Central America to Panama, put the car
on a boat and went on to Colombia.
In Colombia, on the Pacific Ocean
side, we explored a region that had
Vincent Hascall
large, moss ridden trees alongside a
road. In the moss of one of the trees,
I noticed some small orange dots. They turned out to be what is likely the smallest orchid in
the world. The flower is the size of the head of a pin. It was a new species. Kapuler and Hascall
named the orchid Platystele acutilingua (1) (see Fig. 1). The genus Platystele was known, but
the species name, acutilingua, meaning sharp tongue, was their contribution.
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Classics
However, for his thesis research,
Hascall chose to focus on cartilage
rather than orchids. I ended up in
the basement of one of the Rockefeller
research buildings next to the East
River in a small lab with Stan Sajdera, a fellow graduate student, recalls Hascall. We taught as lab techs
in a summer biochemistry lab course
headed by Dominic Dziewiatkowski,
who had cartilage experiments going.
Both of us then continued to work
with cartilage for our Ph.D. work.
Dziewiatkowski accepted a position
FIG. 1. Platystele acutilinqua.
at the University of Michigan before
Hascall and Sajdera finished their
research at Rockefeller. The two students continued their research without Dziewiatkowski and were able to
develop an extraction and purification procedure for proteoglycans on
their own. This work is the subject of
the first and second Journal of Biological Chemistry (JBC) Classics reprinted here.
At the time this research was published, scientists were using high
speed homogenization in low ionic
strength salt solutions to extract
proteinpolysaccharides (now called
proteoglycans) from tissue. However, this method introduced shear
FIG. 2. Electron micrograph and model of a proteoglycan aggregate
artifacts and denatured and depolypurified from calf epiphyseal cartilage. Micrograph kindly provided by
merized the macromolecules. In a
Joseph Buckwalter, University of Iowa. Model and supplemental threedimensional, rotational view kindly provided by Mark Sabo (Art Departserendipitous experiment, Sajdera,
ment) and Vincent Hascall, Cleveland Clinic Foundation.
who was exploring the effects of reducing agents and denaturants on
the isolation and activity of the proteoglycans, used 4 M guanidine HCl as a control for the
extraction of cartilage slices. To Hascall and Sajderas surprise, almost all of the proteoglycans
were released into the solution whereas the slices remained intact. In a series of follow-up
experiments, they showed that this extraction method caused proteoglycan aggregates in the
cartilage to dissociate, thereby allowing the proteoglycan monomer (now called aggrecan) to
diffuse into the extraction solvent. This procedure, which they termed the dissociative
extraction method for proteoglycans, is still used today. The Classic paper became the basis
for Sajderas Ph.D thesis.
In the second Classic paper, Hascall and Sajdera showed that cartilage proteoglycans in the
dissociative extracts reformed aggregates when dialyzed into lower, associative solvent concentrations (0.4 M guanidine HCl). They also showed that a protein they named the glycoprotein link protein was required for successful aggregation. This paper became the basis for
Hascalls Ph.D. thesis. What Hascall and Sajdera did not know at the time, however, was that
hyaluronan was present in the extracts and also required for aggregation.
After receiving his Ph.D., Hascall joined the faculty of the University of Michigan as an
assistant professor. A couple years later, in 1972, he met Dick Heinegrd at a Gordon
Conference. Heinegrd invited Hascall to spend a year in his laboratory at the University of
Lund in Sweden. Hascall accepted the offer and was off to Sweden the next year. When he
arrived, he learned that Hardingham and Muir had just discovered that by adding small
amounts of hyaluronan to a solution of proteoglycan monomer, they could induce the formation
H35
Classics
of proteoglycan aggregates (2). Curious about this phenomenon, Heinegrd and Hascall began
their own series of investigations.
The final JBC Classic paper reprinted here is the last in a series of three that the two
scientists published (3, 4), which finally put all the pieces together to define the role of
hyaluronan and the link protein in forming the cartilage proteoglycan (aggrecan) aggregates.
By digesting aggregated proteoglycan with proteases, Heinegrd and Hascall were able to
show that two proteins were bound to hyaluronan. One protein was the link protein, and the
other was derived from a domain of the core protein of aggrecan, now referred to as the G1
domain. These results led to a model for the proteoglycan aggregates in which the link protein
and the G1 domain were bound to each other as well as to hyaluronan (see Fig. 2).
After returning from Sweden, Hascall spent 2 more years at the University of Michigan as
an associate professor. In 1975, he became Senior Staff Fellow in the Laboratory of Biochemistry at the National Institute of Dental Research, National Institutes of Health. He eventually
became Chief of the Proteoglycan Chemistry Section in the Laboratory of Biochemistry at the
National Institute of Dental Research, a position he held until he left the NIH in 1994 to join
the Department of Biomedical Engineering at the Cleveland Clinic Foundation. From 2001 to
2005, Hascall was co-director of the Orthopaedic Research Center at the Cleveland Clinic.
Today, he remains at the Cleveland Clinic and also holds a position as a professor in the
Department of Biological Chemistry at Case Western Reserve University.
In addition to his scientific research, Hascall is an Associate Editor for the Journal of
Biological Chemistry, a position he has held since 1995. He also received the NIH Merit Award
in 1979, was President of the Society for Complex Carbohydrates in 1987, and was awarded
the Karl Meyer Award for Glycoconjugate Research from the Society for Complex Carbohydrates in 1992.1
REFERENCES
1. Hascall, V. C., and Kapuler, A. M. (1966) Collecting along a transect: from the rain forest to the Andes in Choco,
Colombia. American Orchid Society Bulletin 35, 540 544
2. Hardingham T. E., and Muir, H. (1972) The specific interaction of hyaluronic acid with cartilage proteoglycans.
Biochim. Biophys. Acta 279, 401 405
3. Hascall, V. C., and Heinegrd, D. (1974) Aggregation of cartilage proteoglycans. I. The role of hyaluronic acid.
J. Biol. Chem. 249, 4232 4241
4. Hascall, V. C., and Heinegrd, D. (1974) Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors
of the proteoglycan-hyaluronic acid interaction. J. Biol. Chem. 249, 4242 4249
5. Hascall, V. C. (2000) Hyaluronan, a common thread. Glycoconj. J. 17, 607 616
Biographical information on Vincent Hascall was taken from Ref. 5.
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Vol. 281, No. 6, Issue of February 10, p. e6, 2006

Printed in U.S.A.
Classics
JBC Centennial
19052005
Lactose Synthesis in the Mammary Gland: Lactose

Synthase and the Work of Robert L. Hill
The Complete Amino Acid Sequence of -Lactalbumin
(Brew, K., Castellino, F. J., Vanaman, T. C., and Hill, R. L. (1970) J. Biol. Chem. 245,
4570 4582)
The Disulfide Bonds of Bovine -Lactalbumin
(Vanaman, T. C., Brew, K., and Hill, R. L. (1970) J. Biol. Chem. 245, 4583 4590)
The Purification and Properties of the A Protein of Lactose Synthetase
(Trayer, I. P., and Hill, R. L. (1971) J. Biol. Chem. 246, 6666 6675)
Robert L. Hill was born in Kansas City, Missouri in 1928. He earned a B.A. in chemistry in
1949 and a Ph.D. in biochemistry in 1954 from the University of Kansas. After receiving his
Ph.D., he went to the University of Utah as a National Institutes of Health (NIH) postdoctoral
fellow, where he worked with Emil L. Smith, who was featured in two Journal of Biological
Chemistry (JBC) Classics (1, 2). Although Hills Ph.D. thesis research concerned bacterial
metabolism, his postdoctoral studies introduced him to protein and enzyme chemistry, research areas he pursued during his subsequent career. At Utah, he published papers on
proteolytic enzymes and human hemoglobin and myoglobin. Hill remained at the University
of Utah as a faculty member of the Department of Biochemistry until 1961. He then joined the
faculty of the Department of Biochemistry at Duke University School of Medicine, where he
remains today.
At Duke, Hill continued his work on human hemoglobins, including several abnormal
variants, but soon turned his attention to the structure-function relationships of other proteins
and enzymes, including human fibrinogen and other blood coagulation factors, immunoglobulins, egg white lysozyme, bacterial acyl carrier protein, and lactose synthase. In subsequent
years he studied glycosyltransferases and worked in several areas of glycobiology. Hills work
on lactose synthase is the subject of the three JBC Classics reprinted here.
Lactose synthase consists of a catalytic galactosyltransferase in the endoplasmic reticulum
of the mammary gland and a regulatory protein, -lactalbumin, secreted in milk. Without
-lactalbumin, galactosyltransferase cannot synthesize lactose and instead catalyzes the
attachment of galactose to N-acetylglucosamine units on glycoproteins. The presence of -lactalbumin changes the specificity of galactosyltransferase so that it can transfer galactose to
glucose.
In the first JBC Classic, Hill, along with Keith Brew, Francis J. Castellino, and Thomas C.
Vanaman, reports the complete amino acid sequence of bovine -lactalbumin. They deduced
the sequence by characterizing the tryptic, chymotryptic, and peptic peptides of -lactalbumin
cleaved with cyanogen bromide. Because the amino acid sequence of -lactalbumin is very
similar to that of egg white lysozyme, Hill and his colleagues aligned the two sequences and
found that 49 of the residues were identical and 23 were conservative replacements. Thus, they
concluded that the three-dimensional structures of the two proteins were probably very similar
(3) and that they most likely arose from a common ancestral gene.
Hill, Brew, and Vanaman confirmed this structural similarity in the second Classic in which
they describe the locations of the four disulfide bonds in -lactalbumin. They found that the
disulfide bonds are arranged in a manner similar to those in egg white lysozyme and proposed
H37
Classics
Robert L. Hill
a three-dimensional structure for -lactalbumin based on the three-dimensional structure of

lysozyme, as shown in Fig. 2. Later, Hill, Brew, and Vanaman showed how -lactalbumin
serves as a regulatory protein for lactose synthase and permits the enzyme to synthesize
lactose in the mammary gland.
In the final Classic reprinted here, Hill and Ian P. Trayer describe the purification of the
galactosyltransferase component of lactose synthase or what they refer to as the A protein.
A key step to their purification was the use of columns of -lactalbumin attached covalently to
Sepharose. In the presence of glucose, galactosyltransferase would bind to the column and
could then be removed by omission of glucose from the elution buffer. Using this method, Hill
and Trayer were able to purify galactosyltransferase from bovine milk about 12,000-fold.
In 1969 Hill became the chairman of the Department of Biochemistry, and in 1974, the
James B. Duke Professor. He served as chairman until 1993. He was President (1976) and
Secretary (19721975) of the American Society of Biological Chemistry and was also on the
Editorial Board (19651970, 19721977) and an Associate Editor (1988-present) of the JBC.
Hill served on the FASEB Board (19721978), was General Secretary of the International
Union of Biochemistry (19821991), and chair of the Organizing Committee of the 17th
International Congress of Biochemistry and Molecular Biology in San Francisco in 1997. He
was elected to the National Academy of Sciences in 1975, the Institute of Medicine in 1978, and
the American Academy of Arts and Sciences in 1974. Hill received the Rose Award from the
American Society for Biochemistry and Molecular Biology in 1991, the North Carolina Gold
Medal (Science-1985), and the Karl Meyer Award from the Society for Glycobiology (2001).
Each of the coauthors on these Classic papers was a student or postdoctoral fellow in Hills
laboratory, and all have had productive, independent careers in biochemistry. Thomas Vanaman was a Ph.D. student who subsequently became Professor of Biochemistry and Chair at the
University of Kentucky. The others were postdoctoral fellows. Keith Brew went to the University of Leeds (UK) before returning to the U. S. where he became Professor of Biochemistry
at the University of Miami. He is now at Florida Atlantic University. Francis J. Castellino is
currently Professor of Biochemistry at the University of Notre Dame, and Ian P. Trayer was
H38
Classics
Fig. 2
Professor and Head of the School of Chemistry at the University of Birmingham (UK). Brew
and Castellino served on the JBC editorial board. Vanaman also served on the JBC board and
currently is an Associate Editor.
Nicole Kresge and Robert D. Simoni
REFERENCES
1. JBC Classics: Smith, E. L., and Bergmann, M. (1944) J. Biol. Chem. 153, 627 651 (http://www.jbc.
org/cgi/content/full/279/47/e6)
2. JBC Classics: DeLange, R. J., Fambrough, D. M., Smith, E. L., and Bonner, J. J. (1969) J. Biol. Chem. 244,
5669 5679 (http://www.jbc.org/cgi/content/full/280/36/e33)
3. Browne, W. J., North, A. C., Phillips, D. C., Brew, K., Vanaman, T. C., and Hill, R. L. (1969) A possible
three-dimensional structure of bovine -lactalbumin based on that of hens egg-white lysozyme. J. Mol. Biol. 42,
65 86
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Vol. 282, No. 20, Issue of May 18, p. e15, 2007

Printed in U.S.A.
Classics
JBC Centennial
19052005
Lysosomal Storage Disease Factors: the Work of

Elizabeth F. Neufeld
The Hurler Corrective Factor. Purification and Some Properties
(Barton, R. W., and Neufeld, E. F. (1971) J. Biol. Chem. 246, 77737779)
The Sanfilippo A Corrective Factor. Purification and Mode of Action
(Kresse, H., and Neufeld, E. F. (1972) J. Biol. Chem. 247, 2164 2170)
The Hunter Corrective Factor. Purification and Preliminary Characterization
(Cantz, M., Chrambach, A., Bach, G., and Neufeld, E. F. (1972) J. Biol. Chem.
247, 5456 5462)
Elizabeth Fondal Neufeld was born in 1928 in Paris, France. Her parents were Russian
refugees who had settled in France after the Russian revolution. However, the impending
occupation of France by the Germans forced the Fondal family to move to New York in 1940.
Consequently, Neufeld attended Queens College in New York, where she received her B.S. in
1948. She then worked briefly as a research assistant to Elizabeth Russell at the Jackson
Memorial Laboratory in Bar Harbor, Maine, before enrolling in graduate school at the
University of Rochester. Due to personal reasons Neufeld moved to Maryland in 1951, where
she served as a research assistant to Nathan Kaplan and Sidney Colowick at the McCollumPratt Institute at Johns Hopkins University. Colowicks research on NADH was the subject of
a previous Journal of Biological Chemistry (JBC) Classic (1), and Kaplan will be featured in
an upcoming JBC Classic.
In 1952, Neufeld enrolled in graduate school again, this time at the University of California,
Berkeley, where she studied with JBC Classic author William Zev Hassid (2). She received a
Ph.D. in 1956 for her work on nucleotides and complex carbohydrates. Neufeld then did several
years of postdoctoral research at Berkeley, first working with Dan Mazia on non-protein
sulfhydryl compounds in mitosis and then returning to Hassids laboratory to pursue research
on substituted sugars, polysaccharides, and glycoproteins.
In 1963, Neufeld moved to the National Institutes of Health (NIH), where she became a
research biochemist at the National Institute of Arthritis, Metabolism, and Digestive Diseases. It was during her time at the NIH that Neufeld began her research on mucopolysaccharidoses (MPS) disorders, a group of lysosomal storage diseases in which mucopolysaccharides cannot be stored or metabolized properly. Hurler syndrome and Hunter syndrome are
two MPS disorders in which partially degraded dermatan sulfate and heparan sulfate accumulate in lysosomes. Affected individuals usually die from the diseases by age 15. It was
accepted dogma in the early 1960s that mucopolysaccharides accumulate in these diseases
because of aberrant regulation of their synthesis resulting in overproduction.
At the NIH, Neufeld began to study mucopolysaccharide turnover in cultured fibroblasts
from people with Hurler or Hunter syndrome by incorporating radioactive sulfate into newly
synthesized mucopolysaccharides and studying their degradation with chase experiments. She
showed that the defects in Hurler and Hunter syndromes were due to decreased degradation
of the mucopolysaccharides and their resulting accumulation in lysosomes rather than an
overproduction of the sugars (3).
Subsequently, Neufeld found that Hurler syndrome could be corrected by a macromolecular
factor derived from fibroblasts or urine from donors who did not have Hurler syndrome. Her
H40
Classics
Elizabeth F. Neufeld
purification and characterization of this factor is discussed in the first JBC Classic reprinted
here. The factor was purified 1000-fold from normal human urine. From her experiments,
Neufeld surmised that the Hurler factor accelerates degradation of stored sulfated mucopolysaccharides. When an assay became available for -L-iduronidase in 1972, Neufeld was able
to show that the corrective factor for Hurler syndrome was, in fact, -L-iduronidase and that
this syndrome, as well as the Scheie syndrome, was due to a deficiency of that enzyme (4).
As reported in the second JBC Classic, Neufeld found a similar factor for Sanfilippo
syndrome, a lysosomal storage disease marked by impaired degradation of heparan sulfate.
The life span of children with this disorder does not usually extend beyond late teens to early
twenties. Sanfilippo patients fall into four subgroups, designated A through D, deficient in one
of four factors. Neufeld purified the Sanfilippo A corrective factor 850-fold from normal human
urine. She found that incubation of stored mucopolysaccharide with the purified factor resulted in release of inorganic sulfate, suggesting that the Sanfilippo A factor was a heparan
sulfate sulfatase. This proved to be correct.
In the final JBC Classic, Neufeld returned to her research on Hunter syndrome and
presented the purification of the Hunter corrective factor from normal human urine. She found
that the factor accelerates the degradation of labeled dermatan sulfate as well as exogenously
added proteodermatan [35S]sulfate. Neufeld eventually determined that the Hunter corrective
factor was iduronate sulfatase (5).
In 1973 Neufeld was named chief of the NIH Section of Human Biochemical Genetics, and
in 1979 she was named chief of the Genetics and Biochemistry Branch of the National Institute
of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK). She served as deputy
director of NIADDKs Division of Intramural Research from 1981 to 1983. In 1984 Neufeld
returned to the University of California, this time the Los Angeles campus, as chair of the
Biological Chemistry Department. She continues to do research at UCLA today.
Neufeld has chaired the Scientific Advisory Board of the National MPS Society since 1988
and was president of the American Society for Biochemistry and Molecular Biology in 1992.
She was elected to both the National Academy of Sciences and the American Academy of Arts
and Sciences in 1977 and was named a fellow of the American Association for Advancement in
Science in 1988. In recognition of her scientific achievements she was awarded the Hildebrand
Award in 1975, the Gairdner Foundation Award in 1981, the Lasker Award in 1982, the
International Society for Clinical Enzymology J. Henry Wilkinson Memorial Award in 1983,
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Classics
the Franklin Institutes Elliot Cresson Medal in 1984, the Wolf Prize in Medicine in 1988, the
National Medal of Science in 1994, and the Christopher Columbus Discovery Award for
Biomedical Research in 1992. She was named California Scientist of the Year in 1990.1
REFERENCES
1. JBC Classics: Pullman, M. E., San Pietro, A., and Colowick, S. P. (1954) J. Biol. Chem. 206, 129 141
2. JBC Classics: Babad, H., and Hassid, W. Z. (1964) J. Biol. Chem. 239, 946 948; Babad, H., and Hassid, W. Z.
(1966) J. Biol. Chem. 241, 26722678 (http://www.jbc.org/cgi/content/full/280/34/e31)
3. Fratantoni, J. C., Hall, C. W., and Neufeld, E. F. (1968) The defect in Hurlers and Hunters syndromes: faulty
degradation of mucopolysaccharide. Proc. Natl. Acad. Sci. U. S. A. 60, 699 706
4. Bach, G., Friedman, R., Weissmann, B., and Neufeld, E. F. (1972) The defect in the Hurler and Scheie syndromes:
deficiency of -L-iduronidase. Proc. Natl. Acad. Sci. U. S. A. 69, 2048 2051
5. Bach, G., Eisenberg, F., Cantz, M., and Neufeld, E. F. (1973) The defect in the Hunter syndrome: deficiency of
sulfoiduronate sulfatase. Proc. Natl. Acad. Sci. U. S. A. 70, 2134 2138
6. Hirschhorn, K. (1983) The William Allan Memorial Award. Am. J. Hum. Genet. 35, 10771080
Biographical information on Elizabeth F. Neufeld was taken from Ref. 6.
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Classics
JBC Centennial
19052005
The Biosynthesis of Membrane Glycoproteins: the Work of

William J. Lennarz
Membrane Glycoproteins. I. Enzymatic Synthesis of Mannosyl Phosphoryl Polyisoprenol and Its Role as a Mannosyl Donor in Glycoprotein Synthesis
(Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973) J. Biol. Chem. 248, 7570 7579)
William Joseph Lennarz was born in New York City in 1934. He received his B.S. in
chemistry from Pennsylvania State University in 1956 and his Ph.D. in organic chemistry
from the University of Illinois in 1959. Subsequently, he carried out postdoctoral research on
fatty acid biosynthesis at Harvard University with Journal of Biological Chemistry (JBC)
Classic author Konrad Bloch (1). In 1962 Lennarz moved to Baltimore where he was appointed
Assistant Professor in the Department of Physiological Chemistry at the Johns Hopkins
School of Medicine. He was later promoted to Associate Professor of Biochemistry in 1966 and
Professor in 1971.
The focus of Lennarzs work at Johns Hopkins was lipids and bacterial cell surfaces. He
found that mannosyl phosphoryl undecaprenol participates in the biosynthesis of mannolipids
in Micrococcus lysodeikticus (2). This work showed that lipid-linked sugars were biosynthetic
precursors of envelope-associated bacterial polysaccharides. Curious as to whether polyisoprenol phosphates also served as glycosyl carriers in mammalian tissue, Lennarz and his colleagues looked at the enzymatic transfer of mannose from GDP-mannose to endogenous
acceptors in cell-free preparations of bovine thyroid and hen oviduct. This is the subject of the
JBC Classic reprinted here.
Lennarz found that the preparations did catalyze the transfer of mannose to several
endogenous acceptors including mannosyl phosphoryl polyisoprenol. Two other receptors were
discovered and named soluble mannosylated endogenous acceptor (mannosyl s-acceptor) and
residual mannosylated endogenous acceptor (mannosyl r-acceptor). Lennarz and his colleagues then showed that mannosyl phosphoryl polyisoprenol serves as the mannosyl donor for
the synthesis of both mannosyl s- and r-acceptor. He further postulated that mannosyl
s-acceptor mediates the transfer of mannosyl residues from mannosyl phosphoryl polyisoprenol to glycoproteins. From these results Lennarz concludes, it seems possible that in eukaryotic systems, as in bacterial systems, activated lipid-linked sugars mediate the synthesis of
glycose-containing macro-molecules that are associated with the membranous components of
the cell.
Lennarz left Baltimore for Texas in 1983 when he was appointed Robert A. Welch Professor
and Chairman of the Department of Biochemistry and Molecular Biology at the University of
Texas Cancer Center, M. D. Anderson Hospital in Houston. In 1989, he joined the faculty of the
State University of New York at Stony Brook and became Distinguished Professor and
Chairman of the Department of Biochemistry and Cell Biology, a title he still holds today. In
1990 he founded and became Director of the Institute for Cell and Developmental Biology at
Stony Brook.
Lennarz continues to focus on glycoproteins, and his more recent efforts have been on the
structure, biosynthesis, and function of cell surface glycoproteins and the role of cell surface
proteins in fertilization and embryonic development in the sea urchin and frog. Currently, he
is studying the steps involved in glycoprotein synthesis, including N-glycosylation and protein
folding, as well as the functions of the glycan chains.
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William J. Lennarz
Lennarz served as president of the American Society for Biochemistry and Molecular
Biology in 1989 and was also president of both the Biochemistry Chairmans Organization and
the Society for Glycobiology. He was awarded the Society for Glycobiologys Karl Meyer Award
in 2004. Lennarz was a member of the Executive Committee of the International Union of
Biochemistry and Molecular Biology for almost a decade. He served as co-editor-in-chief for the
Encyclopedia of Biological Chemistry and was a member of the editorial board for Biochemical
and Biophysical Research Communications. He was elected to the National Academy of
Sciences in 1989.
REFERENCES
1. JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625 636; Rittenberg, D., and Bloch, K.
(1945) J. Biol. Chem. 160, 417 424; Bloch, K. (1945) J. Biol. Chem. 157, 661 666
2. Lahav, M., Chiu, T. H., and Lennarz, W. J. (1969) Studies on the biosynthesis of mannan in Micrococcus
lysodeikticus. II. The enzymatic synthesis of mannosyl-1-phosphoryl-undecaprenol. J. Biol. Chem. 244,
5890 5898
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Vol. 281, No. 43, Issue of October 27, p. e34, 2006

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Classics
JBC Centennial
19052005
Hepatic Carbohydrate Binding Proteins and Glycoprotein

Catabolism: the Work of Gilbert G. Ashwell
The Isolation and Properties of a Rabbit Liver Binding Protein Specific for
Asialoglycoproteins
(Hudgin, R. L., Pricer, W. E., Jr., Ashwell, G., Stockert, R. J., and Morell, A. G. (1974)
J. Biol. Chem. 249, 5536 5543)
Isolation and Characterization of an Avian Hepatic Binding Protein Specific for
N-Acetylglucosamine-terminated Glycoproteins
(Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536 6543)
G. Gilbert Ashwell was born in Jersey City, New Jersey, in 1916. He attended the University
of Illinois where he earned his B.A. in 1938 and his M.S. in 1941. Ashwell then went to
Columbia University and received his M.D. in 1948. After graduating, he remained at Columbia University as a research fellow for 2 years. In 1950, Ashwell joined the National Institute
of Arthritis, Metabolism, and Digestive Diseases at the National Institutes of Health. The
Institute later split into the National Institute of Arthritis and Musculoskeletal and Skin
Diseases and the National Institute of Diabetes and Digestive and Kidney Diseases, where
Ashwell remains today as emeritus scientist.
Ashwell is perhaps best known for his work with Anatol G. Morell in which they proposed
that membrane lectins remove senescent circulating glycoproteins and discovered one of the
earliest known carbohydrate receptors. Ashwell met Morell when he was on sabbatical leave
at Columbia University in 1965. Morell, who was at the Albert Einstein College of Medicine in
the Bronx, was interested in devising a method for labeling serum glycoproteins to study the
role of ceruloplasmin in Wilson disease. Together, Ashwell and Morell devised a labeling
procedure (1) that involved enzymatic removal of the glycoproteins terminal sialic acid
residue, thereby exposing galactose which was then treated with galactose oxidase and
tritiated borohydride, resulting in the incorporation of tritium into the protein.
When they injected their radioactive ceruloplasmin into rabbits, Ashwell and Morell noticed
that the asialoglycoproteins rapidly disappeared from the serum and appeared in parenchymal
cells in the liver (2). Further investigations showed that this phenomenon occurred with a
variety of naturally occurring plasma glycoproteins (3) and that the plasma membranes of the
liver were the primary site of binding for the circulating glycoproteins (4). This led to the
hypothesis that the exposure of terminal, nonreducing galactosyl residues by the removal of
sialic acid provides a means by which the liver recognizes and removes the defective molecules
from circulation as part of their normal catabolic pathway.
As described in the first Journal of Biological Chemistry (JBC) Classic reprinted here,
Ashwell and Morell eventually isolated the asialoglycoprotein binding protein from rabbit liver
using an affinity column composed of asialoorosomucoid covalently linked to Sepharose 4B.
Several years later, Ashwell and Toshisuke Kawasaki isolated an avian hepatic binding
protein that was specific for terminal N-acetylglucosamine residues on glycoproteins. This is
the subject of the second JBC Classic reprinted here. They compared the avian and rabbit
proteins and found that they had many properties in common, such as similar carbohydrate
constituents and a requirement for calcium. However, the two proteins also differed in many
ways. For example, the avian protein, in contrast to the mammalian protein, exhibited only
minimal binding activity for asialoglycoproteins but interacted strongly with agalactoglycoH45
Classics
Gilbert G. Ashwell
Anatol G. Morell
proteins. The structures of the two proteins also differed. The rabbit protein consisted of two
different subunits that were 48,000 and 40,000 daltons. The avian protein contained a single
subunit with an estimated molecular weight of 26,000.
Ashwells work on hepatic binding proteins has served as a stimulus for the identification of
a host of carbohydrate-specific receptors on various cell surfaces and has inaugurated the
current concept of a cellular lectin. In recognition of his contributions to science, Ashwell has
received many awards including the Gairdner Foundation International Award in 1982, the
Merck Prize from the American Society for Biological Chemists (now the American Society for
Biochemistry and Molecular Biology) in 1984, and the Senior Scientist Award from the
Alexander von Humboldt Foundation in 1989. He was elected to the National Academy of
Sciences in 1979.
REFERENCES
1. Morell, A. G., Van Den Hamer, C. J. A., Scheinberg, I. H., and Ashwell, G. (1966) Physical and chemical studies
on ceruloplasmin. IV. Preparation of radioactive, sialic acid-free ceruloplasmin labeled with tritium on terminal
D-galactose residues. J. Biol. Chem. 241, 37453749
2. Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H., and Ashwell, G. (1968) Physical and chemical studies
on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J. Biol. Chem. 243, 155159
3. Morell, A. G., Gregoriadis, G., Scheinberg, I. H., Hickman, J., and Ashwell, G. (1971) The role of sialic acid in
determining the survival of glycoproteins in the circulation. J. Biol. Chem. 246, 14611467
4. Pricer, W. E., and Ashwell, G. (1971) The binding of desialylated glycoproteins by plasma membranes of rat liver.
J. Biol. Chem. 246, 4825 4833
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Vol. 282, No. 11, Issue of March 16, p. e8, 2007

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Classics
JBC Centennial
19052005
The Pathway of Complex Oligosaccharide Biosynthesis:

the Work of Stuart A. Kornfeld
Processing of High Mannose Oligosaccharides to Form Complex Type Oligosaccharides on the Newly Synthesized Polypeptides of the Vesicular Stomatitis Virus G
Protein and the IgG Heavy Chain
(Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716 722)
The Synthesis of Complex-type Oligosaccharides. I. Structure of the Lipid-linked
Oligosaccharide Precursor of the Complex-type Oligosaccharides of the Vesicular
Stomatitis Virus G Protein
(Li, E., Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 77627770)
The Synthesis of Complex-type Oligosaccharides. II. Characterization of the Processing Intermediates in the Synthesis of the Complex Oligosaccharide Units of the
Vesicular Stomatitis Virus G Protein
(Kornfeld, S., Li, E., and Tabas, I. (1978) J. Biol. Chem. 253, 77717778)
Stuart A. Kornfeld was born in St. Louis in 1936. He attended Dartmouth College and
received his A.B. in 1958 and then went to the Washington University School of Medicine
where he earned an M.D. in 1962. Kornfeld then spend a year as an intern at Barnes Hospital
in St. Louis, 2 years as a research associate at the National Institute of Arthritis and Metabolic
Diseases, National Institutes of Health, and an additional year as an assistant resident at
Barnes Hospital. In 1966, he joined the faculty of the Washington University School of
Medicine where he has remained since. He became a professor of medicine in 1972 and of
biochemistry in 1976, the same year he began co-directing the hematology division. Kornfeld
also directed the Medical Scientist Training Program from 1991 to 1997. He is currently the
David C. and Betty Farrell Distinguished Professor of Medicine at Washington University.
In his early research at Washington University, Kornfeld focused on the synthesis of
complex oligosaccharides. This is the subject of the three Journal of Biological Chemistry
(JBC) Classics reprinted here. At the time the Classics were published, it was believed that the
glycosylation of glycoproteins containing asparagine-linked oligosaccharides occurred by en
bloc transfer of preformed oligosacchride chains from a lipid-linked oligosaccharide intermediate to an asparagine residue in a newly synthesized polypeptide chain. As reported in the
first Classic, Kornfeld and his student Ira Tabas, in collaboration with the virologist Sondra
Schlesinger, were able to isolate this lipid-linked oligosaccharide intermediate from vesicular
stomatitis virus-infected Chinese hamster ovary cells. They determined that the intermediate
was a high molecular weight mannose-rich oligosaccharide and that as the viral glycoprotein
matured in the cell, the excess mannose residues were removed and the outer sugars were
added. This indicated a single pathway for the formation of both high mannose and complextype glycans on glycoproteins.
In the second JBC Classic, Kornfeld and his students Ellen Li and Ira Tabas determined the
complete structure of the lipid-linked oligosaccharide intermediate. They did this by growing
cells on radioactive precursors, isolating the lipid-linked oligosaccharide, and then specifically
digesting the molecule. Prior to this paper, only partial structures of the molecule were
published. The structure of the oligosaccharide is conserved from yeast to man.
H47
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Stuart A. Kornfeld
After the lipid-linked intermediate is transferred to the nascent protein, the oligosaccharide
is processed to give rise to the completed complex oligosaccharide units. In the final JBC
Classic, Kornfeld and his students characterized the major processing intermediates and
proposed a scheme for the pathway of complex oligosaccharide biosynthesis.
Kornfeld has received numerous honors, including the Passano Award in 1991, the E.
Donnall Thomas Lectureship and Prize in 1992, the Karl Meyer Award from the Society of
Glycobiology in 1999, the UCSD/Nature Medicine Mentorship Award in 2002, the Washington University Gerty & Carl Cori Faculty Recognition Award in 2002, and the Washington
University Second Century Award in 2002. An author or co-author of more than 200 scientific
articles, he is a member of several honorary societies including the National Academy of
Sciences, the Institute of Medicine, the American Academy of Arts and Sciences, and the
Association of American Physicians. Kornfeld was an Associate Editor for the JBC from 1982
to 1987 and Editor of the Journal of Clinical Investigation from 1981 to 1982. He has also
served on numerous editorial boards including those of the Archives of Biochemistry and
Biophysics, the Proceedings of the National Academy of Sciences, the Journal of Cell Biology,
and Molecular Biology of the Cell.
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JBC Centennial
19052005
The Isolation and Localization of Laminin

by Rupert Timpl
Laminina Glycoprotein from Basement Membranes
(Timpl, R., Rohde, H., Robey, P. G., Rennard, S. I., Foidart, J. M., and Martin, G. R.
(1979) J. Biol. Chem. 254, 99339937)
Rupert Timpl (1936 2003) was widely known for his work on extracellular matrix proteins.
As a graduate student Timpl, who already had an impressive list of highly cited publications,
led a group of connective tissue immunologists started by Carl Steffen at the Institute for
General and Experimental Pathology at the University of Vienna Medical School. In this role,
Timpl supervised three postdoctoral fellows. He and his colleagues isolated collagen type I and
published several papers on the production and specificity of the first antibodies to extracellular matrix proteins.
Timpl earned his Ph.D. in chemistry from the University of Graz in 1966, and in 1967 he
became an Assistant in the Department of Immunology at the University of Vienna, Austria.
In 1969 he moved to Germany to become Head of the Research Group in the Department of
Connective Tissue Research at the Max-Planck-Institut for Biochemistry in Martinsried/
Munich. Timpl remained at Max-Planck for the rest of his scientific career, eventually
becoming Scientific Member and Director of the Department of Protein Chemistry in 1992.
Timpl also served as the Executive Director of the Max-Planck-Institut from 1995 to 1997.
At the Max-Planck-Institut, Timpl continued to study extracellular matrix proteins, focusing on the identification of epitopes of collagenous and non-collagenous extracellular matrix
proteins, and became one of the first scientists to apply immunofluorescence to the analysis of
normal and fibrotic tissues. For example, he showed that the tissue distribution of procollagen
type I differs from that of mature type I collagen and also clarified that type III collagen
production precedes that of type I collagen in any type of fibrosis.
The Journal of Biological Chemistry (JBC) Classic reprinted here is the result of a collaboration between Timpl and George R. Martin in which they delineated basement membranes
under normal and pathologic conditions. For their studies, Timpl and Martin used a transplantable mouse tumor, the EHS sarcoma, which produced an extracellular matrix of basement membrane. From the tumor, they extracted type IV collagen and, using antibodies,
localized it to the basement membrane of normal tissues (1, 2). In this paper, Timpl, Martin,
and their colleagues isolated a high molecular weight non-collagenous glycoprotein that was
also a major constituent of the tumors. They determined that the protein, which they named
laminin, consisted of at least two polypeptide chains joined to each other by disulfide bonds.
Using purified antibody against laminin, they showed that the glycoprotein is produced by a
variety of cultured cells and is a constituent of the basement membranes of these tissues.
In addition to his research on collagen and laminin, Timpl performed some unorthodox
experiments. These included analyzing the distribution of extracellular matrix proteins in
tzi), and in 50 million1500-year-old Peruvian mummies, in tissues of the Tyrolean Iceman (O
year-old fossils using immunohistochemical and immunofluorescent methods, thus creating a
new scientific discipline, paleoimmunology.
In recognition of his scientific achievements, Timpl received many honors. These include
the1984 Barbara Robert Medal, the 1991 Max Planck Research Award, the 1997 Wenner-Gren
H49
Classics
Rupert Timpl. Photo reprinted from Ref. 3, published by S. Karger AG, Basel.
Distinguished Lectureship, and the 1998 Lennox K. Black Award from Thomas Jefferson
University in Philadelphia.1
Timpls collaborator on the Classic, George R. Martin, is known for his studies on the
structure and function of connective tissue and alterations with disease. At the time the paper
was written, Martin was chief of the National Institute of Dental Researchs Laboratory of
Developmental Biology and Anomalies. In 1988 he was named Scientific Director of the
National Institute on Aging, a position he held until 1994. Martin has been involved in two
biotech startups, including the South San Francisco-based FibroGen.
Martin received his undergraduate degree in chemistry from Colgate University and his
Ph.D. from the University of Rochester. He has been the recipient of several honors including
the International Association of Dental Research Award in Basic Science, the Department of
Health and Human Services Distinguished Service Award, the Alexander von Humboldt
Senior Scientist Award, and the Federal Meritorious Executive Rank Award in 1987.
REFERENCES
1. Timpl, R., Martin, G. R., Bruckner, P., Wick, G., and Wiedemann, H. (1978) Nature of the collagenous protein in
a tumor basement membrane. Eur. J. Biochem. 84, 4352
2. Timpl, R., Martin, G. R., and Bruckner, P. (1979) Frontiers in Matrix Biology, Vol. 7, pp. 130 141, Karger, Basel
3. Wick, G. (2004) Rupert Timpla Personal Account. Int. Arch. Allergy Immunol. 134, 89 92
Biographical information Rupert Timpl on was taken from Ref. 3.
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Classics
JBC Centennial
19052005
The Formation of N-Glycosidic Linkages: the Work of

Phillips W. Robbins
Temperature-sensitive Yeast Mutants Deficient in Asparagine-linked Glycosylation
(Huffaker, T. C., and Robbins, P. W. (1982) J. Biol. Chem. 257, 32033210)
Phillips Wesley Robbins was born
in 1930, in Barre, Massachusetts. He
graduated from DePauw University
in 1952 with an A.B. and received his
Ph.D. in biochemistry from the University of Illinois in 1955. After finishing his Ph.D. research, he became a
research associate for Nobel Laureate
and Journal of Biological Chemistry
(JBC) Classic author Fritz Lipmann
(1) at the Massachusetts General
Hospital. Working with Lipmann,
Robbins studied how ATP was used in
the activation and transfer of sulfate
and showed that the thermodynamic
potential for the process came directly
from ATP. This discovery made a
large contribution to the general understanding of the global sulfur cycle,
and as a result Robbins received the
Eli Lilly Award in Biochemistry in
1956. The next year, Lipmann moved
to the Rockefeller Institute, and Robbins joined him as Assistant Professor
where he continued his work on sulfur activation.
In 1960, Robbins became Assistant
Professor of Biochemistry at the MasPhillips W. Robbins
sachusetts Institute of Technology.
There, he met Nobel Laureate Salvador Luria, who had just discovered that the structure of
Salmonella lipopolysaccharide (LPS) was controlled by lysogenic bacteriophage. Over the next
several years, Robbins solved the structure of LPS and showed that the repeating units of the
polysaccharide were preassembled on a polyisoprenoid lipid carrier that was eventually named
bactoprenol. Robbins then elucidated the cycle involved in polysaccharide assembly.
After working on bactoprenol, Robbins turned to mammalian N-linked glycosylation. The
formation of N-glycosidic linkages of eukaryotic glycoproteins was known to involve the
transfer of a common precursor oligosaccharide from a lipid carrier (dolichol) to an asparagine
residue in the nascent polypeptide chain. In many organisms, the lipid-linked precursor had
the composition Glc3Man9GlcNAc2. Robbins set out to identify and characterize the genes
involved in the early steps of the dolichol-linked oligosaccharide assembly. To do this, he used
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Classics
Saccharomyces cerevisiae mutants that were blocked in the synthesis of the lipid-linked
oligosaccharide, its co-translational transfer to protein, and the first steps of post-translational
processing.
The JBC Classic reprinted here describes Robbins procedures for isolating temperaturesensitive mutants in asparagine-linked glycosylation as well as his characterization of one of
these mutants (algl-1). Robbins and Tim C. Huffaker showed that algl-1 cells were able to
synthesize GlcNAc2-lipid but were unable to synthesize any mannose-containing oligosaccharide-lipids. The algl-1 cells were also unable to elongate exogenous GlcNAc2-lipid but were able
to convert Man1GlcNAc2-lipid to Man5GlcNAc2-lipid. These results indicated that the algl-1
mutant was blocked at the addition of the fist mannose residue to the oligosaccharide-lipid.
Characterization of the Glc3Man9GlcNAc2-lipid had shown that only the mannose residue
attached to GlcNAc exists in a -D-linkage, thus indicating that the mutant had a deficiency
in the enzyme involved in this process.
Robbins subsequently identified and characterized several other genes in this pathway.
These yeast genes were eventually shown to have orthologs in mammals. The enzymology and
genetics of the dolichol pathway enzymes represent classical pieces of glycobiology history.
Robbins later research turned to the dynamics of yeast cell wall synthesis and remodeling,
focusing on chitin synthesis. In 1998, he joined the newly formed Department of Molecular and
Cell Biology at the Boston University School of Dental Medicine (BUSDM) where he continued
his research on chitin synthesis as well as the evolution of N-linked glycosylation.
In recognition of his contributions to science, Robbins has received many awards and honors.
These include the Karl Meyer Award for Lifetime Achievement in Glycobiology (2000) and
election to the National Academy of Sciences (1986).
REFERENCES
1. JBC Classics: Lipmann, F. (1945) J. Biol. Chem. 160, 173190 (http://www.jbc.org/cgi/content/full/280/21/e18)
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JBC Centennial
19052005
The Transient Glucosylation of Glycoproteins: the Work of

Armando J. Parodi
Protein Glycosylation in Trypanosoma cruzi. The Mechanism of Glycosylation and
Structure of Protein-bound Oligosaccharides
(Parodi, A. J., Lederkremer, G. Z., and Mendelzon, D. H. (1983) J. Biol. Chem. 258,
5589 5595)
Transient Glucosylation of Protein-bound Man9GlcNAc2, Man8GlcNAc2, and
Man7GlcNAc2 in Calf Thyroid Cells. A Possible Recognition Signal in the Processing
of Glycoproteins
(Parodi, A. J., Mendelzon, D. H., and Lederkremer, G. Z. (1983) J. Biol. Chem. 258,
8260 8265)
Evidence That Transient Glucosylation of Protein-linked Man9GlcNAc2,
Man8GlcNAc2, and Man7GlcNAc2 Occurs in Rat Liver and Phaseolus vulgaris Cells
(Parodi, A. J., Mendelzon, D. H., Lederkremer, G. Z., and Martin-Barrientos, J. (1984)
J. Biol. Chem. 259, 6351 6357)
Armando J. Parodi was born in Buenos Aires, Argentina, in 1942. As a
teenager in a Latin American country
in the late 1950s, he was much more
interested in politics than in science
but nonetheless enrolled at the School
of Sciences at the University of Buenos Aires after graduating from high
school. The professors at the university were mostly young, dynamic scientists who had just returned from postdoctoral training in the United States
and Europe, and their enthusiasm was
contagious. As a result, Parodi decided
to obtain a Ph.D. after graduating from
the university. His father, who had
been a graduate student along with
Luis Leloir in Bernardo A. Houssays
laboratory at the University of Buenos
Aires, encouraged his son to attend the
Instituto de Investigaciones Bioqumicas Fundacion Campomar (now the
Fundacion Instituto Leloir), a private
institute created by Leloir in 1947. Being an obedient son, Parodi followed his
fathers advice.
Once at the Campomar Institute,
Parodi joined Leloirs research group.
Armando J. Parodi
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Classics
Leloir, who was featured in a previous Journal of Biological Chemistry (JBC) Classic (1), was
interested in the biosynthesis of saccharides, and Parodi was assigned a problem relating to
the synthesis of high molecular weight glycogen. He was able to publish five papers based on
this research and earned his Ph.D. in 1970, the same year that Leloir was awarded the Nobel
Prize in Chemistry.
Several years prior to 1970, Philips Robbins (who will be featured in an upcoming JBC
Classic) and JBC Classic author Jack Strominger (2) had shown that lipid (polyprenol)-linked
glycans were intermediates in the synthesis of several components of the bacterial cell wall.
Leloir decided to study whether or not this phenomenon occurred in eukaryotes as well and
was able to show that incubating rat liver microsomes with UDP-Glc led to the synthesis of
dolichol-P-Glc. After defending his Ph.D. thesis, Parodi joined this project and was part of a
team that elucidated the role of dolichol in transferring glycans to asparagine residues during
protein N-glycosylation.
After spending 2 years as a postdoctoral fellow at the Pasteur Institute in Paris, Parodi
returned to the Leloir Institute where he remains today. Between 1975 and 1978 he was able
to show that the dolichol-P-dependent pathway of protein N-glycosylation was also present in
yeast. A report in 1980 claiming that the pathway was not present in trypanosomatid protozoa,
based on the fact that neither free nor sugar-bound dolichol-P was present in the organisms,
led Parodi to challenge this claim. By incubating trypanosomatids with [14C]glucose, he was
able to show that the protozoans did indeed synthesize dolichol-P-P-glycans. However, the
glycans that were formed, Man9GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2, lacked glucose.
Parodi decided to further investigate the processing of protein-linked glycans in the trypanosome Trypanosoma cruzi, which is the subject of the first JBC Classic reprinted here. He and
his colleagues found that short pulses with [14C]glucose produced three protein-linked glycans
that were identified as Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2. These
compounds disappeared upon chasing cells with unlabeled glucose, and after a certain period,
Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2 were found in mature, fully
processed glycoproteins. Because an unglucosylated glycan was transferred to protein in this
protozoon, the only way in which Glc1Man9GlcNAc2 could have been synthesized was by the
transfer of glucose units to protein-linked Man9GlcNAc2.
The same glucosylated compounds, Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and
Glc1Man7GlcNAc2, had been detected by other groups in mammalian cells. Curious as to
whether or not glucose was transferred to Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 to
form the respective monoglucosylated compounds in these organisms, Parodi carried out
studies with calf thyroid slices that confirmed that transient glucosylation of glycoproteins
occurred not only in trypanosomatids but in mammalian cells as well. As reported in the
second JBC Classic reprinted here, after pulsing the slices with [14C]glucose, label in the
glucose of Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 appeared instantaneously whereas, as in T. cruzi, label in the mannoses of the last two compounds appeared
after a considerable delay.
Similar results were found in experiments with rat liver slices and Phaseolus vulgaris cells,
as reported in the third JBC Classic. This confirmed that protein-bound Glc1Man9GlcNAc2,
Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 were formed by glucosylation of unglucosylated
oligosaccharides. Parodis experiments also indicated that UDP-Glc was the donor of the
glucose residues and that the rough endoplasmic reticulum was the main subcellular site of
protein glucosylation.
These three JBC Classics established the occurrence of transient glucosylation of glycoproteins in the endoplasmic reticulum. Parodi later showed that the glucosyltransferase involved
in these reactions preferentially used acceptor glycoproteins not displaying their native threedimensional structure, suggesting that the enzyme might be involved in the quality control of
glycoprotein folding. It was later discovered that the endoplasmic reticulum resident chaperone calnexin interacted specifically with glycoproteins bearing monoglucosylated glycans, that
is, with the structures created by the endoplasmic glucosyltransferase, thus confirming the
role of transient glucosylation in quality control of glycoprotein folding.
In recognition of his contributions to science, Parodi has received many awards and honors
including the 1994 Award in Biology from the Third World Academy of Sciences. He has
received fellowships from the Eleanor Roosevelt-International Union against Cancer and the
Guggenheim Memorial Foundation. He was elected a member of the Third World Academy of
H54
Classics
Sciences in 1997, a foreign associate of the U. S. National Academy of Sciences in 2000, a
foreign member of the Brazilian Academy of Sciences and a fellow of the American Academy
of Microbiology in 2001, and a member of the National Academy of Sciences (Argentina) in
2003.1,2
REFERENCES
1. JBC Classics: Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333350;
2. JBC Classics: Suginaka, H., Blumberg, P. M., and Strominger, J. L. (1972) J. Biol. Chem. 247, 5279 5288;
Blumberg, P. M., and Strominger, J. L. (1972) J. Biol. Chem. 247, 8107 8113 (http://www.jbc.org/cgi/content/
full/282/31/e25)
3. Parodi, A. J. (2007) How I became a biochemist. IUBMB Life 59, 361363
1
2
Biographical information on Armando J. Parodi was taken from Ref. 3.

We would like to thank Armando J. Parodi for providing background material for this Introduction.
H55

Vol. 276, No. 45, Issue of November 9, pp. 4152741542, 2001

Printed in U.S.A.
Reflections
A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
19052005
Reflections on Glycobiology*1
Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.R100053200
Saul Roseman
From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University,
Baltimore, Maryland 21218
Glycobiology has become a hot subject,2 a timely one for Reflections. The primary reason,
I think, is illustrated in Fig. 1, which shows the surface of an erythrocyte in cross-section. Just
outside the plasma membrane of this and nearly all cells is a coat of fuzzy material called the
glycocalyx, consisting of a myriad of carbohydrate-rich molecules, polysaccharides, proteoglycans, glycoproteins, and glycolipids. If the cell shown here was a fibroblast or an intestinal
epithelial cell that secretes polysaccharides or mucins, it would be difficult to determine the
location of the cell boundary; the polymers begin on the cytoplasmic face of the lipid bilayer,
within it, or on its periphery, but it is not clear where they end. These extensive, complex
structures must serve essential roles in cell surface phenomena, but we are only beginning to
understand what some of these functions are. I believe that the glycoconjugates or glycans can
serve as important informational macromolecules.
In this remarkable age of genomics, proteomics, and functional proteomics, I am often asked
by my colleagues why glycobiology has apparently lagged so far behind the other fields. The
simple answer is that glycoconjugates are much more complex, variegated, and difficult to
study than proteins or nucleic acids. To understand where we are and to appreciate what it has
taken to get here requires some background, so this article will briefly survey the history of
glycobiology from early studies on fermentation to the beginning of the contemporary era.
The Past
Although glycobiology antedates biochemistry by many millenniums, their histories are
inextricably linked. The principal foundations of both fields lie in the development of organic
chemistry during the 19th century and in studies on the process of fermentation of glucose and
sucrose.
* This work was supported by Grant GM51215 from the National Institutes of Health.
1
The original title of this article was: Glycobiology: Past, Present, and a Very Bright Future. It was intended to
show something of the development of major concepts and to recognize the excellent contributions of pioneers in the
field. I had no problem until the modern era, when I realized that it would take volumes to adequately describe the
diversity of glycans and to cover, however briefly, current work and investigators. So, with regret, I am limited to the
past, where I tried to capture some of the flavor of the field, the origins of some contemporary ideas, and how they may
tie to the future. Insofar as the chemistry is concerned, I have chosen to emphasize the cell surface and extracellular
matrix because these are where most of the glycoconjugates are found. The focus is on two examples, the cartilage
aggrecan aggregate because it illustrates the enormous complexity that is possible with glycans and the erythrocyte
surface and the ABO blood groups, which in some respects, at least, may be a model for other cell surfaces.
2
A recent issue of Science features glycobiology (1), and the April, 2001 meeting of the Carbohydrate Division of the
American Chemical Society emphasizes glycobiology as a major subject; their prestigious C. S. Hudson Award was
presented to a well known glycobiologist, Y. C. Lee. How times have changed! In the 1950s, glycobiology was not a
popular subject. There were a few interested biochemists at the meetings, and we had an annual lunch (Karl Meyer,
Al Dorfman, Dick Winzler, Roger Jeanloz, Ward Pigman and a few others). After lunch, one might as well go home.
My papers (glucosamine metabolism) were invariably scheduled as either last or next to last on Friday afternoon at
the Federation Meetings in Atlantic City. The most hilarious incident was when my paper (next to last) was
announced at one of these sessions. When I reached the platform, the chairman of the session apologized because he
had to leave to make a train. My audience consisted of the next speaker and the slide projectionist. I stayed for the
last paper, but unfortunately I never asked the projectionist how he liked the presentations. I had the same experience
H56
Reflections: Reflections on Glycobiology
FIG. 1. The erythrocyte glycocalyx. This is revealed in electron microscopy by special staining methods. It is up
to 1400 thick, and the oligosaccharide filaments are 1225 in diameter. (Taken from Voet and Voet, Biochemistry,
with permission of the publisher. Original was by courtesy of Harrison Latta, UCLA).
FermentationFermentation was known to the cave man and has been the subject of
intense study ever since. The Old Testament has many references to wine and libations, the
first being to Noah (Genesis, 9:20 21): Noah, the husbandman, began and planted a vineyard.
And he drank of the wine and was drunk.3 Treatises and philosophical discourses were
published on the process during and after the Middle Ages.4 Fermentation was not confined to
making alcohol but has been used for thousands of years to make cheese, soy sauce, etc.
The first chapter of the biochemical classic Alcoholic Fermentation by Arthur Harden (1st
edition, 1911) reviews the history. (a) The most important early study was that of Lavoisier
(1789) who quantitatively established the stoichiometry of the process and concluded that the
sugar was split into two parts, one of which was oxidized (carbonic acid) at the expense of the
other (alcohol). Furthermore, if it were possible to recombine these two substances, sugar
would result. The methodology was insufficient to permit him to see an increase in the weight
of the yeast or of other products that were formed. (b) Yeast at the time was regarded as a
catalyst, much like alumina or diatomaceous earth, and during the first 60 years of the 19th
century, this was the prevailing view of the leading chemists and journal editors of the time
(Liebig, Berzelius, Wohler). This was despite the fact that in 1837 three independent investigators, Cagniard-Latour, Schwann, and Kutzing, presented evidence that yeast was a living
organism, an idea that was ridiculed by the establishment. (c) In 1860, the pivotal experiments
of Louis Pasteur finally laid this ghost to rest (5), and he showed unequivocally that yeast was
a living organism. He also did careful stoichiometry. The balance sheet showed that about 95%
of the C,H,O of the sugar was converted to CO2 and ethanol. The remainder, from 1.6 to 5%,
at the American Chemical Society meetings. Starch chemistry was a big thing for members of the Carbohydrate
Division, and they walked out on papers devoted to the glycoconjugates or hexosamines. At one such meeting, my
audience consisted of other members of the laboratory waiting to drive back to Ann Arbor with me. It was, however,
a great time to do this kind of research. There was virtually no pressure. The handful of us in this country who worked
in the field were supported by the National Institutes of Health. I can capture a little of the intellectual flavor of the
times by my experience when I submitted my first independent application. It stated that I would work on the
enzymatic synthesis of one of the monosaccharides in the glycoconjugates, but I did not know which to choose. I then
listed about four monosaccharides (glucosamine, fucose, glucuronic acid, and galactosamine) and possible preliminary
experiments for each; I would work on whichever problem appeared most fruitful. I was funded, and a short time later
I met one of the members of the Study Section (Ef Racker) who told me that it was the best application he had read.
What would happen today with an application that was so unfocused and with such nonspecific aims? Equally
important to the National Institutes of Health support, we received unsparing help from a number of farsighted
physicians such as Walter Bauer at Massachusetts General Hospital, who not only created a high caliber research unit
(Roger Jeanloz, Jerome Gross, Karl Schmid, Morris Soodak, and others) but was also instrumental in the Helen Hay
Whitney Foundation. In my case, it was William Robinson and Ivan Duff at the Rackham Arthritis Unit at the
University of Michigan. Only once (when I was first interviewed) did I have to explain to Bill Robinson how work on
Escherichia coli might relate to arthritis. Thus, we had the luxury of following our noses and serendipity wherever the
work took us. We started with studies on the intermediary metabolism of glucosamine, which led in turn to the
structure of the sialic acids and the discovery of N-acetylmannosamine, to the intermediary metabolism of these
compounds, to CMP-sialic acid and its enzymatic synthesis, to the glycosyltransferases, and finally to the phosphotransferase system for sugar uptake by bacteria (reviewed in Refs. 2 and 3). In recent years, the complex process of
chitin catabolism by marine bacteria has become a major project (4).
3
This reference was kindly called to my attention by Dr. Michael Edidin.
4
One that struck a chord was a 74-page treatise by John Richardson (1790) entitled Theoretic Hints on an
Improved Practice of Brewing Malt-liquors . . . . He defines fermentation as: A spontaneous internal motion of
constituent parts, which occasions a spontaneous separation and removal from their former order of combination, and
a remarkable alteration in the subject, by a new arrangement and re-union. Not a bad definition of intermediary
metabolism and the thermodynamics of glycolysis.
H57

consisted of substances that the yeast had taken from the sugar. The result of this and
subsequent work by Pasteur led to his famous dictum, no fermentation without life. In an
extension of this work, he came to the conclusion (1875) that fermentation was the result of life
without oxygen, the cells being able under anaerobic conditions to avail themselves of the
energy liberated by the decomposition of substances containing combined oxygen (i.e. anaerobic glycolysis). (d) Enzymes (called ferments), generally hydrolases, were known during the
19th century; indeed, invertase (i.e. sucrase) had been extracted from yeast. In 1858, Traube
proposed that fermentation resulted from the action of ferments secreted by cells on sugar.
Many attempts were made to extract yeast cells and obtain cell-free fermentation of sugar but
without success. Finally, while attempting to preserve yeast extracts for therapeutic purposes,
Eduard Buchner succeeded in 1897. The preservative was sugar, and he noted that carbon
dioxide was formed. This fortunate and serendipitous result marks the beginning of biochemistry as we know it today. It is interesting to note that the Journal of Biological Chemistry was
founded only a few years later by Christian Herter.
The story would not be complete without summarizing what was learned between Buchners
landmark result in 1897 and the publication of Hardens monograph in 1911. Kinetic experiments were conducted using yeast extracts and glucose, and the rate of fermentation was
followed by measuring the rate of CO2 evolution. The following results, especially by Harden
and Young, were obtained. (i) Fructose and mannose were fermented as well as glucose, but
the yeast had to be trained (i.e. adapted) for the extract to ferment galactose. They speculated
that different ferments were required for galactose utilization. (ii) Inorganic phosphate was
required. (iii) A hexose diphosphate was isolated, characterized as fructose-di-P, and was
shown to be an intermediate in the process. (iv) The extract was pressure filtered through a
gelatin film, giving a dialysate and a residue. Neither alone supported fermentation, but it
was restored by mixing the two. The residue contained the heat-labile zymase, and the
dialysate contained the heat-stable coenzyme(s) or cozymase. Soon after, it was shown that
yeast anaerobic glycolysis was closely connected to anaerobic glycolysis by muscle and muscle
extracts. The cozymase, of course, was the source of ATP, NAD, etc.
Development of Organic and Carbohydrate ChemistryThe close connection between the
development of organic chemistry and biochemistry in the 19th century is summarized in an
exemplary, early textbook (6). However, carbohydrate history goes back many centuries
earlier. Cellulose in the form of cotton, for instance, was known from ancient times, and
sucrose was one of the first organic substances to be crystallized (300 A.D., from the juice of
sugar cane in India). Because the climate in Europe was not favorable for growing sugar cane,
alternative sweetening agents were sought early in the 19th century, leading to the discovery
of new sugars (glucose, fructose, mannose, galactose, etc.), all with the same elementary
composition (CH2O)n. Clarifying the structural relationships between these compounds occupied carbohydrate chemists for most of the century. Finally, the structure of D-glucose was
established by Emil Fischer in 1891, which marks the beginning of modern carbohydrate
chemistry. Fischers multitudinous and brilliant contributions were likewise in the fields of
amino acid and purine/pyrimidine chemistry. It is worth reminding the reader that
chromatography and electrophoresis were unknown at the time, and substances were purified
by fractional crystallization and characterized by elemental analyses and their
physical properties (melting point, optical rotation, solubility, etc.). 5 In this age of
5
My interest in carbohydrate chemistry began as a graduate student working in the laboratory of Karl Paul Link
at the University of Wisconsin. He was both a carbohydrate and natural products chemist, with very high standards
and an ability to inspire the best in us. The laboratory had isolated and characterized dicumarol as the hemorrhagic
factor in spoiled sweet clover hay prior to my arrival (warfarin is a synthetic analogue). My project was to study the
metabolism of its parent compound, 4-OH-coumarin, which was not toxic. Four large dogs used as subjects were fed
the drug and maintained in very large metabolic cages so that their urine could be collected. (In those days, graduate
students took complete and very good care of their animals, including feeding, exercising them, and cleaning their
cages.) The metabolic product turned out to be 4-OH-coumarin -D-glucuronide. However, this had to be established
by synthesis and also by elemental analysis. I spent a very muggy, frustrating summer in Madison recording the
swings on a microbalance and learning how to do microanalyses before the standards finally came out right.
Somewhat later, I developed considerable experience with fractional crystallization, particularly of anomeric glycosides. They were being synthesized for Joshua Lederberg, a young faculty member in a neighboring department
(genetics), who was using them for assaying the expression of glycosidases, such as -galactosidase in E. coli.
Fractional crystallization, like elemental analysis, is tedious work, but above all it is a real art and when it works, it
is most gratifying. In doing this kind of work, we even invoked the help of the Lord. To this day my children remember
that my wife Martha (who is not a scientist) concluded the evening prayers over the Sabbath candles with the
following phrase: and may Daddy have crystals. It worked!
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electronics and the internet, one always thinks that science moves forward too slowly, but it
is mind boggling to realize how far we have come since the 1890s (Fischer, Buchner).
Glycobiology in the 20th Century: Chemistry
Structural Studies on Glycosaminoglycans (Mucopolysaccharides)Although mucins from
various sources were studied by organic chemists as early as 1846 (see reviews by Blix,
Gottschalk, and Morgan (7)) and were thought to contain sugars, there was always an
unresolved question of purity. In 1925, the distinguished chemist, P. A. Levene, who had made
fundamental contributions to the structures of the nucleic acids, published a monograph
entitled Hexosamines and Mucoproteins. Chondroitin sulfate had been isolated in 1884 from
cartilage, but the nature of its monosaccharides and structure were controversial until Levene
showed conclusively that the constituents were D-glucuronic acid, chondrosamine (D-galactosamine), acetic acid, and sulfuric acid in equimolar ratios. He depicted the structure as GalNAc
linked to GlcUA and sulfated at C-6 on the GalNAc. As might be expected from the available
methodology and misinformation on sugar ring structures, there were major errors in the
structural assignment, including the fact that it was a tetrasaccharide. Similarly, mucoitin
sulfate (i.e. hyaluronic acid) was depicted as a tetrasaccharide containing GlcNAc but also
sulfate. He also questioned whether substances such as ovalbumin were glucosidoproteins or
whether such substances even existed.
In 1934 (8), hyaluronic acid was isolated in pure form from vitreous humor, and its correct
composition was determined. This groundbreaking paper was the first of many from Karl
Meyers laboratory, creating a science from chaos. His laboratory subsequently isolated and
characterized the chondroitin sulfates, keratan sulfate, and various hyaluronidases.6
Establishing the structures of heteropolysaccharides can be exceptionally difficult, and the
problems can be summarized as follows: (i) identification and quantitation of the monosaccharides; (ii) D- or L-configurations; (iii) branched or unbranched; (iv) sequence; (v) or
anomers; (vi) pyranose or furanose rings; (vii) positions of the linkages; (viii) many of these
polymers are derivatized (e.g. phosphate, sulfate, acetate, etc.), and polymers with different
biological and chemical properties are formed, depending on the position of the linkage in the
derivative; and (ix) to complicate matters even further, some of the polymers and oligosaccharides are covalently linked to proteins or lipids.
One of the major problems confronting workers in this field was protein and how to get rid
of it because it was regarded as a contaminant of the mucopolysaccharides, now called
glycosaminoglycans or GAGs.7 Protein was not easily removed.8 Meyer, for instance, thought
that the protein formed ionic bonds with the polysaccharides. In the 1950s, Maxwell Schuberts
laboratory showed that cartilage chondroitin sulfate was linked to protein, thus opening a new
chapter in the chemistry of these polymers, now called proteoglycans. The next essential step
was to characterize the linkage region between the GAG and the protein. Work on different
polymers around the same time (late 1950s) by Pigman (mucins), Kabat (blood group
substances), and Muir (chondroitin sulfate) suggested that the sugars were linked to
6
Karl Meyer was a delightful person with a keen sense of humor. His exchanges with Albert Dorfman at the
meetings were the highlight for many of us. For instance, at one meeting Al gave a talk, and in the questioning period
Karl asked Al, How did you quantitate the keratosulfate? Al responded that he had not. In a stage whisper, Karl
said: I thought as much. Al, with whom I did my postdoctoral work, was a principal figure in the field. He held both
M.D. and Ph.D. degrees but what made him really unusual was his expertise in both fundamental biochemical
research and in clinical practice (pediatrics). He was a leader in the University of Chicago Medical School and later
became Chair of Pediatrics. Al came around to see me every day, and we would get into the most vigorous discussions
on how to interpret results, the next experiments, etc. He had to be the most tolerant person, considering that I was
fresh out of graduate school and was convinced that I knew everything there was to know (it has been downhill ever
since). My paying job was to direct the pediatric blood chemistry laboratory, which was actually very interesting
because one had to develop ultramicroanalytical methods, especially for samples from the newborn, which were often
obtained by heel puncture. Most of my research was conducted late in the afternoon and evening. Al lived across the
Midway and could see the laboratory window (top floor of Bobs Roberts Hospital) from his bedroom. I always left the
lights on when I went home.
7
The abbreviations used are: GAG, glycosaminoglycan; PAPS, 3-phosphoadenosine-5-phosphosulfate; MGT, multiglycosyltransferase; GT, glycosyltransferase; ST, sialyltransferase.
8
At the University of Chicago we were fortunate to have the large meat packing houses close by, which were sources
of necessary tissues, such as bovine eyes (for vitreous humor), testis (for hyaluronidase), etc. The isolation of
chondroitin sulfate started with bovine nasal septa, which were obtained by working on the line and cutting them out
of the skulls as they came by on a belt (very hard on the hands). The cartilage was ground and extracted with about
0.1 N NaOH for several days in the cold with constant stirring. The alkaline extract was then deproteinized and the
polysaccharide isolated. By hindsight we know now that the alkaline extraction procedure split the polysaccharide
from its O-serine (or threonine) linkage in the protein by -elimination.
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9
serine. In 1964, Lindahl and Roden found that the linkage fragment in heparin was
O--D-xylopyranosyl-L-serine (reviewed in Ref. 10). They later showed that the sequence at the
linkage region in these polymers (chondroitin sulfates, dermatan sulfate, and heparan sulfate)
to which the polysaccharide is attached is GlcUA-Gal-Gal-Xyl-Ser. In skeletal keratan sulfate,
the O-linkage is to -GalNAc in place of the Xyl.
At the same time, a different class of complex carbohydrates, now call glycoproteins, was the
subject of intensive study. Neubergers laboratory in England showed by isolation and synthesis that the linkage region in ovalbumin is -GlcNAc3 Asn, i.e. to the amide N of asparagine. There are, of course, a wide variety of N-linked glycoproteins, particularly the glycoproteins in serum. Since the overriding question in these early studies was purity, the isolation
and characterization of the major serum glycoprotein, 1-acid glycoprotein (orosomucoid), by
Karl Schmid was a key breakthrough. The protein (44 kDa) contained 17% hexose and 12%
hexosamine.
A characteristic of carbohydrate polymers is that they are polydisperse or microheterogeneous. The template mechanisms of protein and nucleic acid synthesis do not apply to the
carbohydrate polymers, thereby resulting in polydispersity. Human orosomucoid, for instance,
contains 6 oligosaccharide chains per molecule, but the chains are different from each other.
In the collection of molecules called orosomucoid, at least 8 oligosaccharides have been
identified (11). Each oligosaccharide can contain up to 5 different kinds of sugars, a given
sugar can occur several times in the chain, and the number of possible combinations is
overwhelming (see below).
Aggrecan AggregateThe major components of cartilage are collagen and a huge macromolecular complex called the aggrecan aggregate. An electron micrograph of one such aggregate
is shown in Fig. 2A, and Fig. 2B presents a schematic view of 6 aggrecan monomers bound to
hyaluronan. Determining the details of these structures is an extraordinary achievement in
this field, equivalent (at least) to delineating the structure of collagen. The structure was
developed through work in the laboratories of Hascall, Muir, and Heinegard and has recently
been reviewed (12, 13). This unusually complex molecule can have an apparent mass of 6
109 Da and is a composite of all of the structural units described above. The relationship
between the structure of the aggregate and its function is briefly discussed below.
The Erythrocyte Surface, Human Blood Group Activity, and Erythroglycan (Poly-Nacetyllactosamine)The frequent incompatibility of the blood of a donor and recipient was
recognized in the 17th century. Starting with the work of Landsteiner (1900), who defined the
ABO group, we now know that there are at least 27 such families of human blood group
substances expressed on the surfaces of erythroid cells and often other cells as well. The
general characteristic of these antigens is that they comprise integral membrane glycoproteins, both O- and N-linked, and in some cases, glycolipid. Thus far, it has been shown that the
glycan units are the epitopes in four of the systems, ABO, Lewis, P, and H/h.10 Some aspects
of the ABO system will be discussed here.
Work on the ABO family was greatly aided by finding these activities in water-soluble form
in various secretions and mucins, such as ovarian cysts. The major antigenic determinants
were established by Morgan and his co-workers (particularly Watkins and Aminoff) and by
Kabat and his co-workers (reviewed in Ref. 14). These determinants were sugars at the
non-reducing termini of oligosaccharide chains linked via Ser and Thr to polypeptides, similar
to the mucins. Blood group O chains were terminated by a trisaccharide Gal(,1 4)[Fuc-(,1
2)]GlcNAcX. Blood group A activity was expressed by linking an -GalNAc to C-3 of the Gal,
whereas in B activity a Gal is substituted for the GalNAc.
The erythrocyte membrane was quite another problem. Although Yamakawa showed that
red blood cell glycolipids exhibited such activity (1953), this conclusion was disputed as late as
1956 (14). It is now clear that the antigens are carried on the erythroid surface by both lipids
and polypeptides (see review by Hakomori (15)).
These structures are closely related to the glycosaminoglycan keratan (desulfated keratan
sulfate). The repeating unit in this GAG is N-acetyllactosamine: Gal-(,1 4)-GlcNAc-(,13)
9
In the alkaline -elimination step, the oligo- or polysaccharides glycosidically linked to serine or threonine are first
released from the protein and then degraded by the alkali at the reducing end of the chain, a reaction called peeling.
An important advance in the field was Carlsons alkaline borohydride procedure, which reduced the aldehyde group
as the glycosidic bond was cleaved and protected the oligomer from alkaline degradation (9).
10
I am very grateful to Dr. Olga Blumenfeld (Department of Biochemistry, Albert Einstein Medical School) for
helpful discussions on the blood group substances.
H60
FIG. 2. Electron micrograph of a proteoglycan aggregate purified from calf epiphyseal cartilage. A, the
aggregate was spread as a monolayer in a cytochrome c film. Under these conditions the chondroitin sulfate chains of
the proteoglycan collapse onto the core protein so that each monomer (aggrecan) of the aggregate is distinct. The
nearly uniform length of the monomers is characteristic of proteoglycans from young cartilages, with each 3.5 million
Da in molecular mass. This aggregate contains 180 aggrecan monomers, and the overall molecular mass of the
aggregate is 6.5 billion Da. The central strand of hyaluronan is 5500 nm in length. The boxed area encloses 6
monomers, the number depicted in the model in B. (Micrograph kindly provided by Joseph Buckwalter, University of
Iowa.) B, model of a portion of the proteoglycan aggregate showing 6 aggrecan monomers (see box in A). Each of the
six monomers is depicted with a central core protein strand to which the glycosaminoglycans are covalently linked,
giving the appearance of bristles. The core protein (250,000 Da) contains a midregion with 100 chondroitin sulfate
chains (blue) of 30,000 average molecular weight and a nearly equal number of keratan sulfate chains (red) of
10,000 average molecular weight. The monomers are anchored (non-covalently) to the central hyaluronan strand
(orange) by: (a) a hyaluronan-binding site in the N-terminal globular-1 (Gl) domain (pink sphere) of the core protein,
and (b) a link protein (crystal sphere) that binds to both hyaluronan and to the G1 domain of aggrecan. The core protein
of aggrecan contains two other globular domains, G2 and the C-terminal G3, shown as green and lavender spheres,
respectively. The functions of G2 and G3 are not known. The globular domains of aggrecan also contain N-linked
oligosaccharides (red Y symbols). The model is fully extended as it would appear in dilute solution. In the tissue the
structure would be compressed to approximately one-tenth its extended size. See Ref. 13 for additional details. (Model
and supplemental three-dimensional, rotational view kindly provided by Mark Sabo (Art Department) and Vincent
Hascall, Cleveland Clinic Foundation.)
linked to the next Gal in the chain. The same structural unit but in shorter chains than the
polysaccharide, called poly-N-acetyllactosamine or polylactosamine, is found both O- and
N-linked to integral membrane proteins on many cell surfaces and is also found linked to
ceramide. Polylactosamine can be straight chain or branched and can be decorated with Fuc
or sialic acid residues. Apparently the first references to Gal-, GlcNAc-, and Fuc-rich glycopeptides in cell membranes came from work by the eminent geneticist/molecular biologist
Francois Jacob and his group on the cell surface antigens found in early embryonic differentiation (reviewed in Ref. 16). At about the same time (17, 18), Laine and co-workers isolated
erythroglycan by extensive Pronase digestion of lipid-free red blood cell stroma and characterized the large branched oligosaccharides (7,000 10,000 Da) by methylation, etc. Band 3,
the major red blood cell integral membrane protein and the anion transporter, is the source of
the polylactosamine, and it accounts for more than 30% of the total Gal and GlcNAc in the red
blood cell membrane. Further, at its non-reducing termini the polymer can carry Fuc and Gal
or GalNAc, thereby becoming an antigenic determinant for A, B, or O activity. The large
quantity of polylactosamine peptide derived from the red blood cell membrane corresponds to
most of the antigenic sites in the intact erythrocyte (about 2 106). There is now an extensive
literature on polylactosamine, its enzymatic synthesis (19), and how branching occurs during
development, tumorigenesis, etc.
Blood group activity is also carried by glycolipids, which are present in small quantities in
the red blood cell membrane (reviewed by Hakomori (15). They consist of a large number of
compounds derived from N-acetyllactosamine oligomers. This family comprises oligosacchaH61

rides, both straight and branched chain, linked to glucosylceramide and terminated by one of
the antigenic determinant sugars. The glycolipids change, especially with respect to branching, during the development of erythroid cells.
Glycobiology in the 20th Century: Biosynthesis
Isotope ExperimentsThe complex carbohydrates contain up to 8 different monosaccharides, including D-xylose, hexoses, hexosamines, and hexuronic acids, in addition to various
sialic acids, such N-acetylneuraminic acid (NAN or NeuAc). Until 1950, we did not know how
most of these monosaccharides were biosynthesized. For the 6-carbon sugars, the theories
ranged from (a) direct conversion of the 6-carbon skeleton of D-glucose to the sugar to (b)
fragmentation of glucose through glycolysis and other catabolic cycles and recombination of
suitable fragments. It was suggested, for example, that the GlcNH2-6-P carbon skeleton was
formed by condensing glyceraldehyde-3-P (G3P) and serine, with subsequent reduction of the
carboxyl to the aldehyde. And how could L-fucose possibly arise directly from D-glucose without
inversion of the carbon skeleton by 180o, which would give the L- from a D-sugar?
These problems were addressed by treating an appropriate biological system with specifically labeled glucose, such as 1-[14C]- and 6-[14C]glucose in companion experiments. The pure
polymer was isolated and hydrolyzed, and the monosaccharides were isolated and dissected
carbon by carbon to determine the specific activity at each C-atom in the skeleton.11 If the
origin of the 6-carbon hexoses was via fragmentation of the Glc 6-carbon chain, followed by
recombination, isotope scrambling would result.12 The results were conclusive, showing that
the 6-carbon skeleton of Glc was converted intact to GlcNH2, glucuronic acid, galactose, and
mannose. Surprisingly, D-Glc was converted to L-Fuc without inversion (20).
Enzyme ExperimentsThe next step, of course, was to determine the pathways of synthesis
and degradation using appropriate ferments. A review (21) published in 1959 shows how
rapidly the field grew in 10 years. Many of the anabolic/catabolic pathways were established,
and although they have since been added to and somewhat modified, the essential elements
remain the same today.
Sialic acid was a separate problem in that enzymatic studies could not proceed until after its
correct structure was established (22), and N-acetyl-D-mannosamine was found to occur
naturally and to be a precursor of NeuAc. Glucosamine-6-P is the precursor of all nitrogencontaining sugars and is formed from Fru-6-P and glutamine (23), although the catabolic
enzyme, GlcN-6-P deaminase (24), which gives Fru-6-P and NH3 as products, is reversible and
can be utilized anabolically when the synthase is mutated in bacteria.
Sugar Nucleotides, Dolichol, and PAPSAside from establishing the intermediary metabolism of the monosaccharides, there were five major developments in the field over the course
of the next 20 years (listed in order of the discussion): (a) isolation of active sulfate or PAPS
(1958); (b) recognition of lectins (sugar-binding proteins) in animal tissues; (c) identification of
dolichol-linked oligosaccharides as intermediates in the synthesis of the Man-rich core oligosaccharides of N-linked glycoproteins (1976); (d) isolation of the sugar nucleotides (1950); (e)
elucidation of the pathways of synthesis and degradation of the complex carbohydrates and of
the number and specificities of the glycosyltransferases.
The precursor of the sulfated glycoconjugates, such as chondroitin sulfate, is 3-phosphoadenosine-5-phosphosulfate (or PAPS) characterized and enzymatically synthesized by Robbins
and Lipmann (25). PAPS is, of course, high energy or active sulfate.
Ricin was apparently the first lectin (proteins that bind carbohydrates) recognized more
than a century ago. Early in their history, lectins were found to agglutinate erythrocytes
depending on blood type. Lectins by now have become a field unto themselves, and the work
of I. J. Goldstein, who developed quantitative methods for accurately defining specificity, as
11
My major postdoctoral project was to determine the modes of synthesis of the glucosamine and glucuronic acid
moieties in hyaluronic acid. The biological system was a strain of Group A streptococcus that secreted the polysaccharide, and the organism was grown (in a rich medium) on 1-[14C]- and 6-[14C]glucose. One of the many problems was
the cost of the labeled sugars (far too expensive for these experiments). [14C]NaCN was more reasonable, and the
labeled sugars were synthesized from this starting material. In the experiments, because of the rich medium, the
labeled glucose, acetate, and lactate were isolated from the medium, as well as the polysaccharide, and were dissected
as well. Konrad Bloch, who was a Professor in the Department of Biochemistry, was of enormous help to me during
this phase of the work.
12
For instance, at the triose-P level, because of triose-P isomerase 1-[14C]glucose would become 1,6-[14C]hexose, and
the specific activity at C-1 would be half that of the 1-[14C]glucose used for the experiment.
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well as in isolating new lectins, is especially significant. The plant lectins are not only powerful
tools for analyzing macromolecules and cell surfaces, but the field became particularly interesting to cell biologists when it was realized that animal cells express lectins.
In 1968, Ashwell and co-workers (26) discovered that liver hepatocytes bind and take up
asialoglycoproteins (the asialoglycoprotein endocytosis receptor). This receptor is a Gal-specific lectin in mammals and GlcNAc-specific in birds. It is called the Ashwell protein in what
follows. Animal lectins, such as the Siglecs (bind to sialic acids), Ig superfamily lectins,
selectins, the integrins, CAMs (cell adhesion molecules), collectins, CD44, and others, have
now become major areas of research.13
Lipid-linked intermediates were discovered around 1964 1965 by three groups (reviewed by
Osborn (27)). These studies were conducted in the laboratories of Horecker, Robbins, and
Strominger, who were working on the enzymatic synthesis of bacterial lipopolysaccharides and
the peptidoglycan cell wall. This work led to similar studies in a number of laboratories on
lipid-linked intermediates in the biosynthesis of complex carbohydrates in eukaryotic organisms, including yeast, plants, and higher animals (for review, see Ref. 28). A polyisoprenoid,
dolichol, was known to occur in animal tissues and was identified as the lipid carrier of
the carbohydrate groups. This early work led to the well established dolichol pathway for the
synthesis of the N-linked glycoproteins (29). The dolichol pathway does not apply to the
O-linked glycoproteins or to the glycolipids.
The isolation and characterization of sugar nucleotides is one of the most important achievements in the field of carbohydrate metabolism in the 20th century. They were discovered in
two laboratories at about the same time, those of Luis Leloir in Argentina and of James Park
in this country. Leloirs group (Caputto, Cardini, Paladini, and Cabib) was working on the
enzymatic synthesis of Glc-6-P from Gal-1-P using yeast extracts and found that a heat-stable
cofactor (cozymase) was required. One of the factors was isolated and fully characterized as
UDP-Glc (30). This was followed by isolation of UDP-Gal and recognition that the galactowaldenase reaction (epimerization at C-4) occurred at the level of the sugar nucleotides. In an
independent discovery, Park found that Staphylococcus aureus treated with penicillin (which
inhibits cell wall synthesis) accumulated considerable quantities of UDP derivatives and
showed that they contained the cell wall sugar muramic acid and amino acids (31). The Park
compounds are intermediates in cell wall biosynthesis.
The list of sugar nucleotides is huge (32). It includes virtually every naturally occurring
monosaccharide, purines, pyrimidines, and in some cases, 2-deoxyribose in place of ribose.
They are most frequently formed by the action of pyrophosphorylases, which catalyze the
reaction (N indicates nucleoside): NTP glycose-1-P 3 PPi N-P-P-glycose. As usual, the
sialic acids are exceptions in that the nucleotide is CMP (not the diphosphate). CMP-sialic
acid, originally isolated from E. coli (33), is formed by condensation of NeuAc or N-glycolylneuraminic acid and CTP (34, 35). A similar nucleotide (36) was obtained with 2-keto-3deoxyoctanoic acid (KDO), a component of bacterial lipopolysaccharides.
Functions of the Sugar NucleotidesSome sugars in glycoconjugates (Man, GlcNAc, NeuAc)
are synthesized from Fru-6-P, whereas others (Gal, GlcUA, Xyl) are synthesized as nucleotide
sugars from UDP-Glc or, in the case of L-Fuc, from GDP-Man. Aside from their participation
in the biosynthesis of monosaccharides, the sugar nucleotides serve as glycose donors in the
biosynthesis of oligo- and polysaccharides. Enzymes that utilize sugar nucleotides as donors
are designated glycosyltransferases and are the major catalysts for generating the glycosidic
bond (also formed by transglycosidases and phosphorylases).
Glycosyltransferases were first reported by the Leloir group (37, 38). The enzymes catalyzed
the synthesis of disaccharides (trehalose, sucrose) and of the ,134 linkage in glycogen. At the
time, it was generally believed that the ,134 linkage in glycogen was synthesized from
Glc-1-P by reversing the glycogen phosphorylase-catalyzed reaction.
The following general glycosyltransferase catalyzed reaction occurs in animal tissues.
13
In the 2-year period 1999 2000, SciFinder lists 1400 papers on selectins and 5900 on the integrins. Early in my
service on National Institutes of Health Study Sections, our section, which comprised a distinguished group of
biochemists, reviewed what I think may have been the first National Institutes of Health application for funds to study
a plant lectin (concanavalin A). A vigorous debate ensued with those opposed asking why a plant protein that binds
carbohydrates should be of any interest to the National Institutes of Health. It should be funded by the National
Science Foundation! Fortunately, the application was funded. In this connection it was this same group that reviewed
applications by Fritz Lipmann and by Luis Leloir, which were of course funded; these applications basically consisted
of describing what the applicants planned to do with very little detail or particular focus. How would they fare today?
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GlycosePP(U or G) HO-acceptor (U or G)DP GlycosideO-acceptor
REACTION 1
In the case of the sialic acids, such as NeuAc, the donor is CMP-NeuAc.
Specificity of the Glycosyltransferases, Biosynthetic Pathways, and Multiglycosyltransferase
SystemsAt the time we began to study macromolecular glycans (around 1960), nothing was
known about the biosynthetic pathways leading to glycoproteins, mucins, and glycolipids. To
undertake this work, we required substrate quantities of the sugar nucleotides. Fortunately
Moffatt and Khorana (39) had just published a method for the synthesis of UDP-Glc. A very
fruitful and enjoyable summer in Vancouver led to a modified, general method for the
synthesis of sugar nucleotides (40),14 giving us the tools for studying glycosyltransferases.
Some 1520 glycosyltransferases were characterized, and it soon became obvious that they
formed families such as sialyl-, Gal-, GlcNAc-, GalNAc-, Glc-, and Fuc-transferases. The
enzymes we studied are involved in the synthesis of the mucins, glycolipids, and terminal
trisaccharides of N-linked glycoproteins, and the results can be summarized as follows (see
Ref. 2 for review). 1) Each glycosyltransferase is specific for both the donor sugar nucleotide
and the acceptor. 2) A different transferase usually catalyzes each step in a pathway. When a
sugar occurs twice in a molecule, such as NeuAc in disialoganglioside, two different transferases are required.15 3) Chain elongation can be at the non-reducing end or can form branch
points. 4) The product of one reaction is the substrate for the next, which leads to the concept
of multiglycosyltransferase (MGT) systems, namely that the transferases required for synthesis of one glycoconjugate are associated. A different MGT system is required for mucins,
glycoprotein trisaccharide termini, and glycolipids (e.g. gangliosides). 5) Polydispersity in
glycoconjugates is explained by the MGT hypothesis. For instance, Svennerholm (41) showed
that there is a particular array of human brain gangliosides of different chain length and
complexity. This array exactly fits the pathway that is predicted by the specificities of the
enzymes in the corresponding MGT. One would expect to find only the final products of the
pathway (e.g. tetrasialoganglioside) if all conditions were optimum for each enzyme and they
are expressed at high enough levels. It should be noted that many of the transferases require
Mn2 for activity and not necessarily at the same concentrations,16 and this may be an
important means of regulating these activities. 6) There is some evidence to support the idea
that glycosyltransferases in an MGT complex bind to one another. In the original work, we
found that all of the transferases in a given MGT were found in the same particulate fraction,
ultimately identified as the Golgi apparatus (42). The Gal-transferase involved in synthesizing
the Gal-O-Xyl-O-Ser (protein) linkage region in chondroitin sulfate was purified by binding to
the immobilized xylosyltransferase, and it coprecipitates with antibody directed at the xylosyltransferase (43). Recent papers present evidence for binding of a glycolipid GalNAc-transferase to a Gal-transferase (44, 45).
The Structure of the Acceptor Can Determine Glycosyltransferase ActivityThe activity of a
GT is not only determined by whether the acceptor is a glycolipid, mucin, or an N-linked
glycoprotein but can also depend on the fine structure of the termini in these potential
acceptors. One example will be given. Enzymatically synthesized, labeled CMP-NeuAc and
CMP-N-glycolylneuraminic acid (34) were used to detect and characterize sialyltransferases
(STs), first from rat mammary gland and then in colostrum (goat, bovine, human), bacterial
extracts (for synthesizing colominic or polysialic acid), submaxillary gland, and embryonic
chicken brain (summarized in Ref. 2). Bovine colostrum and human milk contain mixtures of
14
I arrived with the sugar 1-phosphates and was given space on John Moffatts bench. He measured my waist and
marked off the corresponding width on the bench top. Fortunately, my waist was substantially greater than his.
15
The glycosyltransferases that synthesize the GAGs have exceptional characteristics. (a) Elongation of the
polysaccharide chains in chondroitin sulfate, heparan sulfate, and in one hyaluronan (produced by Pasteurella) takes
place in a stepwise manner at the non-reducing ends of the polymers. In these cases, a single glycosyltransferase
transfers first one and then the other glycose unit from their respective sugar nucleotides to the ends of the chain. (b)
Single glycosyltransferases also catalyze hyaluronan synthesis by eukaryotic and Streptococcal cells, but in these
cases elongation occurs at the reducing ends of the chain by mechanisms that are not entirely clear. One phenomenon
that has always intrigued this reviewer is how the enzymes or cells know when to stop the process of polysaccharide
elongation (see Ref. 12 for review).
16
One mechanism for regulating glycosyltransferase activity could well be via local Mn2 concentrations. A brief
literature search found references to analyses of tissues, mostly autopsy material, for trace metals including Mn2 but
little on its transport. The relevant analyses will require that they be conducted on actively metabolizing cells and
organelles (such as the Golgi) to preserve the in vivo ion gradients.
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3-sialyllactose (NeuAc-2,3-Gal1,4Glc) and 6-sialyllactose (NeuAc-2,6-Gal1,4Glc). The
rat mammary gland ST synthesizes 3-sialyllactose when incubated with CMP-NeuAc and
lactose, whereas the colostrum enzyme yields the 6-isomer. The colostrum enzyme shows
great specificity for its acceptors (46). In quantitative terms, when the efficiency of the enzyme
is expressed as Vmax/Km the following values were obtained (% relative to N-acetyllactosamine): Gal(,1 4)GlcNAc or N-acetyllactosamine, 100; Gal(,1 4)Glc or lactose, 2;
Gal(,13)GlcNAc, 13; Gal(,1 6)GlcNAc, 0.4; and asialoorosomucoid, 1000.
Thus, the nature of the penultimate sugar in an acceptor, the precise linkage of the terminal
to the penultimate sugar, and the size of the acceptor can all play major roles in determining the
activity of a given GT.
The total number of GTs thus far identified exceeds many hundreds (reviewed in Ref. 47).
Many of the structural genes have been cloned, and the enzymes were overexpressed, purified
to homogeneity, and characterized kinetically. At least two have been crystallized and their
three-dimensional structures determined. Insofar as the topics covered here are concerned, the
GlcNAc transferases that act on polylactosamine ((Gal-(1,4)-GlcNAc-(1,3))n), a constituent
of many cell membranes, are of considerable interest (see review by Renkonen (19)). One
GlcNAc transferase is required for increasing the chain length at the non-reducing terminal
Gal. Two others add GlcNAc to internal Gal residues, thereby starting the branching process.
One of the branching enzymes works at the distal end of the chain, and the other acts
centrally. Both are greatly influenced by the presence of Fuc residues on the chains. Thus,
the combination and interplay of the GalT, the three GlcNAc transferases, the FucT, and
possibly the sialyltransferases determine the final structure on the cell surface, but how these
are regulated with respect to each other remains to be determined.
Speculations on Cell-Cell Adhesion
The human brain contains approximately a trillion neurons, and each averages around 103
connections with other cells or about 1015 specific connections. How can this happen given a
total of about 40,000 genes in the human genome? The data banks list 72,000 publications on
cell adhesion, and they report CAMs (cell adhesion molecules), cadherins, catenins, ephrins,
Eph receptor tyrosine kinases, laminins, selectins, integrins, their relationships to the extracellular matrix and the cytoskeleton, to cytokines, and much, much more.
In some instances, the role of carbohydrates is well documented. (a) Leukocyte extravasation
(recruiting leukocytes from the blood to the site of infection, injury, or lymphatic circulation)
involves a sequence of complicated interactions between the leukocytes and the blood capillary
endothelium comprising selectins, other proteins, and carbohydrates (reviewed in Ref. 48). (b)
CD44, a cell surface receptor, binds to hyaluronan (12). (c) Myelin-associated glycoprotein is a
Siglec (sialic acid-specific lectin) that binds to complex gangliosides, an interaction essential
for maintaining normal myelin structure (49, 50).
As indicated below, there is now a rapidly developing interest in the role of glycans in
development and in cell recognition. However, in surveying the literature, it appears that some
old ideas bear repeating. The discussion will be limited to cell-cell recognition.
Specific Intercellular AdhesionThe crucial importance of cell recognition in development
was well established in the late 1800s. In normal embryos, cells exhibit exquisite adhesive
specificity. They know where they are, and they know where they are going. Under in vitro
conditions, cells adhere nonspecifically to many substances, including tissue culture plastic,
glass, serum proteins, etc. Nevertheless, adhesive specificity can be demonstrated in vitro and
was shown in 1907 in a classic case of serendipity. Wilson (51) found that when single-cell
suspensions from two species of marine sponges were mixed they first aggregated to form a
heterologous chimera, but with time they sorted out to yield aggregates of homotypic cells.
Holtfreter (1930s) obtained the same results with cell suspensions from different embryonic
tissues. Although cadherins are thought to be involved in cell sorting, the underlying biochemical basis is very complex, and yet to be fully explained.17 It was subsequently demonstrated
that adult cells, such as hepatocytes and mycocytes (55, 56), exhibit adhesive specificity and
17
Humphreys (52) showed that dissociation of the sponges to single cells released species-specific, heat-labile, large
molecular weight aggregation factors. These observations were followed by a series of studies from many laboratories, particularly by Burgers group. Polysaccharides, sulfated polysaccharides, proteoglycans, and lectins have been
invoked as participants in a multistep process, some of which require Ca2. In a recent paper (53), a unique
supramolecular circular proteoglycan complex is described as one of the components involved in the process. One of
the N-linked glycans contains glucuronic acid, fucose, mannose, galactose, N-acetylglucosamine, and sulfate (54).
H65
FIG. 3. Scanning electron micrographs of chicken hepatocytes and immobilized chicken plasma membrane glycolipid. A quantitative procedure was devised for assaying the effects of immobilized glycolipids and
similar substances on the rate of adhesion of chicken hepatocytes (81). A glycolipid, present in trace quantities in
chicken liver, was the only substance of many pure and mixed lipids and glycolipids tested that stimulated the
adhesion of these (but not rat) hepatocytes. The scanning electron micrographs are as follows. A, mixture of chicken
hepatocytes and polystyrene beads (red balls) previously immersed in a dilute solution of the specific glycolipid. No
such structures were seen with beads treated with the inactive lipids. B and C, cell and bead. The filopodia-like
structures were observed in all cases of cell-bead adhesion. D, cell-cell adhesion. Filopodia-like structures are evident
in the regions of contact. These experiments were conducted with purified but not homogenous preparations of the
glycolipid. The specific glycolipid has recently been purified to apparent homogeneity by Dr. Ming Chuan Shao and
Barbara Rauch. (The photographs were kindly prepared by Michael McCaffery of the Integrated Imaging Center,
Department of Biology, Johns Hopkins University.)
that in liver homogenates, the specific factor was localized to the plasma membranes (56). The
active factor(s) in the chick membranes is a trace glycolipid (Fig. 3).
A quantitative assay (57) was used to study the kinetics of homologous adhesion (58) and
showed that the process is multistep. The first step does not require metabolic energy; the cells
form a loose association that dissociates even by simple dilution of the suspension. In the
second energy-requiring step, the aggregate is stabilized and can only be dissociated by
vigorous treatment, e.g. proteases. In the third step, the stable aggregates synthesize collagen
and sulfated GAGs. All of this takes minutes at 37 C.
Insofar as the underlying biochemical mechanisms are concerned, there are two obvious
questions. (a) What cell surface molecules participate in the process? (b) How is the information transmitted to the interior of the cell? Two hypotheses were suggested to answer these
questions, as indicated in Figs. 4 and 5.
Hypothesis I: Carbohydrates Are Involved in Specific Intercellular AdhesionTwo mechanisms were proposed (2) for carbohydrate participation as indicated in Fig. 4. 1) Cell adhesion
is mediated by hydrogen bonds between carbohydrates on neighboring cells. That hydrogen
bonds can be important in maintaining carbohydrate structures is exemplified by polysaccharides such as cellulose and chitin. 2) Cell adhesion is mediated by the binding of carbohydrates
to cell surface proteins and enzymes. There were two reasons for extrapolating from proteins
to enzymes and in particular to the glycosyltransferases. (a) The glycosyltransferases as a
class appeared to be much more specific than the lectins, a critical requirement for specific
intercellular adhesion. (b) If glycosyltransferases are involved, then one cell could also modify
the surface of its neighbor. However, extracellular modification requires an extracellular cell
surface or soluble enzyme and a source of sugar nucleotides and/or PAPS, either from the
cytoplasm or extracellularly. Is any of this possible? 1) Enzymatically active, soluble extracellular glycosyltransferases do occur in the fluid surrounding intact embryonic chicken brain and
in embryonic and adult chicken serum, vitreous humor, and human spinal fluid (59). 2) Cell
surface glycosyltransferases may occur. Chick embryonic neural retina cells transferred Gal
from UDP-Gal to soluble high molecular weight acceptors (60), suggesting that the reaction
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FIG. 4. Hypothesis I: carbohydrates are involved in specific intercellular adhesion. The suggested mechanisms are as follows. (a) Hydrogen bonding between oligosaccharide chains on adjacent cell surfaces. The scheme is
not meant to imply that hydrogen bonds can only form between identical monosaccharides. (b) Enzyme-substrate
complex. The model is meant to suggest that cells can bind to and/or modify adjacent cells or extracellular matrix
through the action of cell surface glycosyltransferases. See text for discussion.
FIG. 5. Hypothesis II: membrane messengers. An extrapolation of the Sutherland second messenger idea. A
variety of extracellular signals are received by cell membrane receptors, which in turn send specific messages to the
cytoplasm or nucleus. The membrane is a transducer, and the membrane messengers were suggested (82, 83) to
comprise both low and high molecular weight substances, such as proteins. Additionally, it was suggested that in some
cases the messenger molecules would act stoichiometrically (e.g. repressors of operons), whereas in others, they could
be enzymes.
was catalyzed by a cell surface Gal-transferase. Evidence for and against this conclusion has
been presented by other laboratories, and at this time, it remains controversial. However, Fig.
3 suggests that only a vanishingly small percent of the cell surface appears to be involved early
in specific cell-cell interactions. If cell surface glycosyltransferases participate in these interactions, they may be present in traces and difficult to detect by any method, including
immunological procedures. 3) Sugar nucleotides may be secreted. In a recent paper (61), a G
protein-coupled plasma membrane receptor for UDP-Glc was identified in a wide variety of
human tissues, including many regions of the brain. Thus, extracellular sugar nucleotides may
indeed occur.
Hypothesis II: Membrane MessengersIn 1958 1962, a series of studies by Sutherland and
co-workers (62, 63) led to the characterization of cAMP, adenylate cyclase, and the effects of
certain hormones on this enzyme. Sutherland designated cAMP as the second messenger
(hormones were the first messenger). This seminal work surely ranks as one of the most
important biochemical findings of the past century. Somewhat later, Rasmussen invoked Ca2
as another second messenger. These hypotheses were obviously correct, but it seemed to us
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FIG. 6. Effect of quantitative changes in carbohydrate concentration on cell binding in a model system.
A variety of carbohydrates were covalently linked to polyacrylamide gels and tested with chicken and rat hepatocytes.
As expected, the cells bound to the gels in accord with the known specificities of the Ashwell receptor, chicken to
GlcNAc, and rat to Gal. In this model system, the concentrations of the sugars in the gels were varied, as indicated.
A threshold or critical concentration effect is observed. Below this concentration, there is no binding, and above it, all
the cells bind to the gels.
that they were insufficient. Could the diverse stimuli received by a cell and the many
responses that these signals elicited be explained by only two second messengers? A membrane messenger hypothesis was therefore devised in 1974 as illustrated in Fig. 5. The
membrane acts as a transducer containing multiple receptors that respond to external
signals by releasing specific intracellular messengers. Signal transduction by the plasma
membrane is now well established, and a section of each issue of this Journal is devoted to
papers in this field.
Quantitative Changes in Carbohydrate Ligands Can Have Global Effects on Cellular Phenotypic BehaviorQualitative changes in carbohydrate composition of the cell surface or the
substrata to which the cell adheres can have far reaching effects on cell behavior, but what of
quantitative changes? Although the Ashwell protein catalyzes receptor-mediated endocytosis
of glycoproteins in hepatocytes, it does not participate in intercellular adhesion. Nevertheless,
it served as a useful model to answer this question.
We have often tried to mimic cell surfaces by adsorbing or covalently linking potential
carbohydrate ligands to solid matrices (e.g. Fig. 3). This approach was used to test hepatocytes
(64, 65) with sugar derivatives covalently linked to polyacrylamide gels. Chicken hepatocytes
specifically adhered to GlcNAc-derivatized gels and rat hepatocytes to Gal-derivatized gels, in
accord with the known specificities of the Ashwell receptors in the two cell types. However,
there was a remarkable threshold or critical concentration effect of the sugars as shown in Fig.
6. Below this concentration of sugar in the gel, the cells did not bind to the gels. At the
threshold, 15% increases in GlcNAc and Gal concentrations, respectively, in the gels resulted
in 100% cell binding to the gels.18
18
An interaction between a protein and its monovalent ligand may be weak, but if the ligand is polyvalent such that
many protein molecules can interact, the binding affinity for the polyvalent ligand greatly increases. An excellent
example of this is CD44, a cell surface receptor that binds to hyaluronan (12). Hyaluronan oligosaccharides with 6 10
sugars are sufficient to interact with CD44 monovalently, and relatively high concentrations of these oligosaccharides
can prevent binding of macromolecular hyaluronan, which otherwise binds with high affinity. However, the interaction of the monovalent oligosaccharides with CD44 is sufficiently weak that they do not remain bound through a
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The physiological implications are plain if this model represents what can happen in cell
adhesion. A non-adherent cell can become adherent by a slight change in the cell surface
concentration of the appropriate ligand and/or its receptor or in the extracellular matrix. Even
more likely, the grouping of receptors or ligands into microdomains in the plasma membrane
results in binding, and the size of these domains is apparently affected (regulated?) by other
factors, such as cholesterol.
The Future: Glycans as Informational Macromolecules
The particular advantage of carbohydrates is that they have enormous potential for serving
as informational macromolecules, starting with their de novo biosynthesis. Laine (67), for
instance, has calculated that a hexasaccharide has 1012 isomeric permutations. Second, the
glycans are readily modified after synthesis of the core structure. A few such modifications are
sulfation (thought to be essential for leukocyte extravasation), O-acetylation of individual
sugars such as sialic acid, addition of a few sugar residues that can convert blood group O to
A or B, or initiating a branch point by the action of the branching GlcNAc transferases on
polylactosamine. For instance, in the neural retinotectal system where neuronal pathfinding
is essential, immunological methods have shown a dorsoventral gradient in a cell surface
antigen of the rat embryonic neural retina (68). The antigen was identified as 9-O-acetyl-GD3.
At the same time, there was no apparent gradient of the parent ganglioside GD3. Thus,
relatively few enzymes can create a large number of molecular variants.
There is no doubt that the molecular events underlying embryogenesis, especially of the
nervous system, will be the major goal of biology well into the foreseeable future. Experiments
in a number of laboratories are now in progress to elucidate the roles of glycans in these
processes, and some of these are cited above. However, other examples can be given. (a) By
constructing specific glycosyltransferase mutants in mice and other organisms, the synthesis
of specific glycans or classes of glycans can be eliminated. This approach has shown that
gangliosides and glycoproteins of the N-glycan type are essential for the survival of the embryo
and/or its normal development in the mouse (69) and in the nematode Caenorhabditis elegans
(70).19 (b) A number of papers have reported that proteoglycans and glycosaminoglycans,
especially heparan, are essential for normal development of Drosophila and C. elegans. The
affected genes include Wingless, tout-velu, sugarless, sulfateless, dally, and sqv 3,7,8 (7177).
(c) Notch receptors are highly conserved intercellular signaling pathways that direct embryonic cell-fate decisions. The activities of these receptors are regulated by Fringe proteins, and
recent evidence (78, 79) shows that Fringe is a fucose-specific GlcNAc-transferase.
To summarize, the huge gap between the 1015 specific connections in the brain and the
number of genes in the human genome can readily be filled by the glycans.
It is presumptuous to try to predict the future. Who, in the 1960s, could have predicted what
happened to the field we called genetics? At the moment, primary interest seems to be shifting
from genomics to proteomics and functional proteomics. But as others have said, glycobiology
is the field of the future. However, the problems are formidable, as I have tried to indicate in
this brief overview.
One problem is nothing more than a false perception. On several occasions I have heard
structural biologist colleagues state that the glycan units in a glycoprotein, for instance,
cannot be important because they are too flexible to be seen in an x-ray crystal structure or by
NMR. In other words, if they dont have structure, how can they have function? That this
conclusion is gratuitous requires no more than a moments reflection.
For instance, one important physiological property of cartilage is that it is reversibly
compressible, acting like a spring to the application of a force. This feature emanates from the
flexibility of the aggrecan aggregate, which can be compressed to one-tenth its volume.
Hyaluronan provides another example. It is essential as a lubricant in joint fluids where it has
high viscosity and an extended helical or possibly random coil structure. It is more restricted
in the aggregcan aggregate but must still be flexible, and it forms a gel in cumulus cell-oocyte
complexes (12).
routine washing step (66). The cytoplasmic tail of CD44 interacts with anchorin in the cytoskeleton. Therefore,
interaction of CD44 with its polyvalent, linear ligand can contribute to alignment and stabilization of the cytoskeleton
and consequently influence cell behavior.
19
Unpublished data: on the N-type glycoproteins by Schachter et al; on the gangliosides by Sandhoff, Proia et al.
H69

These should be sufficient to make the point. Glycans are different because frequently they
are flexible and adjust to physiological need. In other words, in these substances, function
defines structure, not vice versa. Certain glycans clearly have highly favored conformations,
and lectins may have evolved to reflect those particular three-dimensional structures. Furthermore, whereas energy minimization methods can yield the thermodynamically favored
conformers, the less favored conformers may be the biologically active structures that bind to
their ligands.
It would not be a big surprise if different conformers of a single oligosaccharide interacted
with different ligands or receptors or enzymes or possibly even other carbohydrates under
different physiological conditions. It is this interplay between proteins and different conformers that likely allows a single carbohydrate structure, such as hyaluronan, to be used in many
different ways. In the excellent book by Cantor and Schimmel on the conformation of macromolecules (80), they raise a number of questions about carbohydrate polymers similar to those
discussed above and then say: These are all interesting questions, but it will probably take
much hard work to answer most of them. Amen to that! What is lacking is adequate
biophysical methodology.
The problem is much more complicated when we deal with membranes. Trying to assign
structure or even distribution (if it is not random) of a particular glycolipid on the surface
seems impossible at this point because of fluidity of the external monolayer of the lipid bilayer.
If glycolipids do exist primarily in rafts or domains, these domains are in a constant state of
flux and motion within the monolayer, and their sizes, frequency of formation, etc., depend on
the lipid composition of the remainder of the monolayer and whether they are or are not
associated with membrane-bound signaling proteins, such as the Src family of kinases. The
same problem exists with cell surface glycoproteins, except possibly for those tethered to
cytoplasmic components, such as the cytoskeleton. Even in the latter case, publications
suggest that perturbation of the cell can rapidly result in drastic reorganization of the
cytoskeleton.
Thus, it appears that present methods will permit us only to obtain snapshots of limited
areas of the cell surface. There is no doubt that the task ahead of us is difficult, but if cells
talk to other cells via cell surface substances such as the glycans, the problem cannot be
avoided. I am optimistic. Breakthrough technological advances are produced at an astonishing
rate these days.
Who could have predicted the development of polymerase chain reaction and its
consequences?
AcknowledgmentsI am especially grateful to Drs. Ronald Schnaar and Mark Roseman for critical reading of this
manuscript and for many helpful suggestions. The sections on aggrecan and the aggrecan aggregate and Fig. 2 could
not have been written without the help of Dr. Vincent Hascall, who also provided numerous other insightful
comments.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
(2001) Science 291, 22632502

Roseman, S. (1970) Chem. Phys. Lipids 5, 270 297
Roseman, S. (1989) FEMS Microbiol. Rev. 63, 312
Keyhani, N. O., and Roseman, S. (1999) Biochim. Biophys. Acta 1473, 108 122
Conant, J. B. (1952) Pasteurs Study of Fermentation, Harvard University Press, Cambridge, MA
Fruton, J. S., and Simmonds, S. (1953) General Biochemistry, John Wiley & Sons, Inc., New York
Gottschalk, A. (1972) Glycoproteins: Their Composition, Structure and Function, Elsevier Science Publishers B.V.,
Amsterdam
Meyer, K., and Palmer, J. W. (1934) J. Biol. Chem. 107, 629 634
Carlson, D. M. (1966) J. Biol. Chem. 241, 2984 2986
Roden, L. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 267371, Plenum
Press, New York
Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., and Kobata, A. (1981) J. Biol. Chem. 256, 8476 8484
Hascall, V. C. (2000) Glycoconj. J. 17, 599 608
Wight, T. N., Heinegard, D. K., and Hascall, V. C. (1991) in Cell Biology of Extracellular Matrix (Hay, E. D., and
Olson, B., eds) pp. 4578, Plenum Press, New York
Kabat, E. A. (1956) Blood Group Substances, Academic Press, New York
Hakomori, S.-I. (1999) Biochim. Biophys. Acta 1473, 247266
Jacob, F. (1979) Curr. Top. Dev. Biol. 13, 117137
Jarnefelt, J., Rush, J., Li, Y.-T., and Laine, R. A. (1978) J. Biol. Chem. 253, 8006 8009
Laine, R. A., and Rush, J. S. (1988) Adv. Exp. Med. Biol. 228, 331347
Renkonen, O. (2000) Cell. Mol. Life Sci. 57, 14231439
Heath, E. C., and Roseman, S. (1958) J. Biol. Chem. 230, 511519
Roseman, S. (1959) Annu. Rev. Biochem. 28, 545578
Comb, D. G., and Roseman, S. (1960) J. Biol. Chem. 235, 2529 2537
Ghosh, S., Blumenthal, H. J., Davidson, E., and Roseman, S. (1960) J. Biol. Chem. 235, 12651273
H70

24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
Comb, D. G., and Roseman, S. (1958) J. Biol. Chem. 232, 807 827
Robbins, P. W., and Lipmann, F. (1957) J. Biol. Chem. 229, 837 851
Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H., and Ashwell, G. (1968) J. Biol. Chem. 243, 155159
Osborn, M. J. (1969) Annu. Rev. Biochem. 501538
Waechter, C. J., and Lennarz, W. J. (1976) Annu. Rev. Biochem. 45, 95112
Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631 664
Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. (1950) J. Biol. Chem. 184, 333350
Park, J. T. (1952) J. Biol. Chem. 194, 877904
Gabriel, O. (1982) Methods Enzymol. 83, 332353
Comb, D., Watson, D., and Roseman, S. (1966) J. Biol. Chem. 241, 56375642
Roseman, S. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 437 441
Kean, E. L., and Roseman, S. (1966) J. Biol. Chem. 241, 56435650
Ghalambor, M. A., and Heath, E. C. (1963) Biochem. Biophys. Res. Commun. 10, 346 351
Cabib, E., and Leloir, L. F. (1958) J. Biol. Chem. 231, 259 275
Leloir, L. F., Olavarria, J. M., Goldemberg, S. H., and Carminatti, H. (1959) Arch. Biochem. Biophys. 81, 508 520
Moffatt, J. G., and Khorana, H. G. (1958) J. Am. Chem. Soc. 80, 3756 3761
Roseman, S., Distler, J. J., Moffatt, J. G., and Khorana, H. G. (1961) J. Am. Chem. Soc. 83, 659 663
Svennerholm, L. (1963) J. Neurochem. 10, 613 623
Schachter, H., Jabbal, I., Hudgin, R., Pinteric, L., McGuire, E. J., and Roseman, S. (1970) J. Biol. Chem. 245,
1090 1100
Schwartz, N. B., and Roden, L. (1975) J. Biol. Chem. 250, 5200 5207
van Meer, G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13211323
Giraudo, C. G., Daniotti, J. L., and Maccioni, H. J. F. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 16251630
Bartholomew, B. A., Jourdian, G. W., and Roseman, S. (1973) J. Biol. Chem. 248, 575l-5762
Varki, A., and Marth, J. (1995) Semin. Dev. Biol. 6, 127138
Ebnet, K., and Vestweber, D. (1999) Histochem. Cell Biol. 112, 123
Collins, B. E., Kiso, M., Hasegawa, A., Tropak, M. B., Roder, J. C., Crocker, P. R., and Schnaar, R. L. (1997) J. Biol.
Chem. 272, 16889 16895
Collins, B. E., Yang, L. J. S., Mukhopadhyay, G., Filbin, M. T., Kiso, M., Hasegawa, A., and Schnaar, R. L. (1997)
J. Biol. Chem. 272, 1248 1255
Wilson, H. V. (1907) J. Exp. Zool. 5, 245258
Humphreys, T. (1967) in The Cell Surface and Specific Cell Aggregation (Davis, B. B., and Warren, L., eds)
pp. 195210, Prentice-Hall, Englewood Cliffs, NJ
Jarchow, J., Jurgen, F., Anselmetti, D., Calabro, A., Hascall, V. C., Gerosa, D., and Burger, M. M. (2000) J. Struct.
Biol. 132, 95105
Misevic, G. N., and Burger, M. M. (1990) J. Biol. Chem. 265, 2057720584
Albanese, J., Kuhlenschmidt, M. S., Schmell, E., Slife, C. W., and Roseman, S. (1982) J. Biol. Chem. 257,
31653170
Obrink, B., Kuhlenschmidt, M. S., and Roseman, S. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 10771081
Orr, C. W., and Roseman, S. (1969) J. Membr. Biol. 1, 109 124
Umbreit, J., and Roseman, S. (1975) J. Biol. Chem. 250, 9360 9368
Den, H., Kaufman, B., McGuire, E. J., and Roseman, S. (1975) J. Biol. Chem. 250, 739 746
Roth, S., McGuire, E. J., and Roseman, S. (1971) J. Cell Biol. 51, 536 547
Chambers, J. K., Macdonald, L. E., Sarau, H. M., Ames, R. S., Freeman, K., Foley, J. J., Zhu, Y., McLaughlin,
M. M., Murdock, P., McMillan, L., Trill, J., Swift, A., Aiyar, N., Taylor, P., Vawter, L., Naheed, S., Szekeres, P.,
Hervieu, G., Scott, C., Watson, J. M., Murphy, A. J., Duzic, E., Klein, C., Bergsma, D. J., Wilson, S., and Livi,
G. P. (2000) J. Biol. Chem. 275, 1076710771
Rall, T. W., and Sutherland, E. W. (1962) J. Biol. Chem. 237, 1228 1232
Hardman, J. G., Robison, G. A., and Sutherland, E. W. (1971) Annu. Rev. Physiol. 33, 311336
Schnaar, R. L., Weigel, P. H., Kuhlenschmidt, M. S., Lee, Y. C., and Roseman, S. (1978) J. Biol. Chem. 253,
7940 7951
Weigel, P. H., Schnaar, R. L., Kuhlenschmidt, M. S., Schmell, E., Lee, R. T., Lee, Y. C., and Roseman, S. (1979)
J. Biol. Chem. 254, 10830 10838
Lesley, J., Hascall, V. C., Tammi, M., and Hyman, R. (2000) J. Biol. Chem. 275, 2696726975
Laine, R. A. (1994) Glycobiology 4, 759 767
Sparrow, J. R., and Barnstable, C. J. (1998) J. Neurosci. Res. 21, 398 409
Kawai, H., Allende, M. L., Wada, R., Kono, M., Sango, K., Deng, C., Miyakawa, T., Crawley, J. N., Werth, N.,
Bierfreund, U., Sandhoff, K., and Proia, R. L. (2001) J. Biol. Chem. 276, 6885 6888
Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999) J. Biol. Chem. 274, 288 297
Bulk, D. A., Wei, G., Toyoda, H., Kinoshita-Toyoda, A., Waldrip, W. R., Esko, J. D., Robbins, P. W., and Selleck,
S. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10838 10843
Toyoda, H., Kinoshita-Toyoda, A., Fox, B., and Selleck, S. B. (2000) J. Biol. Chem. 275, 21856 21861
Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S. B. (2000) J. Biol. Chem. 275, 2269 2275
Nakato, H., Futch, T. A., and Selleck, S. B. (1995) Development 121, 36873702
Perrimon, N., and Bernfield, M. (2000) Nature 404, 725728
Baeg, G. H., Lin, X., Khare, N., Baumgartner, S., and Perrimon, N. (2001) Development 128, 8794
Toyoda, H., Kinoshita-Toyoda, A., Fox, B., and Selleck, S. B. (2000) J. Biol. Chem. 275, 21856 21861
Bruckner, K., Perez, L., Clausen, H., and Cohen, S. (2000) Nature 406, 411 415
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D.,
Haltiwanger, R. S., and Vogt, T. F. (2000) Nature 406, 369 375
Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry: The Conformation of Macromolecules, First
Edition, W. H. Freeman, San Francisco
Park, L., Kuhlenschmidt, M., and Roseman, S. (1983) Fed. Proc. 42, 2129 (abstr.)
Roseman, S. (1974) in Cell Surface in Development (Moscona, A. A., ed) pp. 255271, John Wiley & Sons, Inc., New
York
Roseman, S. (1974) in Neuroscience, Study Program (Schmitt, F. O., ed) 3rd Ed., pp. 795 804, MIT Press,
Cambridge, MA
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Vol. 277, No. 50, Issue of December 13, pp. 4796547971, 2002
Printed in U.S.A.
Reflections
JBC Centennial
19052005
The Pentose Phosphate Pathway

Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.X200007200
Bernard L. Horecker
From the Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021
I received my basic training in enzymology as a graduate student in the laboratory of

Professor T. R. Hogness at the University of Chicago from 1936 to 1939. Hogness had constructed a photoelectric spectrophotometer modeled after the one in Otto Warburgs laboratory
in Berlin-Dahlem. I was assigned a problem on succinic dehydrogenase from beef heart, using
the Warburg manometric apparatus, and did not get to use the spectrophotometer until Erwin
Haas arrived from Warburgs laboratory in 1939. Haas asked me to join him in the search for
an enzyme that would catalyze the reduction of cytochrome c by reduced TPN (now NADP).
This reaction was thought to be the missing link in the electron transport chain from substrate
to oxygen and marked the beginning of my interest in what was then thought to function as
a direct oxidative pathway for the metabolism of carbohydrate but is now known as the pentose
phosphate pathway.
After I left Chicago during the Second World War, my experience with the spectrophotometer landed me a job at the National Institute of Health (NIH) in Frederick S. Bracketts group
in the Division of Industrial Hygiene. Brackett had assembled an automatic recording spectrophotometer in the basement of Building 2 that I was assigned to use to develop a method
for the determination of carbon monoxide hemoglobin in the blood of Navy pilots returning
from combat missions. That and a number of other war-related projects kept me occupied for
the next 4 years.
In 1945, after the end of the war with Japan, I was advised by the Director of the Laboratory,
Dr. Paul Neal, that I was free to return to research in enzymology. I began studies on the
reduction of cytochrome c by the succinic dehydrogenase system, using what was now my own
Beckman spectrophotometer. One day, which I consider to be a turning point in my career,
Arthur Kornberg, who had been working in Building 4 on the biological role of folic acid,
appeared in my laboratory. Arthur had become convinced that enzymes were the key to an
understanding of intracellular biochemical processes and suggested that we work together. We
began with studies on the effect of cyanide on the succinic dehydrogenase system, because
cyanide was known to bind to and be a general inhibitor of enzymes containing the heme
group. An exception was cytochrome c, which had been reported to be resistant to the action
of cyanide. Contrary to these early reports, we found that cyanide did react with cytochrome
c and in 1946 published our first paper together, in the Journal of Biological Chemistry,
entitled The Cytochrome c Cyanide Complex.
Making History in Building 3
Two years later in 1948 when Arthur returned from a study leave in the laboratories of
Severo Ochoa in New York and Carl Cori in St. Louis, he invited Leon Heppel and me to join
him in setting up a new Section on Enzymes in the Laboratory of Physiology to be housed in
Building 3, which was being completely renovated. Leon and I were about to be transferred, it
having been discovered that the Industrial Hygiene Research Laboratory in Building 2 had
H72
Reflections: The Pentose Phosphate Pathway

never been officially part of the NIH but was in the Bureau of State Services, which was
moving to new headquarters in Cincinnati.
In the fall of 1948 while we waited for the of the renovation of laboratories in Building 3 to
be completed, we all three worked in Building 2. Arthur and I, both of whose planned research
projects would depend heavily on assays using the pyridine nucleotide coenzymes DPN and
TPN, collaborated in their isolation. In those early years of American biochemistry there were
no vendors that supplied these materials. We isolated them from sheep liver, using the
unpublished procedure from Warburgs laboratory that Erwin Haas and I had used in Chicago.
In 1948 Arthur and I possessed the worlds supply of TPN, and when Warburg visited our
laboratory in 1948, we were able to present the discoverer of TPN with a gift of 25 mg of that
coenzyme.
The new Enzyme Section in the Division of Physiology in Building 3 provided an exciting
and stimulating atmosphere. Together with Herbert Tabor from the Laboratory of Pharmacology in Building 4, we organized a daily lunch hour journal club, during which we reviewed
the literature on every facet of enzymology and intermediary metabolism. This was the
beginning of a great history for Building 3, continuously occupied by scientists who were to
make notable contributions in biomedical science. The first NIH recipients of the Paul Lewis
Laboratories Award in Enzyme Chemistry, then one of the most prestigious awards in
biological research, were all from Building 3: Arthur Kornberg in 1951, myself in 1952, and
Earl Stadtman in 1953. Later, our Section on Enzymes became part of the new Experimental
Biology and Medicine Institute, which Henry Sebrell, then the NIH Director, proposed to
function as the basic research arm of the NIH. It was later renamed the National Institute of
Arthritis and Metabolic Diseases, a change that had no effect on the nature of our research but
resulted in increased funding by Congress. We continued to work in the laboratories in
Building 3.
Use of the pyridine nucleotides in enzyme assays with the Beckman spectrophotometer
required knowledge of the exact extinction coefficients of the 340 nm peaks of the reduced
forms. The published values for DPNH showed considerable variation, and there was scant
information for TPNH. In the new laboratories in Building 3, Arthur and I designed experiments to determine the true extinction coefficients at 340 nm of both coenzymes, which proved
to be identical. That work, published in 1948, made possible quantitative spectrophotometric
measurements in reactions involving the pyridine nucleotides and became one of the most
frequently cited papers in the biochemical literature.
In the new laboratories in Building 3 Leon Heppel and I also collaborated in the
purification of xanthine oxidase from milk after we found that this enzyme could reduce not
only methylene blue, a reaction that I had studied in Chicago, but also cytochrome c.
However, this reduction occurred only if oxygen was present, a curious observation that was
quickly picked up by Fridovich and Handler, who were working at Duke University on the
formation of the superoxide anion. The reduction of cytochrome c by superoxide anion
became a widely used assay for this species of active oxygen. I also returned to the study
of cytochrome c reductase, which Haas and I had isolated from yeast, and accomplished the
first isolation of this flavoprotein from mammalian liver. By then it had become apparent
that these cytochrome c reductases did not function in mitochondrial respiration but rather
as components of the cytochrome P-450 system for the metabolism and detoxification of
drugs and other xenobiotics.
The Pentose Phosphate Pathway
The Oxidation of Glucose 6-PhosphateWhen Otto Warburg discovered TPN as the coenzyme required for the oxidation of glucose 6-phosphate to 6-phosphogluconate, the role of the
other pyridine nucleotide, DPN, as the coenzyme required for the fermentation of glucose to
ethanol in yeast or the glycolysis of glucose to lactic acid in muscle had been well established.
The finding that the new coenzyme was required for the oxidation of glucose 6-phosphate and
also for the further oxidation of the product, 6-phosphogluconate, led Warburg and also Frank
Dickens in England and Fritz Lipmann, then working in Denmark, to propose that there
existed an alternate pathway that functioned as a direct oxidative pathway. They had
obtained evidence that the products formed in the oxidation of 6-phosphogluconate by TPN
were carbon dioxide and an unidentified pentose phosphate. Because carbon dioxide was one
of the products, it seemed reasonable to regard this alternate pathway as the one responsible
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for the oxidation of carbohydrate. Haas and I had already shown that TPN could serve as an
electron transport link to the cytochromes and therefore to molecular oxygen.
Twenty years after the pioneering work of Warburg, Dickens, and Lipmann, I began studies
(with a new laboratory technician, Pauline (Polly) Smyrniotis) on the enzymes involved in the
oxidation of 6-phosphogluconate and the metabolic intermediates formed in this pathway. We
were joined by J. E. Seegmiller, my first postdoctoral student, and he and I worked out a new
method for the preparation of glucose 6-phosphate and 6-phosphogluconate, which were not
yet commercially available. We purified the enzyme, 6-phosphogluconate dehydrogenase, from
brewers yeast, the richest source we could find, and by coupling the reduction of TPN to its
re-oxidation by pyruvate in the presence of lactic dehydrogenase showed that the first product
of the oxidation of 6-phosphogloconate, in addition to carbon dioxide, was a new pentose ester,
ribulose 5-phosphate, which was then converted to ribose 5-phosphate by a pentose-phosphate
isomerase present in our purified dehydrogenase preparations. The separation of ribulose
phosphate from ribose phosphate and the demonstration that their interconversion was
catalyzed by a pentose-phosphate isomerase were made possible by the recent development at
the Oak Ridge National Laboratory of a separation technique for nucleotides called ionexchange chromatography.
The identification of the sugar in the new pentose ester as ribulose was based on a number
of criteria, including comparison with the authentic sugar, prepared by the method of
Glatthaar and Reichstein, using a number of chemical and physical criteria, which included
x-ray diffraction of the crystalline nitrophenyl hydrazones. In those days discoveries of new
sugar phosphate esters were rare events, and I felt that it was necessary to establish its
identity beyond a shadow of doubt. The results were first presented at the American Chemical
Society meeting in Boston in 1951 at a symposium honoring Arthur Kornbergs Paul Lewis
Laboratories Award, and I recall the warm reaction at that meeting to the work reported from
our laboratories in Building 3.
During the following year Jay Seegmiller and I showed that the same products were formed
in the metabolism of 6-phosphogluconate by enzymes from mammalian tissues.
The Further Metabolism of the Pentose PhosphatesAn important clue to the further steps
in what was later to become known as the pentose phosphate pathway was already in the
literature. In 1938 Zaccharias Dische had demonstrated that red cell lysates catalyzed the
conversion of the 5-carbon sugar, ribose 5-phosphate to hexose monophosphate, an observation
that Seegmiller and I confirmed in 1952 with rabbit bone marrow extracts. These observations
gave rise to the hypothesis that the oxidative pathway was really a cyclic mechanism for the
direct oxidation of carbohydrate. With each turn of the cycle one molecule of carbon dioxide
would be produced, and the pentose phosphates formed would be metabolized back to hexose
phosphates to start another cycle. Six turns of the cycle would result in the complete oxidation
of one molecule of glucose.
However, the reactions involved in the conversion of the 5-carbon pentose phosphates to the
6-carbon hexose phosphates were completely unknown. What ensued was a race involving a
number of laboratories, including ours at the NIH and those of Ephraim Racker at the New
York City Research Laboratories, later at Cornell University in Ithaca, Seymour Cohen at the
University of Pennsylvania in Philadelphia, Oliver Lampen at Washington University in St.
Louis, and Frank Dickens in England, to identify the reactions and metabolic intermediates
involved.
It had already been established from the work of Dische and others that one of the products
of pentose phosphate metabolism was the 3-carbon sugar, glyceraldehyde 3-phosphate, which
suggested cleavage of a 5-carbon sugar, probably ribulose 5-phosphate, between carbon atoms
2 and 3. The 3-carbon fragment, glyceraldehyde 3-phosphate, was a known intermediate in
glycolysis, but what was the fate of the remaining 2-carbon fragment? Polly Smyrniotis and I,
now joined by my first foreign postdoctoral fellow, Hans Klenow from Copenhagen, set out to
purify the enzyme(s) involved in the cleavage of pentose phosphate, using rat liver as the
enzyme source and an assay that measured the appearance of glyceraldehyde 3-phosphate.
When we also followed the disappearance of the 5-carbon sugar, using Disches orcinol
reaction, we detected the formation of a new product that also reacted with orcinol but
produced a different color and a visible absorption spectrum distinctly different from that
produced in the reaction with pentoses. Our clue to the identity of this product came from
Melvin Calvins laboratory in Berkeley, where, using radioactive carbon dioxide, they had
H74

identified both ribulose diphosphate and a 7-carbon sugar, sedoheptulose monophosphate, as
early intermediates in the fixation of carbon dioxide in photosynthesis. Authentic sedoheptulose was available from Nelson Richtmyers laboratory in Building 4, and on Christmas Eve,
1951, when everybody else had gone home, I sprayed a paper chromatogram with orcinol and
up came the blue spot characteristic of sedoheptulose. I rushed up and down the laboratory
hallway clutching the paper chromatogram, but there was nobody there to show it to, so I took
it home and hung it on the Christmas tree, singing the little ditty: Its sedoheptulose, its
sedoheptulose, tra la la boom deay, tra la la boom deay, much to the amusement of my young
daughters.
We adopted the name transketolase, first suggested by Racker and his co-workers, because
it catalyzed the transfer of a 2-carbon fragment from the ketopentose, ribulose 5-phosphate, to
the other 5-carbon sugar, ribose 5-phosphate, to generate the new ketol linkage in the 7-carbon
sugar, sedoheptulose 7-phosphate. Thus the unknown 2-carbon fragment never occurred as a
free entity. Later, simultaneously with Racker, we showed that the coenzyme carrier for the
2-carbon fragment by transketolase was thiamine pyrophosphate.
Two puzzling observations remained to be explained. One was that the configuration of the
hydroxyl group on the third carbon atom of the new product, sedoheptulose 7-phosphate, was
opposite that on the third carbon atom of the presumed substrate, ribulose 5-phosphate. This
lack of stereospecificity, particularly because we had demonstrated that the reaction was
readily reversible, was highly improbable for an enzyme-catalyzed reaction. The other unexplained observation was that the cleavage of pentose phosphate by our purified transketolase
preparations from rat liver required the presence of aldolase, a crystalline and supposedly
pure enzyme from rabbit muscle that catalyzed the condensation of two triose phosphates to
form the hexose, fructose 1,6-bisphosphate. The answer to both of these puzzling observations
came from the discovery by Paul Stumpf, while on sabbatical in my laboratory, and reported
almost simultaneously from the laboratories of Dickens, Racker, and Ashwell, of another
enzyme, an epimerase, that catalyzed the conversion of ribulose 5-phosphate to its 3epimer, xylulose 5-phosphate, which had the same stereo-configuratuion at the 3-carbon atom
as sedoheptulose phosphate. With this substrate, we confirmed the earlier report by Racker
and his co-workers that xylulose phosphate, rather that ribulose phosphate, was the true
substrate for cleavage by transketolase and the true donor of the 2-carbon fragment. The
requirement for aldolase was explained when we found that crystalline preparations of this
enzyme from rabbit muscle contained the epimerase as a contaminant, which could only be
removed by many re-crystallizations.
Thus three different pentose phosphates were now shown to be involved in the new pathway:
ribulose 5-phosphate, the first product of the oxidation of 6-phosphate gluconate, and xylulose
5-phosphate and ribose 5-phosphate, both formed from ribulose 5-phosphate, one serving as
the 2-carbon donor and the other as the acceptor in the reaction catalyzed by transketolase.
The addition of any one of these pentose phosphates to crude tissue extracts would result in the
formation of an equilibrium mixture of all three.
Completion of the CycleStill to be discovered, however, was a mechanism that would
convert the products of the transketolase reaction, sedoheptulose phosphate and glyceraldehyde phosphate, to the 6-carbon sugars, fructose 6-phosphate and glucose 6-phosphate, and
complete the cycle. In particular, what was the fate of sedoheptulose phosphate? We found that
purified enzyme preparations from liver or yeast would catalyze the formation of hexose
monophosphate from sedoheptulose monophosphate but only if triose phosphate was also
present. When I described this finding at one of our luncheon journal club meetings, Horace
(Nook) Barker, who was visiting from the University of California at Berkeley and working
in Kornbergs laboratory, suggested that we consider the possibility of another transfer, this
time of a 3-carbon fragment from sedoheptulose phosphate to triose phosphate to generate
fructose 6-phosphate. When we carried out an experiment with carbon-14-labeled triose
phosphate, we found that, as predicted, the fructose 6-phosphate formed had radioactivity in
the last three carbon atoms with the first three unlabeled. We named the enzyme transaldolase because it catalyzed the transfer of an aldol linkage rather than the hydrolytic cleavage
catalyzed by aldolase.
What remained was to account for the fate of the remaining 4 carbon atoms of sedoheptulose
7-phosphate. For this work Polly Smyrniotis and I were joined by Paul Marks and Howard
Hiatt, two young M.D.s working as Clinical Associates in the new Clinical Center in Building
H75

10, who had asked to join my group to learn enzymology in their spare time, which turned
out to be from 5 p.m. to midnight. We identified the missing fragment as another new sugar
ester, the 4-carbon sugar erythrose 4-phosphate, in a number of tests, including its conversion
to the 7-carbon sugar sedoheptulose 1,7-diphosphate in a condensation with dihydroxyacetone
phosphate, catalyzed by fructose bisphosphate aldolase. It was also converted to fructose
6-phosphate in the reaction catalyzed by transketolase.
The elucidation of the pentose phosphate pathway had now been accomplished. It consisted
of two branches, an oxidative branch in which the hexose, glucose 6-phosphate, was converted
to pentose phosphate and carbon dioxide with the reduction of two molecules of TPN, and a
non-oxidative branch, in which three molecules of pentose phosphate (15 carbon atoms) were
reconverted to two and one-half molecules of hexose phosphate (15 carbon atoms) in a series
of fully reversible reactions. Our contributions included the discovery of three new sugar
phosphate esters, ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phosphate, and three new enzymes, transketolase, transaldolase, and pentose-phosphate 3-epimerase. We shared with Racker the discovery of transketolase and confirmed his finding that
xylulose 5-phosphate, rather than ribulose 5-phosphate, was the 2-carbon donor in the reaction catalyzed by that enzyme. We also shared with McLean and Dickens, working in England,
the discovery that fructose 6-phosphate was also a substrate for transketolase. If the pathway
operated as originally envisioned, six turns of the cycle would result in the oxidation of one
molecule of 6-carbon sugar to six molecules of carbon dioxide.
Functions of the Pentose Phosphate PathwayThe function(s) of the new pathway, however,
turned out to be quite different from the pathway for the direct oxidation of carbohydrate that
we had expected. It provides two mechanisms for the production of ribose 5-phosphate. One is
the oxidative branch of the pathway, which also generates 2 eq of TPNH (NADPH). Ribose
5-phosphate can also be formed directly from hexose and triose phosphates by the nonoxidative rearrangements catalyzed by transketolase and transaldolase. Where large quantities of NADPH are required, as in the synthesis of fatty acids or sterols, the excess pentose
phosphates produced would be recycled back to hexose monophosphates.
To assist medical students in memorizing the reactions, someone composed the following
song.
THE PENTOSE PHOSPHATE SHUNT
(Tune: MacNamaras Band)
If youre converting carbohydrate into triglyceride,
If you need pentose moieties to make nucleotide,
Youll find that Embden-Meyerhof is not the game to play
And youll do your biosynthesis the pentose phosphate way.
Chorus:
With transaldolase, transketolase, G6PDH too,

Six times six gives five times six plus six of CO2
Carbons passing to and fro, the back becomes the front,
Did you ever see a pathway like the pentose phosphate shunt?
First G6P is oxidized, NADP reduced

To give gluconolactone (as might have been deduced).
The lactone is then hydrolyzed to make the gluconate
And decarboxylated to its metabolic fate.
There ends the oxidative phase, now multiply by three,
An intermediate balance sheet by way of summary,
Six NADPH are formed, three CO2 set free,
Three ribulose 5-phosphate formed from three of G6P.
One isomerization from ketose to aldose
Turns ribulose 5-phosphate to the phosphate of ribose
The other two epimerized, inverted at C3,
Two xylulose 5-phosphates formed (hence called Xu5P).
H76

Two carbons from Xu5P transferred from the ketose to aldose
(Transketolase needs TPP as everybody knows),
Thus three plus seven made to meet transaldolase attack,
Three Cs from sedoheptulose the GAP gets back,
Glyceraldehyde 3-phosphate thus becoming F6P
Leaves erythrose 4-phosphate looking for some company,
But Xu5P number two has two top Cs to spare,
Transketolase negotiates their transfer as a pair.
So weve made another F6P, a triose phosphate too,
To see what we have now achieved lets multiply by two,
Four F6Ps, two GAPs, by glycolytic tricks,
Give five glucose 6-phosphates, when we started out with six!
(Author unknown)
The first discovery relevant to the new pentose phosphate pathway, namely the formation of
ribulose and ribose phosphates as products of the oxidation of 6-phosphogluconate, was
announced in the spring of 1952 at the annual meeting of the American Chemical Society in
Chicago. The outline of the complete pentose phosphate cycle, including the reactions catalyzed by the new enzymes transketolase and transaldolase, was published in 1955 in a review
written in collaboration with I. C. (Gunny) Gunsalus and W. A. (Woody) Wood for Bacteriological Reviews entitled Pathways of Carbohydrate Metabolism in Microorganisms. The
existence of the cycle in mammalian liver and in plant leaves was confirmed in experiments
with carbon-labeled ribose 5-phosphate in two papers published with Martin Gibbs of the
Brookhaven National Laboratory, describing work carried out there during the summer of
1953.
The Path of Carbon in Photosynthesis
When, in 1952, Calvins group at the University of California at Berkeley reported evidence
for ribulose 1,5-diphosphate as the CO2 acceptor for the formation of 3-phosphoglyceric acid,
the first CO2 fixation product in photosynthesis, we were excited by the possibility that the
pentose phosphate pathway might serve as the mechanism for regenerating this key intermediate from hexose monophosphates. Art Weissbach, a newly arrived postdoctoral student
from Columbia, and Polly Smyrniotis carried out the first experiments to identify the enzymatic mechanisms involved. They were able to show that with crude extracts from spinach
leaves ribose 5-phosphate was a unique substrate for the formation of phosphoglyceric acid,
and they purified a kinase from spinach leaves that they used to prepare the barium salt of
ribulose 1,5-bisphosphate (RUDP).
In the fall of 1954 we moved from Building 3 to new laboratories on the 9th floor of the NIH
Clinical Center, where, joined by Jerry Hurwitz, we isolated the enzyme phosphoribulokinase,
responsible for that reaction, as well as the enzyme ribulose-bisphosphate carboxylase, which
catalyzed the formation of 2 mol of phosphoglyceric acid from ribulose bisphosphate and CO2.
Working in laboratories across the hall from each other, Art, Jerry, and I divided responsibilities. Jerry was charged with the purification of phosphoribulokinase, I took on the task of
preparing pure ribulose 1,5-bisphosphate, and Art went after the most important enzyme, the
ribulose-bisphosphate carboxylase. The last effort deserves a special comment. Although the
enzyme was purified only 10-fold from the crude spinach leaf extracts, by all the criteria that
we could apply it appeared to be a pure protein, which meant that it constituted about 10% of
the soluble protein in the spinach leaf. Later work in other laboratories around the world
confirmed this finding, and this enzyme, now known as Rubisco for ribulose-bisphosphate
carboxylase/oxygenase is now considered to be the most abundant protein on earth.
Our work was published in three back-to-back papers in the February 1956 issue of the
Journal of Biological Chemistry, entitled: Spinach Phosphoribulokinase, The Enzymatic
Synthesis and Properties of Ribulose 1,5-Diphosphate, and The Enzymatic Formation of
Phosphoglyceric from Ribulose Diphosphate and Carbon Dioxide. With this work and our
earlier demonstration of the reversible reactions for the interconversion of pentose and hexose
H77

phosphates, with sedoheptulose phosphate as a prominent intermediate, all of the enzymes for
the reactions of the Calvin Cycle were identified.
Conclusion
The pentose phosphate pathway in animals, as discussed earlier, fulfills two important cell
requirements: 1) for ribose 5-phosphate for the synthesis of nucleotides and nucleic acids; and
2) for reducing power in the form of NADPH. In photosynthesis, it functions to regenerate the
primary CO2 acceptor, ribulose bisphosphate, from the hexose phosphates produced. Chloroplasts utilize radiant energy to produce ATP, required for the production of ribulose 1,5bisphosphate from ribulose 5-phosphate and also for the reduction of 3-phosphoglyceric acid to
glyceraldehyde 3-phosphate. The reducing agent for the latter reaction, NADPH, is also
generated by the action of light in the chloroplasts. In both animals and plants, NADP rather
than NAD appears to function as the coenzyme for reductive synthesis.
Comment
Because these were personal reflections they have mainly described work from my laboratory. Calvin and his co-workers provided the first clues leading to the development of the
photosynthetic cycle and also the conclusive evidence for its function as the path of carbon in
intact photosynthesizing cells. These pioneering experiments, as well as important contributions from many other laboratories, are cited in a review that I published in 1957 with Wolf
Vishniac and Severo Ochoa in Advances in Enzymology (see last entry of the Bibliography).
Address correspondence to: blhorecker@aol.com.
BIBLIOGRAPHY
Horecker, B. L., and Kornberg, A. (1948) The extinction coefficients of the reduced band of the pyridine nucleotides.
J. Biol. Chem. 175, 385390
Horecker, B. L., Smyrniotis, P. Z., and Seegmiller, J. E. (1951) The enzymatic conversion of 6-phosphogluconate to
ribulose 5-phosphate and ribose 5-phosphate. J. Biol. Chem. 193, 383396
Horecker, B. L., and Smyrniotis, P. Z. (1953) The coenzyme function of thiamine pyrophosphate in pentose phosphate
metabolism. J. Am. Chem. Soc. 75, 1009 1010
Horecker, B. L., and Smyrniotis, P. Z. (1953) Transaldolase: the formation of fructose 6-phosphate from sedoheptulose
7-phosphate. J. Am. Chem. Soc. 75, 2021
Horecker, B. L., Smyrniotis, P. Z., and Klenow, H. (1955) The formation of sedoheptulose phosphate from pentose
phosphate. J. Biol. Chem. 205, 661 682
Weissbach, A., Smyrniotis, P. Z., and Horecker, B. L. (1954) Pentose phosphate and CO2 fixation with spinach
extracts. J. Am. Chem. Soc. 76, 3611
Horecker, B. L., and Smyrniotis, P. Z. (1955) The purification and properties of yeast transaldolase. J. Biol. Chem. 212,
811 825
Horecker, B. L., Smyrniotis, P. Z., Hiatt, H., and Marks, P. (1955) Tetrose phosphate and the formation of sedoheptulose diphosphate. J. Biol. Chem. 218, 827 836
Hurwitz, J., Weissbach, H., Horecker, B. L., and Smyrniotis, P. Z. (1956) Spinach phosphoribulokinase. J. Biol. Chem.
218, 769 783
Horecker, B. L., Hurwitz, J., and Horecker, B. L. (1956) The enzymatic synthesis and properties of ribulose 1,5diphosphate. J. Biol. Chem. 218, 785794
Weissbach, A., Horecker, B. L., and Hurwitz, J. (1956) The enzymatic formation of phosphoglyceric acid from ribulose
diphosphate and carbon dioxide. J. Biol. Chem. 218, 795 810
Horecker, B. L., Smyrniotis, P. Z., and Hurwitz, J. (1956) The role of xylulose 5-phosphate in the transketolase
reaction. J. Biol. Chem. 223, 1009 1019
Vishniac, W., Horecker, B. L., and Ochoa, S. (1957) Enzymatic aspects of photosynthesis. Adv. Enzymol. XIX, 177
H78

Vol. 279, No. 53, Issue of December 31, pp. 5497554982, 2004
Printed in U.S.A.
Reflections
JBC Centennial
19052005
My Brief Encounter with the Phosphoinositides and IP3

Published, JBC Papers in Press, October 8, 2004, DOI 10.1074/jbc.X400010200
Clinton E. Ballou
From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
For my first independent research project after my appointment to the Berkeley faculty, I
chose to work on the structures of myoinositol-containing phospholipids, a study that led us
eventually to the discovery of D-myoinositol 1,4,5-trisphosphate or IP3. Before describing this
research, however, I should say how that choice came about. While in graduate school at the
University of Wisconsin, I had had the good fortune to study under Karl Paul Link, who was
widely renowned for his discovery of dicumarol and the synthesis of related blood anticoagulants such as warfarin, work that was recognized with two Lasker Awards (1). On the side,
however, Link remained a carbohydrate chemist at heart, a hobby that had grown out of his
studies on plant polysaccharides and uronic acids while a student and then a young faculty
member. In fact, Stanford Moore had completed his doctoral dissertation with Link on a
method for characterizing aldo-monosaccharides as benzimidazole derivatives (2).
I arrived at Madison in the fall of 1946, fresh from a stint in the United States Navy, and
I found Links laboratory bursting at the seams with about 15 ex-GIs, all hard at work trying
to make up for lost time. During earlier investigations on the structure-function relationship
of coumarin anticoagulants, an attempt to synthesize the glucoside of dicumarol had been
frustrated because the acetylated intermediate was degraded in alkali under conditions used
for deacetylation (3). Because glycosides are acetals, which are typically acid-labile and
alkali-stable, I found the anomaly intriguing and decided to study a variety of synthetic
compounds in an effort to understand the structural basis for alkali sensitivity (4). This
research formed the core of my doctoral dissertation, and although I failed to recognize it at the
time, the chance exposure to carbohydrate chemistry was to have a lasting influence on the
direction my career would take.
I continued my indoctrination in sugar chemistry during a postdoctoral year in Edinburgh,
Scotland, with E. G. V. Percival in the new Department of Chemistry at Kings Buildings
headed by Edwin Hirst. This was a time of economic depression in Britain, which was still
suffering the aftermath of the war, and I discovered that I had left a well equipped laboratory
in Madison to engage an unexpectedly primitive research environment. Wisely I did not let
this change in fortunes discourage me. Instead I undertook a project dealing with the structure
of maple sapwood starch and did the best that I could with the available facilities (5). My
efforts were well rewarded because, in the process, I became adept at the uses of analytical and
preparative filter paper and cellulose column chromatography, skills that were to be extremely
valuable in my later research. The greatest challenge to my ingenuity, however, was to
construct an electric stirring device from a small board-mounted motor, a couple of wooden
pulleys, a piece of string, and a glass rod. The speed of the motor was regulated by adjusting
light bulbs that were wired in series with the power cord to draw off electricity, a crude but
effective method of control. I have always enjoyed working with my hands, so this mundane
project even took on a certain appeal.
Living in a new environment always has its fringe benefits. While in Edinburgh, I developed
a special affection for the Scots and a better understanding for the lingering resentment that
H79
Reflections: Reflections on Phosphoinositides and IP3
FIG. 1. Alkaline degradation of soybean monophosphoinositide (15). The products are D-myoinositol 1-phosphate
(I-1-P) and myoinositol 2-phosphate (I-2-P). R is a fatty alkyl chain.
reflects a long history of conflict within the British Isles. Thus, I could understand why one of
my graduate student colleagues was proud to proclaim, at every opportunity, that he had
never been south of the border! It was also during this year that some Scottish separatists
sneaked into Westminster Abbey and made off with the Stone of Scone. This symbol of Scottish
nationalism, which was taken from Scotland to England by Edward I, had long rested beneath
the chair on which British monarchs were crowned. The incident created quite a stir among
the local patriots, but after its recovery the stone was returned to the Abbey. (I was recently
informed that the Stone of Scone has since been returned to Scotland.)
Although I enjoyed the time, when the year ended I was ready to move on to Berkeley, where
I had arranged to study with Hermann O. L. Fischer. Nicknamed Hermannol, probably by
his friend Claude Hudson as a play on the term polyol, Fischer was an expatriate German
scientist who had experienced a turbulent career that eventually led him to the University of
Toronto. Then, when the new Biochemistry and Virus Laboratory was set up in 1948, Wendell
Stanley had recruited him to Berkeley. I was attracted to Fischer in part because of his
research on phosphorylated sugars but also because during graduate school I had drawn
heavily on the published works of his father, Emil Fischer (6). I guess the idea of being
associated with the son of Emil Fischer just seemed real cool to me. As it turned out, it also
proved beneficial that I happened to go to Berkeley just as the University was entering a
period of rapid postwar expansion.
This was a time of active research on biosynthetic pathways that involved short chain
phosphorylated sugars, as exemplified by the studies of Melvin Calvin on photosynthesis, of
P. R. Srinivasan and David Sprinson on shikimic acid biosynthesis, and of Bernard Horecker
on transaldolase. With Fischer and his colleague, Donald MacDonald, I undertook the syntheses of several such metabolic intermediates, including D-glyceric acid 2-phosphate, Dglyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate,
and D-erythrose 4-phosphate (7). The novelty of our approach was to prepare stable dimethyl
acetal derivatives of the inherently unstable phosphorylated compounds with aldehydo or keto
groups. These could be stored indefinitely and then be converted by mild acid hydrolysis of
the acetal to the active metabolites as needed. Our success is documented by the fact that
today, 50 years later, samples of the preparations have survived in pure crystalline usable
form. During these first years in Berkeley, I also became interested in inositol chemistry as
a result of studies on the cyclitols in sugar pine heartwood (8). Then, when Elvin Kabat
came to Berkeley from Columbia University to spend a sabbatical with Fischer and learn
some carbohydrate chemistry we all collaborated on the methylation analysis of galactinol,
an -D-galactoside of myoinositol. This study established that the galactose was linked to
the L-l-position on the inositol ring (9), a fact that I was to put to good use in my later
studies.
After my appointment to the faculty in 1955, I was in a position to set up an independent
program, and this background led me to undertake a project concerned with the characterH80
FIG. 2. Synthesis of L-myoinositol 1-phosphate (16, 17). The starting material for this synthesis was galactinol,
1-O--D-galactopyranosyl-L-myoinositol (9).
ization of inositol-containing phospholipids. In so doing, I was fortunate to have Finn Wold,

Lewis Pizer, and Francis Lane Pizer as my first graduate students. At the time, there was
convincing evidence from a number of studies that the lipid known as phosphoinositide was
a phosphatidylmyoinositol (10), and as expected for such a structure, acid or alkaline hydrolysis of the phosphodiester bond had yielded myoinositol phosphate as one of the degradation
products (1113). Because the chemical hydrolysis of phosphate diesters with neighboring free
hydroxyl groups can lead to phosphate migration, however, the position of attachment of the
phosphatidic acid unit to the myoinositol ring was uncertain. Important studies at Cambridge
University by Brown and Todd (14), showing that the alkaline hydrolysis of the phosphate
diester linkage in nucleic acids proceeds via a cyclic phosphate intermediate, suggested to us
a strategy to resolve this uncertainty. We subjected pure soybean phosphoinositide to alkaline
hydrolysis and isolated the inositol phosphate fraction. It consisted mainly of myoinositol
1-phosphate along with some myoinositol 2-phosphate and other minor products (15). This
result indicated that the putative myoinositol cyclic phosphate intermediate had involved
positions 1 and 2 on the ring.
Because position 2 of the myoinositol ring lies between two adjacent cis-hydroxyls, called D-1
and L-1, the phosphatidyl group in the lipid could have been attached to position 2 or to one of
the adjacent enantiomeric 1-positions.1 The choice between these alternatives was suggested
by the fact that the myoinositol 1-phosphate we isolated was optically active, []D 9.8 (water,
pH 2). This would be expected from the cyclization and reopening of a phosphate diester group
originally on the D-1- or L-1-position, because the intermediate cyclic phosphate would be
asymmetric if the myoinositol in the starting diester were asymmetrically substituted (Fig. 1).
This would not be the result if the original diester involved position 2, which has a plane of
symmetry, unless the asymmetry of the glycerol portion were able to exert a directive influence
during the reaction.
To complete the characterization, we carried out a definitive synthesis of L-myoinositol
l-phosphate, starting from galactinol (9). For this synthesis, we perbenzylated galactinol,
removed the benzylated galactose moiety by acidic methanolysis, and phosphorylated the free
L-1-position of the recovered penta-O-benzylmyoinositol. Deblocking of the product by hydrogenolysis yielded L-myoinositol 1-phosphate (Fig. 2), which showed []D 9.3 (water, pH 2) (16,
17). Because this synthetic L-isomer, which later was found to occur naturally (18), showed
a rotation equal to that of the lipid-derived product, but of opposite sign, it must be the
enantiomer; and consequently, the 1-phosphatidylmyoinositol (15) must have had the
1
For these assignments, the three adjacent cis-hydroxyls of myoinositol are numbered one to three, and the
direction of numbering is selected to give substituted positions the lowest possible number. When the ring is
represented with these three hydroxyls projecting downward and the direction of numbering is clockwise, the
myoinositol configuration is D and if counterclockwise it is L. Note that myoinositol 2-phosphate and 5-phosphate have
a plane of symmetry and are meso compounds.
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FIG. 3. Reactions used to characterize D-myoinositol 1,4,5-trisphosphate (22, 25). P is PO3H2.

2
D-configuration.
In an important parallel study, Brown et al. (19) degraded horse liver

phosphoinositide by the periodate/phenylhydrazine procedure, which avoided the cyclic phosphate intermediate, and they recovered a single myoinositol 1-phosphate with the same optical
activity as the isomer we had obtained from the soybean lipid. Together, these studies firmly
established that the myoinositol ring was substituted on the D-1-position in phosphatidylmyoinositol from both plants and animals. This was an important result, although it was not
surprising because most myoinositol derivatives show chirality.
At the time, I was aware of the important work of Jordi Folch at Harvard Medical School,
who had isolated a complex phosphoinositide from beef brain (20, 21). This isolation was based
on the facts that phospholipids have low solubility in acetone, but they can be extracted from
an acetone powder of brain tissue with chloroform, and the inositides can then be precipitated
selectively by adding ethanol or methanol. Because strong acid hydrolysis of the material had
yielded an inositol metadiphosphate, Folch concluded that the original lipid was a polyphosphoinositide. Because it was known, however, that acid treatment could cause phosphate
groups to migrate around the inositol ring (15), we decided to reexamine this characterization.
A new graduate student from the University of Chile, Carmen Grado, had just joined my
group, and I suggested that she should repeat the brain phosphoinositide preparation according to Folch. When Grado subjected this material to strong acid hydrolysis, she observed that
the resulting inositol phosphate fraction gave a very diffuse unresolved streak on paper
chromatography (22). She then repeated the study, using alkaline degradation of the brain
lipid, and found that chromatography of the inositol phosphate fraction gave a well resolved
pattern of five components, one mono-, two bis-, and two trisphosphates of myoinositol. This
suggested that the Folch brain inositide preparation was a mixture of related substances, and
because the myoinositol trisphosphates predominated, we proposed that the lipid might more
accurately be called a triphosphoinositide (22). At about the same time, Dittmer and Dawson,
at Cambridge University, reported the isolation from ox brain of a lipid fraction with the
composition expected for a triphosphoinositide (23).
Grado then went on to characterize each inositol polyphosphate in the mixture, using a
sequence of periodate oxidation to cleave the inositol ring between free glycol groups, borohydride reduction of the resulting dialdehyde, and dephosphorylation to yield a free polyalcohol.
From an inositol bisphosphate, one could expect a tetritol if the phosphate groups were next
to each other. If they were in a 1,3-position, a pentitol would result; and if they were in a
1,4-position, the ring would be cleaved in two places to give two molecules of malondialdehyde
phosphate, which would be oxidized further by excess periodate to yield inorganic phosphate,
formate, and carbon dioxide. From an inositol trisphosphate with the phosphates adjacent to
each other, a pentitol would be formed; if in a 1,2,4 arrangement, a hexitol would result; and
if in 1,3,5 arrangement, the inositol ring would survive the treatment. Besides indicating the
phosphate positions, the identity and optical activity of the resulting polyol would also reveal
the chirality of the inositol derivative. Using these methods, Grado characterized the two
bisphosphates as D-myoinositol 1,4- and 4,5-bisphosphate and the major trisphosphate fraction
as either the D-1,4,5-isomer or the D-1,4,6-isomer, the uncertainty arising because both of these
trisphosphates would yield the same D-iditol in the above analytical procedure (22). The
2
The convention for assigning configurations to substituted myoinositols was changed during the 1970s, so that
what we designated L-myoinositol 1-phosphate in 1959 (16, 17) was later renamed D-myoinositol 1-phosphate. In this
article, I have assigned all configurations in agreement with the convention now used.
H82
FIG. 4. Products isolated from deacylated brain polyphosphoinositide (27). The intact lipids were found to be
acylated by a mixture of stearic, oleic, and arachidonic acids (28).
uncertainty was resolved by Raymond Tomlinson, a graduate student who had made a
detailed study of the dephosphorylation of phytic acid (myoinositol hexaphosphate) by wheat
bran phytase (24). He observed that alkaline phosphomonoesterase selectively removed phosphate groups flanked by unsubstituted hydroxyls, and he found that the myoinositol trisphosphate isolated by Grado was converted to D-myoinositol 4,5-bisphosphate by this treatment
(25). Thus, the complete characterization of D-myoinositol 1,4,5-trisphosphate can be summarized as shown in Fig. 3.
These studies still left open the question of the true nature of the apparently heterogeneous
brain phosphoinositide. From the composition of his preparation, Folch (21) had postulated
that it could be represented as a meta-diphosphatidylmyoinositol, whereas Hawthorne (26)
proposed a cyclic structure of myoinositol meta-diphosphate with monoacylglycerol. I was
fortunate at the time to be joined by a postdoctoral co-worker, Hans Brockerhoff, who had
studied with Donald Hanahan at the University of Washington. To obtain the water-soluble
component(s) of the brain lipid complex with intact phosphodiester linkages, Brockerhoff
deacylated the phosphoinositide preparation with hydroxylamine and separated the products
on an ion exchange column (27). This yielded three fractions with compositions corresponding
to glycerol myoinositol phosphate (20%), glycerol myoinositol diphosphate (22%), and glycerol
myoinositol triphosphate (58%) (Fig. 4). Further analysis suggested that these products could
be derived from three lipids: l-phosphatidyl-D-myoinositol, 1-phosphatidyl-D-myoinositol 4-phosphate, and l-phosphatidyl-D-myoinositol 4,5-bisphosphate. This conclusion was confirmed when
Stewart Hendrickson, a postdoctoral fellow who had studied with Herbert Carter at the University of Illinois, developed an ion exchange procedure using a homogeneous chloroform/methanol/
water solvent. This solvent dissolved the intact brain phosphoinositide preparation and allowed
its separation into three homologs (28), analysis of which agreed with Brockerhoffs assignments
(27). Hendrickson also found that the three lipids were closely related in that each was predominantly acylated by the same mixture of stearic, oleic, and arachidonic acids. Later, Brown and
Stewart (29) also characterized purified triphosphoinositide, using the selective degradation
procedure Brown and co-workers had exploited so effectively earlier (19).
During the 1960s when we were conducting the above studies, very little was known about
the cellular function(s) of the inositol phospholipids. Mabel and Lowell Hokin at the University
of Wisconsin had investigated the possible role of these lipids in cellular secretion (30), and
they, along with others, had studied the incorporation of 32Pi into the brain lipids (31, 32).
These studies had yielded only limited information owing, in part, to uncertainty about the
actual structure of the brain inositide. From the insight we had gained by our structural work,
it appeared to us likely that the three components would be interconvertible in cells by an
enzyme-catalyzed process of cyclic phosphorylation-dephosphorylation. When Brockerhoff investigated the incorporation of [32P]phosphate, [3H]myoinositol, and [14C]glycerol into the
individual inositides in brain tissue slices, the results proved to be consistent with such a
pathway (33, 34). Thus, the monoester phosphate groups turned over rapidly, whereas the
glycerol, myoinositol, and phosphodiester groups were much more stable. Moreover, turnover
of the monoester phosphate groups was not random, because partial enzymic dephosphorylation of 1-phosphatidylmyoinositol 4,5-bisphosphate to the next lower homolog occurred by the
selective removal of the 5-phosphate group, indicative of a specific 5-phosphomonoesterase in
brain tissue (35).
I became eligible for a sabbatical leave in 1961, and because our work on the brain
polyphosphoinositides was going well, I asked Edgar Lederer if I could spend a year with him
to study the glycophosphoinositides of mycobacteria. He welcomed me in his very gracious
H83
FIG. 5. Structures of mycobacterial mannophosphoinositides (40). M is -D-mannopyranosyl.
manner, and he even arranged the rental of a spacious apartment in Paris on rue Pierre Curie
(later renamed rue Pierre et Marie Curie). Thus, I had only a short stroll to catch the Ligne de
Sceaux at Luxembourg Station for the daily ride to his CNRS laboratory at Gif-sur-Yvette.
Myoinositol, as a lipid constituent, was first reported by R. J. Anderson to occur in the phospholipids of mycobacteria (36), and Lederer subsequently described a dimannosyl phosphoinositide
from the same source (37). While at Gif, I collaborated with his colleague, Erna Vilkas, on
experiments to establish the linkages of both the phosphatidyl and the mannosyl groups to the
myoinositol ring (38). In later investigations by Yuan Chuan Lee, a postdoctoral student from the
University of Iowa, we determined the structures of the family of mannosyl phosphoinositides in
Mycobacterium smegmatis (39, 40). Like the other phosphoinositides, the phosphatidyl group was
found attached to the D-1-position of myoinositol, whereas a single mannose was linked to position
2 and one to four mannoses were linked to the D-6-position (Fig. 5). This phospholipid was later
found to serve as an anchor for the lipoarabinomannan in mycobacteria (41), and it is interesting
that the glycophosphoinositide protein anchor has the analogous structure in which a carbohydrate chain is also attached to position 6 of myoinositol (42).
In a report to the International Congress of Biochemistry on the Structure of Myoinositol
Phospholipids (43), I summarized the results of our studies and observed that: In attempting
to assess the role of phospholipids in cellular metabolism, one can place primary emphasis on
the lipid end of the molecule and its modification according to the type of fatty acid there
esterified. Or, one can direct attention to the hydrophilic end. In the case of the inositol
phospholipids, we find, in the great structural variability of the inositol part, evidence that
herein may lie the prime functional center of these molecules. I have never considered myself
a clairvoyant, and as it turned out, both the polar and nonpolar ends of the inositides have
important regulatory functions. Today, we can look back and see that our earlier studies were
significant mainly in helping to prepare the groundwork for the explosive developments
concerning the cellular functions of the phosphoinositides that followed upon the important
discoveries described in the review by Berridge and Irvine (44).
In these Reflections, I have limited myself to that early period of the 1960s in which I was
directly involved, and I have referred only peripherally to the many subsequent important
developments. I cant avoid reference, however, to the role that has been discovered for
1-phosphatidylmyoinositol 3-phosphate and its derivatives (45), substances we never encountered in our investigations. I should also admit that I am a little disappointed that I never
encouraged my co-workers to pursue a detailed study of the enzymes involved in the metabolism of the polyphosphoinositides. My only excuse is that we were drawn in other directions
by our discovery of the mycobacterial polymethylpolysaccharides (46, 47), which were found
later to act as regulators of fatty acid synthesis in this microorganism (48), and to investigations on the genetic control of yeast mannoprotein structure (49). Both of these developments
are traceable to the sabbatical leave I spent at Gif in 1961, a testament to the unpredictable
influence such an experience can have. Despite the minor doubt expressed above, I must say
that I enjoyed a wonderful ride with the phosphoinositides, and it was all great fun! I am
especially grateful to the many co-workers who shared this journey with me.
Address correspondence to: ceba@berkeley.edu.
H84

REFERENCES
1. Link, K. P. (1959) The discovery of dicumarol and its sequels. Circulation XIX, 97107
2. Moore, S., and Link, K. P. (1940) Carbohydrate characterization. I. The oxidation of aldoses by hypoiodite in
methanol. II. The identification of seven aldo-monosaccharides as benzimidazole derivatives. J. Biol. Chem.
133, 293311
3. Huebner, C. F., Karjala, S. A., Sullivan, W. R., and Link, K. P. (1944) Studies on the hemorrhagic sweet clover
disease. VI. Glucosides of 4-hydroxycoumarins. J. Am. Chem. Soc. 66, 906 909
4. Ballou, C. E. (1954) Alkali-sensitive glycosides. Adv. Carbohydr. Chem. 9, 59 95
5. Ballou, C. E., and Percival, E. G. V. (1952) Wood starches: the structure of the sapwood starch of the maple (Acer
spp.). J. Chem. Soc. 1054 1056
ber einige synthetische glucoside. Ber. Dtsch. Chem. Ges. 27, 2478 2486
6. Fischer, E., and Beensch, L. (1894) U
7. Ballou, C. E., Fischer, H. O. L., and MacDonald, D. L. (1955) The synthesis and properties of D-erythrose
4-phosphate. J. Am. Chem. Soc. 77, 59675970
8. Ballou, C. E., and Anderson, A. B. (1953) On the cyclitols present in sugar pine (Pinus lambertiana Dougl.). J. Am.
Chem. Soc. 75, 648 650
9. Kabat, E. A., MacDonald, D. L., Ballou, C. E., and Fischer, H. O. L. (1953) On the structure of galactinol. J. Am.
Chem. Soc. 75, 4507 4509
10. Hanahan, D. J., and Olley, J. N. (1958) Chemical nature of monophosphoinositides. J. Biol. Chem. 231, 813 828
11. Wooley, D. W. (1943) Isolation and partial determination of structure of soybean lipositol, a new inositolcontaining phospholipid. J. Biol. Chem. 147, 581591
12. Hawthorne, J. N., and Chargaff, E. (1954) A study of inositol-containing lipids. J. Biol. Chem. 206, 2737
13. McKibbin, J. M. (1956) A monophosphoinositide of liver. J. Biol. Chem. 220, 537545
14. Brown, D. M., and Todd, A. R. (1952) Nucleotides. Part X. Some observations on the structure and chemical
behavior of the nucleic acids. J. Chem. Soc. 5258
15. Pizer, F. L., and Ballou, C. E. (1959) Studies on myoinositol phosphates of natural origin. J. Am. Chem. Soc. 81,
915921
16. Ballou, C. E., and Pizer, L. I. (1959) Synthesis of an optically active myoinositol 1-phosphate. J. Am. Chem. Soc.
81, 4745
17. Ballou, C. E., and Pizer, L. I. (1960) The absolute configuration of the myoinositol 1-phosphates and a confirmation
of the bornesitol configurations. J. Am. Chem. Soc. 82, 33333335
18. Eisenberg, F. (1967) Myoinositol 1-phosphate as product of cyclization of glucose 6-phosphate synthase reaction.
J. Biol. Chem. 242, 13751382
19. Brown, D. M., Clark, B. F., and Letters, R. (1961) Phospholipids. Part VII. The structure of a monophosphoinositide. J. Chem. Soc. 3774 3779
20. Folch, J. (1949) Complete fractionation of brain cephalin: isolation from it of phosphatidylserine, phosphatidylethanolamine, and phosphoinositide. J. Biol. Chem. 177, 497504
21. Folch, J. (1949) Brain diphosphoinositide, a new phosphoinositide having inositol metadiphosphate as a constituent. J. Biol. Chem. 177, 505519
22. Grado, C., and Ballou, C. E. (1961) Myoinositol phosphates obtained by alkaline hydrolysis of beef brain
phosphoinositide. J. Biol. Chem. 236, 54 60
23. Dittmer, J. C., and Dawson, R. M. C. (1960) The isolation of a new complex lipid: triphosphoinositide from ox
brain. Biochim. Biophys. Acta 40, 379 380
24. Tomlinson, R. V., and Ballou, C. E. (1962) Myoinositol polyphosphate intermediates in the dephosphorylation of
phytic acid by phytase. Biochemistry 1, 166 171
25. Tomlinson, R. V., and Ballou, C. E. (1961) Complete characterization of the myoinositol polyphosphates from beef
brain phosphoinositide. J. Biol. Chem. 236, 19021906
26. Hawthorne, J. N. (1955) A further study of inositol-containing phospholipids. Biochim. Biophys. Acta 18, 389 393
27. Brockerhoff, H., and Ballou, C. E. (1961) The structure of the phosphoinositide complex of beef brain. J. Biol.
Chem. 236, 19071911
28. Hendrickson, H. S., and Ballou, C. E. (1964) Ion exchange chromatography of intact brain phosphoinositides on
diethylaminoethyl cellulose by gradient salt elution in a mixed solvent system. J. Biol. Chem. 239, 1369 1373
29. Brown, D. M., and Stewart, J. C. (1966) The structure of triphosphoinositide from beef brain. Biochim. Biophys.
Acta 125, 413 421
30. Hokin, L. E., and Hokin, M. R. (1958) Acetylcholine and the exchange of inositol and phosphate in brain
phosphoinositide. J. Biol. Chem. 233, 818 821
31. Hokin, L. E., and Hokin, M. R. (1953) Enzyme secretion and the incorporation of 32P into phospholipids of
pancreas slices. J. Biol. Chem. 203, 967977
32. Dawson, R. M. C. (1954) A measurement of 32P labelling of individual kephalins and lecithin in a small sample
of tissue. Biochim. Biophys. Acta 14, 374 379
33. Brockerhoff, H., and Ballou, C. E. (1962) Phosphate incorporation in brain phosphoinositides. J. Biol. Chem. 237,
49 52
34. Brockerhoff, H., and Ballou, C. E. (1962) On the metabolism of the brain phosphoinositide complex. J. Biol. Chem.
237, 1764 1766
35. Chang, M., and Ballou, C. E. (1967) Specificity of ox brain triphosphoinositide phosphomonoesterase. Biochem.
Biophys. Res. Commun. 26, 199 205
36. Anderson, R. J. (1930) The chemistry of the lipoids of tubercle bacilli. XIV. The occurrence of inosite in the
phosphatide of human tubercle bacilli. J. Am. Chem. Soc. 52, 16071608
37. Vilkas, E., and Lederer, E. (1961) Sur la structure du phosphatidyl-inositol-dimmanosides de Mycobacterium
tuberculosis. Bull. Soc. Chim. Biol. 42, 10131022
38. Ballou, C. E., Vilkas, E., and Lederer, E. (1963) Structural studies on the myoinositol phospholipids of Mycobacterium tuberculosis (var. bovis BCG). J. Biol. Chem. 238, 69 76
39. Ballou, C. E., and Lee, Y. C. (1964) The structure of myoinositol mannoside from Mycobacterium tuberculosis
glycolipid. Biochemistry 3, 682 685
40. Lee, Y. C., and Ballou, C. E. (1965) Complete structures of the glycophospholipids of mycobacteria. Biochemistry
4, 13951404
41. Hunter, S. W., and Brennan, P. J. (1990) Evidence for the presence of a phosphatidylinositol anchor on the
lipoarabinomannan and lipomannan of Mycobacterium tuberculosis. J. Biol. Chem. 265, 92729279
42. Ferguson. M. A. J., Homans, S. W., Dwek, R. A., and Rademacher, T. W. (1988) Glycosylphosphatidylinositol
moiety that anchors Trypanosoma bruccii variant surface glycoprotein to the membrane. Science 239, 753759
43. Ballou, C. E. (1964) Structure of myoinositol phospholipids. 6th International Congress of Biochemistry, pp.
547548, New York
H85

44. Berridge, M. J., and Irvine, R. F. (1989) Inositol phosphates and cell signalling. Nature 341, 197205
45. Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. C. (1988) Type I phosphatidylinositol kinase
makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 332, 644 646
46. Ballou, C. E. (1968) Studies on the structure of a lipopolysaccharide from mycobacterium species. Acc. Chem. Res.
1, 366 373
47. Gray, G. R., and Ballou, C. E. (1970) Isolation and characterization of a polysaccharide containing 3-O-methylD-mannose from Mycobacterium phlei. J. Biol. Chem. 246, 6835 6842
48. Bloch, C. (1977) Control mechanisms for fatty acid synthesis in Mycobacterium smegmatis. Adv. Enzymol. Relat.
Areas Mol. Biol. 45, 1 84
49. Ballou, C. E. (1990) Isolation, characterization and properties of Saccharomyces cerevisiae mutants with nonconditional protein glycosylation defects. Methods Enzymol. 185, 440 470
H86
REFLECTIONS

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 5, pp. 27532764, February 2, 2007
2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Lectins: Carbohydrate-specific
Reagents and Biological
Recognition Molecules
PUBLISHED, JBC PAPERS IN PRESS, DECEMBER 4, 2006, DOI 10.1074/JBC.X600004200
Nathan Sharon
From the Department of Biological Chemistry, The Weizmann Institute of Science,
Rehovot 76100, Israel
he occurrence in nature of proteins with hemagglutinating activity that in later years

were shown to be sugar-specific and eventually named lectins has been known since
the turn of the 19th century, but until about two decades ago they aroused little interest
(for a historical survey, see Ref. 1). My own involvement with these proteins began
inadvertently and initially on a part-time basis in the early 1960s after my return to the Weizmann
Institute from two and a half years of exciting and educational postdoctoral studies in the United
States. During the first of these I worked in the laboratory of Fritz Lipmann at the Massachusetts
General Hospital, Boston. Lipmann, one of the most influential biochemists of the last century,
was then interested in the mechanism of protein biosynthesis. I was assigned to study the amino
acid activation reaction (the first step in this process), work that resulted in two publications (2, 3).
Concurrently, I greatly enriched my knowledge of biochemistry, mainly from my fellow postdoctoral students, and especially from the guest seminars in which ongoing biochemical discoveries
and developments were reported. I spent the second postdoctoral year at the Massachusetts
General Hospital with Roger Jeanloz, a leading carbohydrate chemist, where I got my training in
the subject and also succeeded in isolating an unusual diamino sugar from a Bacillus polysaccharide I had brought with me from Rehovot (4) (see below); the remaining time I worked with Dan
Koshland at Brookhaven National Laboratory on the mechanism of action of myosin ATPase (5,
6). Dan was then starting to make his mark on enzymology with his induced fit concept of
enzyme action, originally greeted with much skepticism (7).
Back at Rehovot my original aim was to establish the structure of that diamino sugar; I was
fortunate to receive for this purpose my first National Institutes of Health (NIH) grant, a modest
one of some $25,000 for 3 years. (This would have been unheard of at the present time because
nothing was known then about the function of the compound.) The task took me (with a couple of
graduate students) over a decade; eventually we were able to prove by degradation and synthesis
that the compound in question, which we named bacillosamine, is 2,4-diamino-2,4,6-trideoxy-Dglucose (8, 9). To my delight, the di-N-acetyl derivative of bacillosamine has recently been found
attached glycosidically to the amide of asparagine or the hydroxyl of serine in the carbohydratepeptide linkage region of several interesting glycoproteins of pathogenic bacteria (10). By a strange
twist of fate, most of these glycoproteins were originally isolated in 2002 by Martin Young and his
colleagues at the National Research Laboratories, Ottawa, from Campylobacter jejuni by affinity
chromatography on immobilized soybean agglutinin (SBA) (11), the first lectin I got involved with
40 years earlier.
From Soy Proteins for Nutrition to Glycoproteins and Lectins

My studies of SBA began together with Halina Lis with whom it has been my good fortune to
collaborate to this very day. It aroused our curiosity not because of its ability to bind sugars
FEBRUARY 2, 2007 VOLUME 282 NUMBER 5
H87
REFLECTIONS: Lectins
specifically and reversibly and to agglutinate cells, the hallmarks of proteins of this class, but because of other reasons that I shall presently mention. We did not have the
slightest idea that lectins would become extremely useful
carbohydrate-specific reagents, that they would be found
to function as mediators of cell recognition, or that they
would make a major contribution to glycobiology (12). In
fact, for a time we were not even aware of the term lectin,
which was originally proposed in 1956 by William C. Boyd
from Boston University for blood type-specific hemagglutinins. Because SBA, like the majority of the hemagglutinins, is not blood group-specific, we began referring to it as
a lectin only in 1970, when it occurred to us that the original definition should be broadened to include all cell-agglutinating and sugar-specific proteins (13).
Our interest in SBA developed in the course of investigations on soybean proteins carried out within the framework of a generous and long term grant from the United
States Department of Agriculture that I received in 1961
jointly with Katchalski-Katzir (14). Katchalski was the
founding Head of the Department of Biophysics at the
fledgling Weizmann Institute, which was officially inaugurated in 1949. I came to the department in 1954 after having received my Ph.D. degree from the Hebrew University,
Jerusalem; Halina, with a Ph.D. degree from Uppsala University, joined the department 5 years later. The purpose
of the above grant was to carry out a fundamental study of
the soy proteins with the aim of providing information for
their improved utilization for human nutrition. Katchalski
and I were persuaded to embark on this project by Tim
(M. L.) Anson and Aaron Altschul, close friends, noted
protein chemists, and enthusiastic believers in these proteins as the best solution to world hunger. After some
time, Katchalski became immersed in his pioneering studies of polyamino acids as protein models and on enzyme
immobilization and turned over the whole project to me,
for which I am extremely grateful.
Halina and I set out by trying to obtain pure proteins
from soybeans by chromatographic techniques, but this
proved to be a difficult task as most of them lack biological
activity, are poorly soluble, and undergo complex association-dissociation reactions. We therefore chose to focus
on SBA, originally isolated and characterized in the 1950s
by Irvin E. Liener at the University of Minnesota, St. Paul.
The main reason for our choice was the evidence presented by Liener that it contained glucosamine, raising the
likelihood that it may be a glycoprotein (15). In those days,
research on glycoproteins was in its infancy, but I became
intrigued by these compounds because of my interest in
carbohydrates, as described elsewhere (16).
Soybean Agglutinin, a Plant Glycoprotein

Working on SBA, Halina and I soon found that it contains not only glucosamine but also mannose. We then
isolated from a proteolytic digest of SBA an asparaginyloligosaccharide that contained all the N-acetylglucosamine and mannose of the lectin (17). Eventually we also
isolated from the lectin N-acetylglucosaminylasparagine
(18), the carbohydrate-peptide linking group, that was
identical with the one originally obtained in 1963 by Albert
Neuberger, the founding father of modern glycoprotein
research, in his pioneering studies of ovalbumin.
As pointed out recently by Liener (19), The fact that
SBA was shown to be a glycoprotein may not be particularly surprising to the modern day biochemists, but at the
time the finding of a sugar moiety in a plant protein was
accepted with reservation. It was thought that glycoproteins were strictly of animal origin and that the finding of a
sugar with a plant protein was most likely because of noncovalent contamination.
In 1981, jointly with Hans (J. F. G.) Vliegenthart from
the University of Utrecht, the complete structure of the
carbohydrate of SBA was established by NMR as the
branched oligomannoside Man9(GlcNAc)2, found in animal glycoproteins too, demonstrating that protein N-glycosylation is a process conserved in plants and animals
(20). A unique feature of SBA is that all its molecules carry
the same oligosaccharide (21) in contrast to essentially all
other glycoproteins, which bear a variety of glycans at each
attachment site, i.e. consist of mixtures of distinct glycoforms. SBA serves therefore as an excellent source of this
oligosaccharide (for an example, see Ref. 22).
Emerging from Obscurity

Our few 1960s publications on SBA attracted little
attention, and we sometimes felt like wanderers in a
desert. Although the studies of lectins were in their eighth
decade and several hundreds of these proteins (almost all
from plants) had already been identified, the handful of
other scientists active in the field at the time did not fare
better. Irwin J. Goldstein from the University of Michigan
at Ann Arbor, still a leading lectin researcher, tells that
when he sent a note in 1963 to Biochemical and Biophysical Research Communications describing the purification
of concanavalin A by affinity chromatography, it was
rejected forthright because this represents a modest
advance in an obscure area. The note was eventually published in the Biochemical Journal (23), and affinity chromatography soon became the method of choice for lectin
isolation.
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VOLUME 282 NUMBER 5 FEBRUARY 2, 2007
However, as the 1960s were folding, the attitude toward

lectins began to change, and a number of leading biochemists and immunologists, among them Gerald Edelman at
Rockefeller University, Mel Greaves at London University,
Elvin Kabat at Columbia University, Jerker Porath at Uppsala, and Jon Singer at University of California, San Diego,
became involved with them. The reasons for this change in
attitude were summarized by Kabat, who had become
intrigued with lectins primarily because their combining
sites seemed similar to those of antibodies and who in 1977
stated: During the past 10 years there has been an extraordinary burst of activity in the study of plant and animal
lectins, stimulated largely by the findings that they have
specific receptor sites for carbohydrates and react with
glycoproteins in solution or on cell membranes . . . (24).
In 1970, affinity chromatography of glycoproteins on
immobilized lectins was introduced (among others) by
Donnely and Goldstein (25). It became a must at one step
or another for the isolation of membrane proteins, all of
which are glycosylated, a classical case being that of the
insulin receptor with the aid of wheat germ agglutinin
(WGA) (26). Lectins proved also to be useful for the separation of purified glycoproteins into their glycoforms, i.e.
differently glycosylated forms of the same protein. A very
recent telling example is of different glycoforms of IgG
with different degrees of sialylation, obtained by fractionation on the sialic acid-specific Sambucus nigra agglutinin
and shown to differ in their anti-inflammatory activity
(27).
Tools for Study of Membranes and Cells

Interest in lectins intensified with the realization that
they are extremely valuable reagents for the investigation
of cell surface sugars, for the assessment of the role of the
latter in cell growth and differentiation, in interactions of
cells with their environment, and also in a variety of pathological processes. In this connection it is instructive to
refer to two classical studies with lectins that provided
very early evidence for the presence of sugars on cell surfaces and their potential role as cell identity markers, a
common theme in modern glycobiology. One came from
the laboratory of James Sumner at Cornell University,
Ithaca, who in 1919 isolated concanavalin A in crystalline
form but only in 1936, together with Howell, reported that
it agglutinates cells such as erythrocytes and yeasts and
that this agglutination is inhibited by sucrose, thus demonstrating for the first time the sugar specificity of lectins
(28). Moreover, with much foresight they suggested that
the hemagglutination induced by the lectin might be a
consequence of its reaction with carbohydrates on the surface of the red cells. The other study was by Walter Mor-
gan and Winifred Watkins at the Lister Institute, London,

who in the early 1950s used blood type-specific hemagglutinins to show that the blood type A immunodeterminant
is -linked N-acetylgalactosamine and that the H(O)
determinant is -L-fucose (reviewed in Ref. 29). This was
the first demonstration that cell surface carbohydrates can
serve as carriers of biological information.
Much excitement was created in the following decade
by the reports of Joseph C. Aub from the Massachusetts
General Hospital (30) and Max Burger from Princeton
University (31), who were both working with WGA (specific for N-acetylglucosamine and N-acetylneuraminic
acid), and of Leo Sachs with Michael Inbar from the
Department of Genetics of our Institute, who used concanavalin A (specific for mannose and glucose) (32), that
these lectins agglutinated malignantly transformed cells
but not their normal parental cells. The reports provided
compelling evidence that cancer might be associated with
a change in cell surface sugars, an idea that only a few years
before had been considered completely unfounded. In collaboration with Leo Sachs and Ben-Ami Sela, we found
soon thereafter that SBA (specific for galactose and
N-acetylgalactosamine) also possesses the remarkable
ability to distinguish between normal and malignant cells
(33). Numerous subsequent studies have demonstrated
that high susceptibility to agglutination by lectins is a
property shared by many, albeit not all, malignant cells.
Several basic features of membranes were revealed, or
their existence confirmed, with the aid of lectins. Thus,
using ferritin-conjugated concanavalin A and ricin as an
electron microscopic probe, Garth Nicolson and Jon
Singer at University of California, San Diego, found that
the lectin derivatives bind specifically to the outer surface
of the human and rabbit erythrocyte membrane and concluded that the oligosaccharides of the plasma membrane
of eukaryotic cells are asymmetrically distributed (34).
Further support for such distribution was obtained by
Vincent Marchesi at Yale University, who used ferritinlabeled phytohemagglutinin (PHA) and showed that glycophorin, the major sialoglycoprotein of the human erythrocyte membrane, is oriented so that its carbohydratecarrying segment is exposed to the external medium,
whereas the other segments of the same molecule are
embedded in the lipid bilayer or protrude into the cytoplasm (35).
Other ultrastructural studies with lectins provided
some of the most convincing evidence for the fluid mosaic
membrane model of Singer and Nicolson, according to
which the membrane consists of proteins and glycoproteins floating in a lipid bilayer (reviewed in Ref. 36). Prom-
H89
inent among these was the finding of the lectin-induced

clustering and patching of the corresponding membrane
receptors on lymphocytes and other kinds of cell, as illustrated for example by the treatment with fluorescein-labeled concanavalin A of rat or mouse lymphocytes (37).
Reorganization of cell surface carbohydrates was later
shown to be required for various activities of lectins on
cells such as mitogenic stimulation and induction of
apoptosis.
The toxicity for animals of certain plant lectins has been
recognized since the earliest days of lectin research, at the
end of the 19th century. However, research on the toxic
action of lectins on cells started only many decades later
with special attention being paid to mammalian cell lines
(e.g. CHO and BHK) resistant to different lectins, primarily the highly toxic ricin and the less toxic PHA and WGA
(reviewed in Ref. 38). Leading the field was one cell phenotype independently isolated in 1974 by Stuart Kornfeld
at Washington University, Colin Hughes at the National
Institute for Medical Research, Mill Hill, London, and
Pamela Stanley at Toronto University, Canada. This phenotype lacked GlcNAc transferase I, the key enzyme in the
biosynthesis of complex and hybrid N-linked carbohydrate units of glycoproteins. Soon thereafter many other
lectin-resistant cell lines with different enzymatic glycosylation defects became available. They proved extremely
valuable for the investigation of the biosynthesis of glycoproteins and glycolipids and of the function of their carbohydrates, especially those expressed on the cell surface.
Currently they also serve for the large-scale production
of pharmacologically useful glycoproteins such as
erythropoietin.
Review Articles
In the fall of 1970 I arrived at the Department of Biochemistry, University of California at Berkeley, for a sabbatical year as Visiting Professor. My host was Clint Ballou
with whom I discussed at length the possibility of using
lectins to examine the ideas on the roles of carbohydrates
as information and recognition molecules. Such ideas had
been entertained by Saul Roseman from Johns Hopkins
University (39) and Victor Ginsburg at the NIH (40).
Although there existed a few books and several reviews on
lectins, none of them dealt with their molecular properties
nor did they indicate their enormous potential as tools for
biological research. Because Dan Koshland from the same
department at Berkeley was then a member of the editorial
board of Science, I approached him with the suggestion
that I write a review on lectins for that journal. This suggestion was readily accepted by Philip Abelson, then editor
of Science.
Writing was started by me in the fall of that year in the

laboratory of Albert Neuberger at St. Marys Hospital in
London, where I arrived for a few months to study
lysozymes, on which Neuberger and I were at that time
working. However, I ended up purifying WGA by ion
exchange chromatography from commercial wheat germ
together with Tony (A. K.) Allen, separating it into three
isolectins and showing that its specificity is similar to that
of lysozyme (41), because it too exhibited a pronounced
affinity not only for oligosaccharides derived from chitin,
as originally demonstrated by Burger and Goldberg (31),
but also of peptidoglycan. In addition we also proved that,
contrary to suggestions in the literature, WGA was not a
glycoprotein. This work further stimulated the interest of
Neuberger in lectins with which he continued to be
engaged for several years into his eighties.
The Science review was completed jointly with Halina
upon my return to Rehovot early in 1972 (13). It summarized the history of the research on lectins since their discovery, their specificity for monosaccharides and cells,
and the properties of concanavalin A and the few other
lectins that had been purified at the time. The changes that
occur on cell surfaces upon malignant transformation, as
revealed by lectins, were discussed, although their significance was not clear and doubts were raised by us, amply
supported later, as to whether they are a distinctive characteristic of malignant cells. Regardless of this, we concluded that lectins, both native and modified, provide a
new and useful tool for the study of the chemical architecture of cell surfaces. Finally, we dealt in brief with the speculations on the role of lectins in nature, about which nothing was known with certainty. Another review on lectins
was published by us in the following year in the Annual
Review of Biochemistry (42) and a third appeared in the
same series in 1986 (43). In these reviews we tried to convey to the readers our fascination and enthusiasm for the
subject.
In 1989 we prepared a monograph on lectins and in
2003 a second edition of the same (44), both of which have
been translated into Japanese. Some 20 years ago I coedited a treatise on lectins to which Halina and I contributed several chapters (45). A related activity of mine was
the publication in 1975 of a book entitled Complex Carbohydrates in which lectins are featured and where I
expressed my firm belief that the specificity of many natural polymers is written in terms of sugar residues, not of
amino acids or nucleotides (46). The book was based on
notes that I prepared for the graduate students taking my
course on the same subject and remained in use for a long
H90
time. I still continue teaching this course, now under the

title Molecular and Cellular Glycobiology.
Structural Diversity of Lectins

The 1970s witnessed the intensification of the study of
the molecular properties of individual lectins, a prerequisite for a deep understanding of their activities at the
molecular level. In 1972 concanavalin A became the first of
these proteins for which the primary and three-dimensional structures have been established, the latter by x-ray
crystallography. This was thanks to the efforts of Gerald
Edelmans group at the Rockefeller University (47) and of
the efforts of Karl Hardman and Clinton F. Ainsworth
at Argonne National Laboratories (48). The fold first
observed in this structure, an elaborate arrangement of
extended beta strands into two sheets, became known as
the jelly roll or lectin fold (126). The publication of the
concanavalin A structure was soon followed by the determination by Christine Schubert Wright at Virginia Commonwealth University of the three-dimensional structure
of WGA as well as of its complexes with ligands even
before the complete amino acid sequence of this lectin had
become available (49). It is worth noting that at present the
structures of close to 100 lectins have been solved, almost
all also in complexes with ligands.
In my laboratory, we continued to be occupied with
SBA, mainly with Reuben Lotan, a talented and hard working graduate student. Among others we demonstrated
that lectin is a tetramer made up of four nearly identical
subunits (50) (all legume lectins consist of two or four
subunits) and that the carbohydrate of SBA is not essential
for its biological activity (51). Final proof for the latter
conclusion came when we obtained the carbohydrate-free
SBA in a bacterial expression system in a fully active form
(52); still, why SBA, like most lectins, is glycosylated
remains an enigma.
Analysis of the amino acid composition of SBA showed
that it is similar to that of other legume lectins, the composition of which was known at the time. In particular, it
was devoid of sulfur-containing amino acids, in striking
contrast to WGA that is rich in such residues. We have
therefore proposed that although lectins have many biological properties in common, they represent a diversified
group of proteins with respect to size, composition, and
structure, which is indeed the case (53).
The primary sequence of SBA was determined in the
early 1980s at the Rockefeller University by conventional
methods (reviewed in Ref. 54). Although homologous with
the sequences of the other two legume lectins known at
the time (from lentil and fava bean), homology with concanavalin A (also a leguminous lectin) could only be
obtained by circular permutation of the latter. This means

by aligning residue 119 of concanavalin A with the aminoterminal residue of the SBA, proceeding to the carboxyl
end of concanavalin A (residue 237), and continuing with
its amino-terminal region along the sequence of SBA. This
kind of circular homology, never observed before, was
shown by Diana Bowles and her co-workers at York University, United Kingdom, to be the result of a unique rearrangement of the peptide chain that occurs in the last step
of the biosynthesis of concanavalin A (55).
Crystals of SBA suitable for x-ray diffraction studies
were obtained by Boaz Shaanan and colleagues from our
Department of Structural Chemistry in 1984 (56), but the
high resolution structure of the lectin, in complex with a
ligand, was solved only a decade later by James Sacchettini
and co-workers at Albert Einstein College of Medicine,
New York (57).
Biological Activities and Functions of Plant Lectins

In 1960 Peter Nowell at the University of Pennsylvania
discovered that PHA, the lectin of the red kidney bean, acts
as a mitogen for lymphocytes, namely that it has the ability
to stimulate these cells to grow and divide (58). This finding shattered the belief, held until then, that lymphocytes
are dead-end cells that could neither divide nor differentiate further. Within a short time several other lectins were
proven to be mitogenic. Of special significance was the
finding, first reported by Werner G. Jaffe at the University
of Venezuela, Caracas, that concanavalin A acts as a mitogen (59) because its binding to the lymphocytes could be
inhibited and reversed by low concentrations of mannose,
in contrast to PHA for which no effective inhibitor was
available at the time. It was thus concluded that mitogenic
stimulation is the result of binding of lectins to cell surface
sugars, providing another early demonstration of a biological function of the latter compounds. Not all lectins that
bind to cells are, however, mitogenic, indicating that
attachment to selected carbohydrates is required for cell
stimulation.
In subsequent years there was an explosive growth in
the use of mitogenic lectins in biological research. Until
the advent of monoclonal antibodies they served as a popular tool in attempts to clarify the mechanism of signal
transmission through the cell membrane and of cell activation. Mitogen-stimulated lymphocytes were found,
among others, to produce many growth factors collectively known as lymphokines or cytokines, the first of
which was discovered by Robert C. Gallo and co-workers
at the National Institutes of Health, Bethesda, as T-cell
growth factor in 1976 and later named interleukin-2 (60).
When the immune system is malfunctioning, the mito-
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genic response of lymphocytes is defective. Stimulation by

PHA, and to some extent also concanavalin A and
pokeweed mitogen, is routinely employed in clinical laboratories as a simple means to assess the immunocompetence of patients suffering from different diseases and to
monitor the effects of various immunosuppressive and
immunotherapeutic treatments.
Studies with mitogenic lectins have provided information on the cell surface sugars and other factors involved in
cell activation. Thus, using SBA, Abraham Novogrodsky
and Ephraim Katchalski found that the lectin stimulated
mouse lymphocytes only after the cells had been treated
with sialidase, which unmasked the subterminal galactose
and N-acetylgalactosamine residues of the surface glycoproteins and glycolipids to which the lectin bound (61),
and we have subsequently demonstrated that peanut
agglutinin (PNA, see below) exhibits the same property
(62). We then found that SBA was mitogenic only in polymerized form (63). It was an early demonstration of the
requirement of receptor cross-linking for cell activation.
The latter findings were published in the European Journal of Immunology over the objection of one of the referees
in whose opinion papers on lectins had no place in an
immunological journal. Luckily, this view was then not
generally held; indeed, at the same time Michael Sela
invited Halina and me to contribute a chapter on lectins
for The Antigens, a treatise he was editing (64). A few years
later I wrote a review for Advances in Immunology on the
application of lectins for lymphocyte identification and
separation (65). At present lectins form an integral part of
immunology because of their pivotal role in innate immunity (see later).
Because lectins do not stimulate plant cells, it is
highly unlikely that they have been selected by evolution for this purpose. Based on findings in my laboratory
that SBA (as well as PNA) inhibits the sporulation and
growth of fungi such as Trichoderma viride, Penicilium
notatum, and Aspergillus niger, we raised the possibility
that lectins may protect plants against pathogenic
microorganisms (66). Work in other laboratories has
subsequently extended this proposal to include the
defense of plants against predatory animals and phytopathogens (reviewed in Ref. 67). According to another
suggestion, plant lectins may be responsible for the specific association between leguminous plants and nitrogen-fixing rhizobia that provide the plants with the
needed nitrogen (for review, see Ref. 68). However, this
suggestion can account for the role of lectins in only one
plant family, the Leguminosae.
For Cell Separation and Bone Marrow

Transplantation
Toward the end of his doctoral research, Reuben Lotan,
with Yehuda Marikovsky and David Danon from our Institute, purified PNA by affinity chromatography (69). The
lectin was also purified at the same time by Toshiaki
Osawa from Tokyo University, friend and pioneer lectin
researcher (70). The detailed specificity of PNA was then
established in collaboration with Miercio Pereira and
Elvin Kabat at Columbia University, confirming among
others that the lectin has a high affinity for Gal3GalNAc
(known also as T antigen), a characteristic glycan of O-glycoproteins (71).
Just as PNA had become available in my laboratory, it
was my good fortune that Yair Reisner joined me as a
doctoral student. Bright and imaginative, he set his mind
to find out whether lectins can serve as markers for lymphocyte subpopulations. He soon found that immature,
cortical mouse thymocytes were agglutinated by PNA but
that the mature, medullar cells were not. This served as the
basis for the development by him of a facile method
(sometimes referred to by us as poor mans cell sorter)
for separation, by selective agglutination with PNA, of the
two thymocyte subpopulations in good yield and with full
viability. A manuscript we sent early in 1976 to Nature
magazine describing the method was promptly rejected by
the editor on the grounds that it was not of general interest. Time proved Nature wrong because soon after it was
published in Cellular Immunology (72) the method
became very popular. This was primarily because it
afforded access to the immature thymocyte subpopulation
needed for the investigation of T lymphocyte maturation
and because it became a classic technique for defining the
cortical and medullar regions of the thymus (73). In our
study, we have found a marked difference in the level of
PNA binding to the thymocyte subpopulations, which is
abolished upon treatment of the mature subpopulation
with sialidase. We have therefore proposed that sialylation
of the PNA receptor may be an important step in the maturation of the thymic cells. Nearly 20 years later evidence
was indeed presented by Linda Baum and her co-workers
from UCLA and UCSD that regulated expression of a single glycosyltransferase, a Gal3GalNAc 2,3-sialyltransferase, can account for this glycosylation change (73). This
enzyme sialylates Gal3GalNAc, the preferred ligand of
PNA, forming the sequence NeuAc2,3Gal3GalNAc
and thus masking the PNA-binding sites; expression of the
enzyme is inversely proportional to that of the PNA receptor. The PNA receptor continues to serve as a differentiation marker for lymphocytes (74). It has also been
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employed as a similar marker in other systems, for example in embryonal carcinoma cells of mice, as shown by Yair
in a joint study with Francois Jacob and Gabriel Gachelin
of the Pasteur Institute, Paris (75).
What proved to be highly significant was Yairs demonstration, together with Asher Meshorer and Lea Itzicovitch from our Experimental Animal Center, that sequential agglutination of mouse bone marrow or spleen cells by
SBA and PNA afforded a cell fraction suitable for transplantation across histocompatibility barriers. In the paper
reporting on these results, we stated that the same
approach may prove useful for bone marrow transplantation in humans (76). For his postdoctoral research, Yair
joined Robert A. Good, then President of the Sloan Kettering Institute, New York, and Richard OReilly, Chief of
Bone Marrow Transplantation at that Institute, with the
express aim of adapting the lectin separation method to
humans. By 1981 he had found that treatment of human
bone marrow with SBA alone removed the bulk of the cells
responsible for the lethal graft-versus-host disease and
that, after additional processing, such bone marrow, even
from haploidentical donors, could be safely transplanted
into children born with severe combined immune deficiency (SCIDs or bubble children) (77). It is a matter of
great pride and satisfaction to Yair, and to me, that over
75% of the hundreds of bubble children who received
since then transplants of bone marrow that had been
purged with SBA have been cured and lead a normal life.
For several years SBA-purged bone marrow was also used
on an experimental basis for treatment of end stage leukemia patients (78).
Asn-linked heptasaccharide Man3(Man6)(Xyl2)Man4GlcNAc4(Fuc3)GlcNAc (80). It was one of the

first examples of this plant-specific oligosaccharide
reported in the literature. The oligosaccharide is allergenic
for humans and presents a major obstacle that needs to be
overcome before plants can be employed for the production of pharmacologically useful glycoproteins such as
monoclonal antibodies.
When we exhausted the supply of E. cristagalli seed
flour that Jose had brought with him from Uruguay, I
turned my attention to Erythrina corallodendron, the
coral tree that grows commonly in Israel, the seed lectin of
which (ECorL) was originally isolated in 1980 by Nechama
Gilboa-Garber at Bar Ilan University, Ramat Gan (81).
Working on ECorL proved to be highly rewarding. Its primary sequence was established by Rivka Adar using conventional methods and by her (jointly with Rafael Arango,
a graduate student from Medeillin, Colombia, and Shmuel
Rozenblatt from Tel Aviv University) by recombinant
techniques (82). The sequence was homologous to that of
other legume lectins, providing further evidence for the
proposal that I made in 1977 with Donny Strosberg, then
at the Free University of Brussels, that despite their distinct sugar specificities, legume lectins are members of a
single protein family and that the genes coding for them
have a common ancestry (83). According to a recent
count, 210 sequences of legume lectins are known, all
homologous (84). In the 1980s sequence similarities were
found for lectins from unrelated taxonomic families,
including lectins from animal sources, starting with the
galectins and C-type lectins (85).
Legume Lectins, a Large Family of Homologous

Proteins
Identical Tertiary Structures, Different Quaternary

Structures
I owe the last turning point in my research on plant

lectins to Jose Luis Iglesias, a young medical student at the
University of Montevideo, Uruguay, who had become fascinated by these proteins and found one in the seeds of
Erythrina cristagalli, an ornamental leguminous tree common in Uruguay. Because of a lack of facilities in his country he was unable to isolate the lectin so he came to my
laboratory for a short stay early in 1981, bringing with him
6 kg of the flour of E. cristagalli seeds. In no time he had
ascertained that his lectin, which we had designated ECL,
is galactose-specific and purified it by affinity chromatography on the immobilized sugar (79). He also demonstrated that ECL is a glycoprotein containing fucose and
xylose in addition to mannose and N-acetylglucosamine
present in SBA. The structure of the carbohydrate of ECL
was established in the laboratory of Raymond Dwek at
Oxford University, United Kingdom, as the branched
In collaboration with Boaz Shaanan, then at the Weizmann Institute, the three-dimensional structure of ECorL
in complex with lactose was established by high resolution
x-ray crystallography (86). Although the tertiary structure
observed was superimposable on that of other legume lectins, the quaternary structure was markedly different from
the canonical one, such as that of concanavalin A (which is
devoid of carbohydrate); the same was found also for the
structure of ECL recently solved in collaboration with Ute
Krengel and colleagues from Goteborg University, Sweden (87). We originally assumed that the bulky N-linked
carbohydrate of ECorL interfered with the formation of
the canonical structure by the lectin. This interpretation
has been proven to be incorrect, because in a recent joint
study with Avadesha Surolia and colleagues from the
Indian Institute of Scientific Research, Bangalore (88) as
well as by K. Ravi Acharya and colleagues at the University
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of Bath, United Kingdom (89), the quaternary structure of

the bacterially expressed ECorL, devoid of carbohydrate,
was identical with that of the native one. The non-canonical mode of dimerization of the Erythrina lectins is therefore most likely because of factors intrinsic to the protein.
Indeed, according to Surolia and his co-workers, legume
lectins are a family of proteins in which small alterations in
essentially the same tertiary structure lead to large variations in quaternary association (90).
An exceptional feature of the three-dimensional structure of the Erythrina lectins is that all the seven proteinlinked monosaccharide residues are seen with extraordinary clarity, whereas in almost all other such structures of
glycoproteins at most 3 or 4 of the protein-proximal monosaccharides have been observed (86 89). The structures
also show that in the crystal lattice the glycosylation site
and the carbohydrate-binding site are involved in intermolecular contacts through water-mediated interactions.
How Lectins Combine with Carbohydrates

To obtain detailed information on the combining site of
ECorL, Rivka Adar together with Hansjorg Streicher, postdoctoral fellow from the University of Konstanz, carried
out extensive site-directed mutagenesis of the lectin. In
addition they made a thorough examination of its specificity in collaboration with Jonas ngstrom and co-workers from the University of Goteborg and with Ray Lemieux
from the University of Alberta, Edmonton. Taking also in
consideration the three-dimensional structure of the
ECorL-ligand complex (87), we have concluded that a
constellation of three key amino acid residues, an aspartic
acid, an asparagine, and an aromatic one, is essential for
galactose binding (9194). The first two residues form
hydrogen bonds with the hydroxyls of the ligand, whereas
the third interacts with it hydrophobically. Moreover, an
identical constellation of amino acid residues is involved
also in binding of mannose by other legume lectins, for
example by concanavalin A. We suggested therefore that
homologous lectins with distinct specificities might bind
different monosaccharides primarily by the same set of
invariant residues that are identically positioned in their
tertiary structures although with the ligand in a different
orientation (95). Quite surprisingly, the constellation
present in the combining sites of legume lectins is also
found in certain animal lectins, such as the mannose-specific ERGIC-53 that serves as a carrier of a specific subset
of nascent glycoproteins between the ER and Golgi compartments (96). Comparison with the combining sites of
lectins from other sources, including of animals, led to an
additional conclusion, namely that structurally different
lectins with similar specificities may bind the same sacchaJOURNAL OF BIOLOGICAL CHEMISTRY
ride by different sets of combining site residues (95). A

survey of the literature showed that lectins bind their
ligands most commonly by hydrogen bonds (some mediated by water) and hydrophobic interactions and that in
rare cases electrostatic interactions (ion pairing) and coordination with metal ions also play a role. On the whole,
most of the side chains of the protein amino acids, as well
as main chain groups, can participate in ligand binding. All
the above seems to indicate that lectins are products of
convergent evolution.
Cell-Cell Recognition Molecules in Microbial

Infection
In the foregoing, I dealt almost exclusively with plant
lectins. However, for the last 30 years I have also been
interested in microbial lectins, which I wish to describe
now. This interest was motivated by the arrival in my laboratory in 1975 of Itzhak Ofek as a postdoctoral fellow.
Through him I learned that primarily thanks to the efforts
of J. P. Duguid of Ninewells Hospital Medical School in
Dundee it had been known that many strains of Escherichia coli possess the ability to agglutinate erythrocytes
and that this activity is inhibited by mannose and methyl
-mannoside (97), but little attention was paid to these
findings. Moreover, the idea that sugar-specific adhesion
to host cells might be a prerequisite for bacterial colonization and infection was not considered at all. In retrospect,
this is all the more surprising because it had already been
established that initiation of infection by influenza virus
required its attachment via its hemagglutinin to a sugar
(sialic acid) on cells (for an early review, see Ref. 98).
Together with David Mirelman, a former graduate student
of mine, and Ofek (who later moved to the Sackler Medical
School, Tel Aviv University) we found that E. coli adheres
readily to buccal epithelial cells and that this adhesion was
inhibited specifically by mannose and methyl -mannoside. The adhesion was also inhibited by pretreatment of
the epithelial cells with concanavalin A but not with other
lectins such as SBA. Extraction of the bacteria afforded a
lectin-like constituent specific for mannose. We concluded that mannose, a common constituent of most
mammalian cell surfaces, acts as a receptor for E. coli (99).
The same conclusion was reached independently and at
the same time by Irving Salit and Emil Gotschlich from the
Rockefeller University, who demonstrated that binding to
monkey cells of purified fimbriae obtained from a mannose-specific strain of E. coli was inhibited by analogues of
mannose or by preincubation of the cells with mannosespecific plant lectins (100). These studies provided a clear
example (I believe the first of its kind) that lectins function
in cell-cell recognition.
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With Ofek and Nurit Firon, a graduate student, we have

mapped the combining site of the type 1 fimbriae, mainly
by the ability of a variety of mannose-containing oligosaccharides and -mannosides to inhibit the agglutination of
yeasts by the bacteria or by the isolated fimbriae (101, 102).
Hydrophobic mannosides in particular were found to be
powerful inhibitors of the agglutination, up to three orders
of magnitude more effective than methyl -mannoside.
Based on our results, we proposed that the site is extended
with an adjoining hydrophobic region. A very recent study
by Julie Bouckaert and colleagues at the Free University of
Brussels of FimH, the carbohydrate-binding subunit of the
fimbriae, has confirmed our results and extended them
(103). By x-ray crystallography and modeling of FimHligand complexes a molecular explanation was obtained
for the high affinity of hydrophobic mannosides to the
fimbriae, which is based on the interaction of their aglycones with aromatic side chains (referred to as hydrophobic gateway) close to the mannose-binding site of the
subunit.
That blocking of the bacterial lectins may prevent infection was proven by us in a study carried out in collaboration with Ofek and Mirelman together with Moshe Aronson from Sackler Medical School, Tel Aviv University.
Infection of mouse bladder with a mannose-specific E. coli
strain was markedly diminished by pre-suspension of the
organism in a solution of methyl -mannoside but was not
affected by glucose, a sugar to which the bacteria do not
bind (104). The prophylactic effects of adhesion-inhibitory saccharides have been demonstrated in many other
animal models, such as pneumococcal pneumonia in rats
(105) and Helicobacter pylori gastric infection in monkeys
(106). In addition to demonstrating unequivocally that
recognition of cell surface carbohydrates is a prerequisite
for infection by lectin-carrying bacteria, the above data
serve as a definitive proof for the validity of the concept of
anti-adhesion therapy of microbial diseases. However,
success of such treatment in humans has not yet been
achieved (for review, see Ref. 107).
The specificity of the bacterial surface lectins is a key
determinant in their animal tropism, as first illustrated by
Victor Ginsburg in the case of E. coli K99 (108). The fimbrial lectin of this organism is specific for glycolipids containing N-glycolylneuraminic acid but not for those containing N-acetylneuraminic acid. The former sialic acid is
present on intestinal cells of newborn piglets but is
replaced by N-acetylneuraminic acid when the animals
develop. It is also not normally formed by humans,
explaining why E. coli K99 can cause lethal diarrhea in
piglets but not in adult pigs or in humans.
In addition to their role in initiation of infection, the

mannose-specific bacterial surface lectins may also function in protection against infectious agents. This is the
case also for surface lectins of phagocytic cells (such as
granulocytes and macrophages). As we have shown some
time ago, bacteria and yeasts may bind to these cells in the
absence of opsonins, leading to uptake and killing of the
organisms. This phenomenon, named by us lectinophagocytosis (109), is an early example of innate immunity,
a phenomenon of great importance, in which lectins are
now known to be major players (reviewed for example in
Refs. 110 and 111).
Concurrent with our studies of the mannose-specific
E. coli, Catharina Svanborg and Hakon Leffler discovered
strains of this organism with galabiose (Gal4Gal)-specific fimbriae that also serve as mediators of attachment of
the bacteria to host tissues as a prelude to infection (112).
Two surface lectins were discovered soon thereafter in the
pathogenic protozoan Entamoeba histolytica. One of
these, specific for N-acetylgalactosamine, was reported by
Jonathan Ravdin and Richard Guerrant from the University of Virginia, Charlottesville (113); the other, specific for
N-acetylglucosamine oligomers, was described at the
same time by David Mirelman and David Kobiler in our
department (114). During the years, compelling evidence
has been obtained for the involvement of the former lectin
in host cell binding, as well as in a variety of other properties of the ameba, that determine the severity of its infection (for review, see Ref. 115).
Animal Lectins in Cell Recognition

Despite the evidence presented above, wide acceptance
of the concept that lectins function as recognition molecules was slow to come and had to await the isolation and
characterization of mammalian lectins. The first of these
was the galactose-specific liver lectin discovered in 1974
by Gilbert Ashwell at NIH with Anatol G. Morell at Albert
Einstein College of Medicine in the course of their investigation of the mechanisms that control the lifetime of
glycoproteins in blood circulation (reviewed in Ref. 116).
This lectin recognizes and binds asialo-glycoproteins and
is responsible for their uptake by the liver and eventual
degradation.
In our department and at about the same time, my former graduate student Vivian Teichberg and Gad Reshef,
another graduate student of mine who was killed in the
October 1973 war, isolated from the electric eel (Electrophorus electricus) and from chicken muscle the first
members of the family of the soluble -galactose-specific
lectins (117) designated galectins, several of which are
known to be mediators of cellular interactions (for
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reviews, see Refs. 118 and 119). One of these, galectin-3,

that is involved in metastatic spread of B16 melanoma cells
was isolated by Reuben Lotan while still in our department
together with Avraham Raz from the Department of
Membrane Research (120, 121). Another member of this
family, galectin-8, has also been isolated at the Weizmann
Institute by Yehiel Zick and co-workers from the Department of Molecular Cell Biology (122); by binding to cell
surfaces, this lectin modulates cell-matrix interactions and
regulates cellular functions in a variety of physiological
and pathological conditions.
What fully convinced the skeptics was the discovery in
about 1990 of the selectins, a class of C-type mammalian
lectins, and the demonstration of their crucial role in the
control of lymphocyte migration (homing) to specific
lymphoid organs and to sites of inflammation (for a recent
review, see Ref. 123). Since then, numerous other mammalian lectins have been discovered, many of which are
cell surface constituents, strategically located to serve as
recognition molecules in a variety of systems, both normal
and pathological (for reviews, see Refs. 110, 111, 122, 124,
and 125). Indeed, recognition by lectins in animal tissues is
undoubtedly one of the major developments in glycobiology during the last part of the 20th century. But that is
another story.
This Reflections article is dedicated to Ephraim Katchalski-Katzir, mentor and friend, on the occasion of his 90th birthday, May 16, 2006.
AcknowledgmentsI am most grateful to my long time colleague, Dr.
Halina Lis, for her help in the preparation of these reflections. Thanks are
also due to my friend, Dr. Uriel Littauer, for his constructive criticism of
the manuscript.
Address correspondence to: nathan.sharon@weizmann.ac.il.
REFERENCES
1. Sharon, N., and Lis, H. (2004) Lectins: from hemagglutinins to biological
recognition molecules. A historical overview. Glycobiology 14, 53R 62R
2. Sharon, N., and Lipmann, F. (1957) Reactivity of analogs with pancreatic
tryptophan-activating enzyme. Arch. Biochem. Biophys. 69, 219 227
3. Hoagland, M. B., Zamecnik, P. C., Sharon, N., Lipmann, F., Stulberg, M. P.,
and Boyer, P. D. (1957) Oxygen transfer to AMP in the enzymatic synthesis of
the hydroxamate of tryptophan. Biochim. Biophys. Acta 26, 215217
4. Sharon, N., and Jeanloz, R. W. (1960) The diaminohexose component of a
polysaccharide isolated from Bacillus subtilis. J. Biol. Chem. 235, 15
5. Levy, H. M., Sharon, N., and Koshland, D. E., Jr. (1959) Purified muscle proteins and the walking rate of ants. Proc. Natl. Acad. Sci. U. S. A. 45, 785791
6. Levy, H. M., Sharon, N., Lindemann, E., and Koshland, D. E., Jr. (1960) Properties of the active site in myosin hydrolysis of adenosine triphosphate as
indicated by the O18-exchange reaction. J. Biol. Chem. 235, 2628 2632
7. Koshland, D. E., Jr. (2004) Crazy, but correct. Nature 432, 447
8. Zehavi, U., and Sharon, N. (1973) Structural studies of 4-acetamido-2-amino2,4,6-trideoxy-D-glucose (N-acetylbacillosamine), the N-acetyl diaminosugar
of Bacillus licheniformis. J. Biol. Chem. 248, 433 438
9. Liav, A., Hildhesheim, J., Zehavi, U., and Sharon, N. (1974) Synthesis of 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine), 2-acetamido2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine) and 2,4-acetamido-2,4,6trideoxy-D-glucose from 2-acetamido-2-deoxy-D-glucose. Carbohydr. Res.
H96
33, 217227
10. Weerapana, E., and Imperiali, B. (2006) Asn-linked protein glycosylation:
from eukaryotic to prokaryotic systems. Glycobiology 16, 91R101R
11. Young, N. M., Brisson, J. R., Kelly J., and Watson, D. C., et al. (2002) Structure
of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277, 45230 45239
12. Taylor, M. E., and Drickamer, K. (2006) Introduction to Glycobology, 2nd Ed.,
Oxford University Press, Oxford, UK
13. Sharon, N., and Lis, H. (1972) Lectins: cell agglutinating and sugar-specific
proteins. Science 177, 949 959
14. Katchalski-Katzir, E. (2005) My contributions to science and society. J. Biol.
Chem. 280, 16529 16541
15. Wada, S., Pallansch, M. J., and Liener, I. E. (1958) Chemical composition and
end groups of soybean hemagglutinin. J. Biol. Chem. 233, 395 400
16. Sharon, N. (2000) Half a century between carbohydrates and proteins. Comprehensive Biochemistry. A History of Biochemistry, Vol. 41, pp. 391 448,
Elsevier Publishing Co., Amsterdam
17. Lis, H., Sharon, N., and Katchalski, E. (1966) Soybean hemagglutinin, a plant
glycoprotein. I. Isolation of a glycopeptide. J. Biol. Chem. 241, 684 689
18. Lis, H., Sharon, N., and Katchalski, E. (1969) Identification of the carbohydrate-peptide linking group in soybean agglutinin. Biochim. Biophys. Acta
192, 364 366
19. Liener, I. E. (2002) A trail of research revisited. J. Agric. Food Chem. 50,
6580 6582
20. Dorland, L., van Halbeek, H., Vliegenthart, J. F. G., Lis, H., and Sharon, N.
(1981) Primary structure of the carbohydrate chain of soybean hemagglutinin.
A reinvestigation by high resolution 1H NMR spectroscopy. J. Biol. Chem.
256, 7708 7711
21. Ashford, D. A., Dwek, R. A., Rademacher, T. W., Lis, H., and Sharon, N. (1991)
The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in
N-linked oligosaccharide expression. Carbohydr. Res. 213, 215227
22. Deras, I. L., Kawasaki, N., and Lee, Y. C. (1998) Quantitative recovery of
Man9GlcNAc2Asn from concanavalin A. Carbohydr. Res. 306, 469 471
23. Agrawal, B. B. L., and Goldstein, I. J. (1965) Specific binding of concanavalin A
to cross-linked dextrans. Biochem. J. 96, 23c25c
24. Kabat, E. A. (1978) Dimensions and specificities of recognition sites on lectins
and antibodies. J. Supramol. Struct. 8, 79 88
25. Donnely, E. H., and Goldstein, I. J. (1970) Glutaraldehyde-insolubilized concanavalin A: an adsorbent for the specific isolation of polysaccharides and
glycoproteins. Biochem. J. 118, 697 698
26. Jacobs, S., Schechter, Y., Bissell, K., and Cuatrecasas, P. (1977) Purification and
properties of insulin receptors from rat liver membranes. Biochem. Biophys.
Res. Commun. 77, 981988
27. Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V. (2006) Anti-inflammatory
activity of immunoglobulin G resulting from Fc sialylation. Science 313,
670 674
28. Sumner, J. B., and Howell, S. F. (1936) Identification of concanavalin A with
the hemagglutinin of jack bean. J. Bacteriol. 32, 227237
29. Morgan, W. T. J., and Watkins, W. M. (2000) Unraveling the biochemical
basis of blood group ABO and Lewis antigenic specificity. Glycoconjugate J.
17, 501530
30. Aub, J. C., Sanford, B. H., and Wang, L. H. (1965) Reactions of normal and
leukemic cell surfaces to a wheat germ agglutinin. Proc. Natl. Acad. Sci.
U. S. A. 54, 400 402
31. Burger, M., and Goldberg, A. R. (1967) Identification of a tumor-specific
determinant of neoplastic cell surfaces. Proc. Natl. Acad. Sci. U. S. A. 57,
359 366
32. Inbar, M., and Sachs, L. (1969) Interaction of the carbohydrate-binding protein concanavalin A with normal and transformed cells. Proc. Natl. Acad. Sci.
U. S. A. 63, 1418 1425
33. Sela, B. A., Lis, H., Sharon, N., and Sachs, L. (1970) Different locations of
carbohydrate-containing sites at the surface membrane of normal and transformed mammalian cells. J. Membr. Biol. 3, 267279
34. Nicolson, G. L., and Singer, S. J. (1971) Ferritin-conjugated plant agglutinins
as specific saccharide stains for electron microscopy: application to saccharides bound to cell membranes. Proc. Natl. Acad. Sci. U. S. A. 68, 942945
35. Marchesi, V. T., Tillack, T. W., Jackson, R. L., Segrest, J. P., and Scott, R. E.
(1972) Chemical characterization and surface orientation of the major glycoprotein of the human erythrocyte membrane. Proc. Natl. Acad. Sci. U. S. A.
69, 14451449
36. Nicolson, G. L. (1974) Interactions of lectins with animal cell surfaces. Int. Rev.
Cytol. 39, 89 190
37. Inbar, M., and Sachs, L. (1973) Mobility of carbohydrate-containing sites on
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
the surface membranes in relation to control of cell growth. FEBS Lett. 32,
124 128
Stanley, P. (1987) Glycosylation mutants and the functions of mammalian
carbohydrates. Trends Genet. 3, 77 81
Roseman, S. (1970) The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion.
Chem. Phys. Lipids 5, 270 297
Ginsburg, V., and Kobata, A. (1971) in Structure and Function of Surface
Components of Mammalian Cells in Structure and Function of Biological
Membranes (Rothfield, L. I., ed) pp. 439 459, Academic Press, New York
Allen, A., Neuberger, A., and Sharon, N. (1973) The purification and specificity of wheat germ agglutinin. Biochem. J. 131, 155162
Sharon, N., and Lis, H. (1973) The biochemistry of plant lectins (phytohemagglutinins). Annu. Rev. Biochem. 42, 541574
Lis, H., and Sharon, N. (1986) Lectins as molecules and tools. Annu. Rev.
Biochem. 55, 35 67
Sharon, N., and Lis, H. (1989) Lectins. Chapman & Hall, London; (2003)
Lectins, 2nd Ed., Kluwer Academic Publishers, Dordrecht, The Netherlands
Liener, I. E., Sharon, N., and Goldstein, I. J. (eds) (1986) The Lectins, Properties,
Functions and Applications in Biology and Medicine, Academic Press,
Orlando, FL
Sharon, N. (1975) Complex Carbohydrates, Their Properties, Biosynthesis and
Functions, Addison Wesley, Reading, MA
Edelman, G. M., Cunningham, B. A., Reeke, G. N., Jr., Becker, J. W., Waxdal,
M. J., and Wang, J. L. (1972) The covalent and three-dimensional structure of
concanavalin A. Proc. Natl. Acad. Sci. U. S. A. 69, 2580 2584
Hardman, K. D., and Ainsworth, C. F. (1972) Structure of concanavalin A at
2.4 resolution. Biochemistry 11, 4910 4919
Wright, C. S. (1977) The crystal structure of wheat germ agglutinin at 2.2
resolution. J. Mol. Biol. 111, 439 457
Lotan, R., Siegelman, H. W., Lis, H., and Sharon, N. (1974) Subunit structure
of soybean agglutinin. J. Biol. Chem. 249, 1219 1224
Lotan, R., Debray, H., Cacan, M., Cacan, R., and Sharon, N. (1975) Labeling of
soybean agglutinin by oxidation with sodium periodate followed by reduction
with sodium [3H]borohydride. J. Biol. Chem. 250, 19551957
Adar, R., Streicher, H., Rozenblatt, S., and Sharon, N. (1997) Synthesis of
soybean agglutinin in bacterial and mammalian cells. Eur. J. Biochem. 249,
684 689
Sharon, N., Lis, H., and Lotan, R. (1974) On the structural diversity of lectins.
Colloq. Int. du C.N.R.S. 221, 693709
Hemperly, J. J., and Cunnigham, B. A. (1983) Circular permutation of amino
acid sequences among legume lectins. Trends Biochem. Sci. 8, 100 102
Bowles, D. J., Marcus, S. E., Pappin, D. J., Findlay, J. B., and Eliopoulos, E., et al.
(1986) Posttranslational processing of concanavalin A precursors in jack bean
cotyledons. J. Cell Sci. 102, 1284 1297
Shaanan, B., Shoham, M., Yonath, A., Lis, H., and Sharon, N. (1984) Crystallization and preliminary x-ray diffraction studies of soybean agglutinin. J. Mol.
Biol. 174, 723725
Dessen, A., Gupta, D., Sabesan, S., Brewer, C. F., and Sacchettini, J. C. (1995)
X-ray crystal analysis of the soybean agglutinin crosslinked with a biantennary
analog of the blood group I carbohydrate antigen. Biochemistry 34,
4933 4942
Nowell, P. (1960) Phytohemagglutinin: an initiator of mitosis in cultures of
normal human leukocytes. Cancer Res. 20, 462 466
Wecksler, M., Levy, A., and Jaffe, W. G. (1968) Mitogenic effects of extracts of
Canavalia ensiformis and concanavalin A. Acta Cient. Venez. 19, 154 156
Mier, J. W., and Gallo, R. C. (1982) The purification and properties of human
T cell growth factor. J. Immunol. 128, 11221127
Novogrodsky, A., and Katchalski, E. (1973) Transformation of neuraminidase-treated lymphocytes by soybean agglutinin. Proc. Natl. Acad. Sci. U. S. A.
70, 25152518
Novogrodsky, A., Lotan, R., Ravid, A., and Sharon N. (1975) Peanut agglutinin,
a new mitogen that binds to galactosyl sites exposed after neuraminidase
treatment. J. Immunol. 115, 12431248
Schechter, B., Lis, H., Lotan, R., Novogrodsky, A., and Sharon, N. (1976)
Requirement for tetravalency of soybean agglutinin for induction of mitogenic stimulation of lymphocytes. Eur. J. Immunol. 6, 145149
Sharon, N., and Lis, H. (1997) Lectins: their chemistry and applications to
immunology. The Antigens, Vol. 4, pp. 429 529, Academic Press, New York
Sharon, N. (1983) Lectin receptors as lymphocyte surface markers. Adv.
Immunol. 34, 213298
Barkai-Golan, R., Mirelman, D., and Sharon, N. (1978) Studies on growth
inhibition by lectins of Penicillia and Aspergilli. Arch. Microbiol. 116,
H97
119 124
67. Peumans, W. J., and Van Damme, E. J. M. (1995) Lectins as plant defense
proteins. Plant Physiol. 109, 347352
68. Kijne, J. W. (1997) Root lectins and Rhizobia. Plant Physiol. 115, 869 873
69. Lotan, R., Skutelsky, E., Danon, D., and Sharon, N. (1975) The purification,
composition, and specificity of the anti-T lectin from peanut (Arachis
hypogaea). J. Biol. Chem. 250, 8518 8523
70. Terao, T., Irimura, T., and Osawa, T. (1975) Purification and characterization
of a hemagglutinin from Arachis hypogaea. Hoppe-Seylers Z. Physiol. Chem.
356, 16851692
71. Pereira, M. E. A., Kabat, E. A., Lotan, R., and Sharon, N. (1976) Immunochemical studies on the specificity of peanut (Arachis hypogaea) agglutinin.
Carbohydr. Res. 51, 107118
72. Reisner, Y., Linker-Israeli, M., and Sharon, N. (1976) Separation of mouse
thymocytes into two subpopulations by the use of peanut agglutinin. Cell.
Immunol. 25, 129 134
73. Gillespie, W., Paulson, J. C., Kelm, S., Pang, M., and Baum. L. G. (1993) Regulation of -2,3-sialyltransferase expression correlates with conversion of
peanut agglutinin (PNA) to PNA phenotype in developing thymocytes.
J. Biol. Chem. 268, 38023804
74. Daniels, M. A., Hogquist, K. A., and Jameson, C. S. (2002) Sweet n sour: the
impact of differential glycosylation on T cell differentiation. Nat. Immunol. 3,
903910
75. Reisner, Y. Gachelin, G., Dubois, P., Nicolas, J. F., Sharon, N., and Jacob, F.
(1977) Interaction of peanut agglutinin, a lectin specific for terminal D-galactosyl residues, with embryonal carcinoma cells. Dev. Biol. 61, 20 27
76. Reisner, Y., Itzicovitch, L., Meshorer, A., and Sharon, N. (1978) Hemopoietic
stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins. Proc. Natl. Acad. Sci. U. S. A. 75, 29332936
77. Reisner, Y., Kapoor, N., Kirkpatrick, D., Pollack, M. S., Cunningham-Rundles,
S., Dupont, B., Hodes, M. Z., Good, R. A., and OReilly, R. J. (1983) Transplantation for severe combined immunodeficiency with HLA-A,B,D,DR-incompatible parental marrow cells fractionated by soybean agglutinin and sheep
red blood cells. Blood 61, 341348
78. Aversa, F., Tabilio, A., Velardi, A., Cunningham, I., Terenzi, A., Falzetti, F.,
and Ruggeri, L., et al. (1998) Treatment of high-risk acute leukemia with
T-cell-depleted stem cells from related donors with one fully mismatched
HLA haplotype. N. Engl. J. Med. 339, 1186 1193
79. Iglesias, J. L., Lis, H., and Sharon, N. (1982) Purification and properties of a
galactose/ N-acetyl-D-galactosamine-specific lectin from Erythrina cristagalli. Eur. J. Biochem. 123, 247252
80. Ashford, D., Dwek, R. A., Welply, J. K., Amatayakul, S., Homans, S. W., Lis, H.,
Taylor, G. N., Sharon, N., and Rademacher, T. W. (1987) The (12)-D-xylose
and (13)-L-fucose substituted N-linked oligosaccharides from Erythrina
cristagalli lectin. Isolation, characterization, and comparison with other
legume lectins. Eur. J. Biochem. 166, 311320
81. Gilboa-Garber, N., and Mizrachi, L. (1981) A new mitogenic D-galactosephilic
lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins.
Can. J. Biochem. 59, 315322
82. Arango, R., Adar, R., Rozenblatt, S., and Sharon, N. (1992) Expression of
Erythrina corallodendron lectin in Escherichia coli. Eur. J. Biochem. 205,
575581
83. Foriers, A., Wuilmart, C., Sharon, N., and Strosberg, A. D. (1977) Extensive
sequence homologies among lectins from leguminous plants. Biochem.
Biophys. Res. Commun. 75, 980 985
84. Chandra, N. R., Kumar, N., Jeyakani, J., Singh, D. D., Gowda, S. B., and Prathima, M. N. (2006) Lectinb: a plant lectin database. Glycobiology 16, 938 946
85. Drickamer, K. (1988) Two distinct classes of carbohydrate recognition domains in animal lectins. J. Biol. Chem. 263, 95579560
86. Shaanan, B., Lis, H., and Sharon, N. (1991) Structure of a lectin with ordered
carbohydrate in complex with lactose. Science 253, 862 866
87. Svensson, C., Teneberg, S., Nilsson, C. L., Schwarz, F. P., Kjellberg, A., Sharon,
N., and Krengel, U. (2002) High-resolution crystal structures of Erythrina
cristagalli lectin in complex with lactose and 2--L-fucosyllactose and their
correlation with thermodynamic binding data. J. Mol. Biol. 321, 69 83
88. Kulkarni, K. A., Srivastava, A., Mitra, N., Sharon, N., Surolia, A., Vijayan, M.,
and Suguna, K. (2004) Effect of glycosylation on the structure of Erythrina
corallodendron lectin. Proteins 56, 821 827
89. Turton, K., Natesh, R., Thiagarajan, N., Chaddock, J. A., and Acharya, K. K.
(2004) Crystal structure of Erythrina cristagalli lectin with bound N-linked
oligosaccharide and lactose. Glycobiology 14, 923929
90. Brinda, K. V., Mitra, N., Surolia, A., and Wishveshwara, S. (2005) Determi-
nants of quaternary association in legume lectins. Protein Sci. 13, 17351749

91. Adar, R., and Sharon, N. (1996) Mutational studies of the combining site
residues of Erythrina corallodendron lectin. Eur. J. Biochem. 239, 668 674
92. Adar, R., ngstrom, J., Moreno, E., Karlsson, K. A., Streicher, H., and Sharon,
N. (1998) Structural studies of the combining site of Erythrina collarodendron
lectin. Role of tryptophan. Protein Sci. 7, 52 63
93. Moreno, E., Teneberg, S., Adar, R., Sharon, N., Karlsson, K.-A., and ngstrom,
J. (1997) Redefinition of the carbohydrate specificity of Erythrina corallodendron lectin based on solid-phase binding assays and molecular modeling
of native and recombinant forms obtained by site-directed mutagenesis.
Biochemistry 36, 4429 4437
94. Lemieux, R., Ling, C. C., Sharon, N., and Streicher, H. (2000) The epitope of
the H-type trisaccharide recognized by Erythrina corallodendron lectin. Evidence for both attractive polar and strong hydrophobic interactions for complex formation involving a lectin. Isr. J. Chem. 40, 167176
95. Sharon, N., and Lis, H. (2001) The structural basis for carbohydrate recognition by lectins. Adv. Exp. Med. Biol. 491, 116
96. Velloso, L. M., Svensson, K., Schneider, G., Pettersson, R. F., and Lindqvist, Y.
(2002) Crystal structure of the carbohydrate recognition domain of p58/ERGIC-53, a protein involved in glycoprotein export from the endoplasmic
reticulum. J. Biol. Chem. 277, 15979 15984
97. Duguid, J. P., and Old, D. C. (1980) in Bacterial Adherence (Beachey, E. H., ed)
pp. 185217, Chapman and Hall, London
98. Schauer, R. (1973) Chemistry and biology of acylneuraminic acids. Angew.
Chem. Int. Ed. Engl. 12, 127138
99. Ofek, I., Mirelman, D., and Sharon, N. (1977) Adherence of Escherichia coli to
human mucosal cells mediated by mannose receptors. Nature 265, 623 625
100. Salit, I. E., and Gotschlich, E. C. (1997) Type I Escherichia coli pili: characterization of binding to monkey kidney cells. J. Exp. Med. 146, 11821194
101. Firon, N., Ofek, I., and Sharon, N. (1983) Carbohydrate specificity of the
surface lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella
typhimurium. Carbohydr. Res. 120, 235249
102. Firon, N., Ashkenazi, S., Mirelman, D., Ofek, I., and Sharon, N. (1987) Aromatic alpha-glycosides of mannose are powerful inhibitors of the adherence
of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells.
Infect. Immun. 55, 472 476
103. Bouckaert, J., Berglund, M., Schembri, E., De Genst, L., Cools, M., and Wuhrer, M., et al. (2005) Receptor binding studies disclose a novel class of highaffinity inhibitors of the Escherichia coli FimH adhesin. Mol. Microbiol. 55,
441 455
104. Aronson, M., Medalia, O., Schori, L., Mirelman, D., Sharon, N., and Ofek, I.
(1979) Prevention of colonization of the urinary tract of mice with Escherichia
coli by blocking of bacterial adherence with methyl -D-mannopyranoside. J.
lnfect. Dis. 139, 329 332
105. Indapaan-Heikkila, I., Simon, P. M., Zopf, D., Vullo, T., Cahill, P., et al. (1997)
Oligosaccharides interfere with the establishment and progression of experimental pneumococcal pneumonia. J. Infect. Dis. 176, 704 712
106. Mysore, J. V., Wigginton, T., Simon, P. M., Zopf, D., Heman-Ackah, L. M.,
and Dubois, A. (1999) Treatment of Helicobacter pylori infection in rhesus
monkeys using a novel anti-adhesion compound. Gastroenterology 117,
1316 1325
107. Sharon, N. (2006) Carbohydrates as future anti-adhesion drugs for infectious

diseases. Biochim. Biophys Acta 1760, 527537
108. Kyogashima, M., Ginsburg, V., and Krivan, H. C. (1989) Escherichia coli K99
binds to N-glycolyl-sialoparagloboside and N-glycolyl-GM3 found in piglet
small intestine. Arch. Biochem. Biophys. 270, 391397
109. Ofek, I., and Sharon, N. (1988) Lectinophagocytosis: a molecular mechanism
of recognition between cell surface sugars and lectins in the phagocytosis of
bacteria. Infect. Immun. 56, 539 547
110. Crocker, P. R., and Varki, A. (2001) Siglecs, sialic acid and innate immunity.
Trends Immunol. 22, 337342
111. Lu, J., Teh, C., Kishore, U., and Reid, K. B. (2002) Collectins and ficolins: sugar
pattern recognition molecules in the mammalian innate immune system.
Biochim. Biophys. Acta 1572, 387 400
112. Leffler, H., and Svanborg-Eden, C. (1981) Glycolipid receptors for uropathogenic Escherichia coli on human erythrocytes and uroepithelial cells. Infect.
Immun. 34, 920 929
113. Ravdin, J. I., and Guerrant, R. L. (1981) Role of adherence in cytopathogenic
mechanisms of Entamoeba histolytica: studies with mammalian tissue culture
cells and human erythrocytes. J. Clin. Invest. 68, 13051313
114. Kobiler, D., and Mirelman, D. (1981) Adhesion of Entamoeba histolytica trophozoites to monolayers of human cells. J. Infect. Dis. 144, 534 546
115. Frederick, J. R., and Petri, W. A., Jr., (2005) Roles for the galactose-/N-acetylgalactosamine-binding lectin of Entamoeba in parasite virulence and differentiation. Glycobiology 15, 53R59R
116. Ashwell, G., and Morell, A. G. (1974) The role of surface carbohydrates in the
hepatic recognition and transport of surface glycoproteins. Adv. Enzymol.
Relat. Areas Mol. Biol. 41, 99 128
117. Teichberg, V. I., Silman, I., Beitsch, D. D., and Reshef, G. (1975) A -D-galactoside binding protein from electric organ tissue of Electrophorus electricus.
Proc. Natl. Acad. Sci. U. S. A. 72, 13831387
118. Hughes, R. C. (2001) Galectins as modulators of cell adhesion. Biochimie
(Paris) 83, 667 676
119. Hernandez, J. D., and Baum, L. G. (2002) Ah, sweet mystery of death! Galectins and control of cell fate. Glycobiology 12, 127R136R
120. Raz, A., and Lotan, R. (1981) Lectin-like activities associated with human and
murine neoplastic cells. Cancer Res. 41, 36423647
121. Meromsky, L., Lotan, R., and Raz, A. (1986) Implications of endogenous tumor cell surface lectins as mediators of cellular interactions and lung colonization. Cancer Res. 46, 5270 5275
122. Hadari, Y. R., Paz, K., Dekel, R., Mestrovic, T., Accili, D., and Zick, Y. (1995)
Galectin-8: a new rat lectin, related to galectin-4. J. Biol. Chem. 270,
34473453
123. Ley, K. (2003) The role of selectins in inflammation and disease. Trends Mol.
Med. 9, 263268
124. Sharon, N., and Lis, H. (1989) Lectins as cell recognition molecules. Science
246, 227234
125. Sharon, N., and Lis, H. (1993) Carbohydrates in cell recognition. Sci. Am. 268,
82 89
126. Srinivasan, N., Rufino, S. D., Pepys, M. B., Wood, S. P., and Blundell, T. L.
(1986) A superfamily of proteins with the lectin fold. Chemtracts Biochem.
Mol. Biol. 6, 149 164
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REFLECTIONS

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 22, pp. 1490114909, May 30, 2008
In Search of the Message

Published, JBC Papers in Press, March 31, 2008, DOI 10.1074/jbc.X800001200
John H. Exton
From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232-0615
was born in Auckland, New Zealand, in 1933. Hitler was on the rise in Germany, but this was
of little concern to most of the inhabitants of this beautiful country so far from the political,
economic, and cultural centers of the world. During my early years, my family lived by the
seaside, and my sister and I led an idyllic existence playing on the beach, exploring rock pools
teeming with life, and climbing the cliffs to see the nests of ocean birds. My exposure to the unique
flora and fauna of New Zealand engendered an early interest in biology. I left all this behind when
my family moved to the city, where my education began in earnest. This was when atomic bombs
were dropped on Japan to end World War II. I did not recognize the moral issues involved in this
act, but it stimulated my intense interest in nuclear physics. I was probably one of few elementary
school pupils in New Zealand who read about Madame Curie, Niels Bohr, Max Planck, Ernest
Lawrence, Enrico Fermi, Walter Heisenberg, Erwin Schrodinger, and, of course, New Zealands
own Ernest Rutherford.
For my secondary education, I was sent to a Church of England preparatory school that was
patterned after an English public school. Discipline was severe, with the cane being liberally
applied and bullying rampant. However, my parents did not pay high fees just for the infliction of
pain on their son, but for an excellent academic program in which Latin was mandatory and Greek
recommended. This classical education was broadened by exposure to history and literature,
almost exclusively British, and to mathematics and the sciences. For sport, we were required to
play cricket and rugby football whatever our competence, which in my case was low. The chemistry teacher frequently put on dramatic explosive displays of chemical reactions, causing us to
crouch behind our desks while he dropped solid sodium or potassium into concentrated nitric,
sulfuric, and hydrochloric acids!
In New Zealand, there is no college system, and at that time, relatively few high school graduates
proceeded to university. I was fascinated by both chemistry and biology and had to choose
between a degree in medicine or the sciences, with my teachers strongly recommending medicine.
So I took the two-day journey from Auckland in the North Island to Dunedin in the South Island,
where the only medical school at that time was located at the University of Otago. I had led a rather
sheltered life and was initially shocked by the riotous and decidedly unintellectual behavior of my
fellow students. Dunedin was founded by dour Scottish settlers, many of them Calvinists, but
somehow they tolerated the frequent and outrageous student pranks. One of Dunedins greatest
assets is its proximity to some of New Zealands most beautiful lakes and mountains, and groups
of us would spend weekends and other breaks hiking in the spectacular scenery.
Medical training in New Zealand takes six years, with almost two years devoted to anatomy,
physiology, and biochemistry. Unlike the vast majority of students, I was fascinated by biochemistry because it fused my interests in chemistry and biology. Another factor was that it was taught
by Norman Edson, one of the unsung heroes of New Zealand Science. He had worked with Hans
Krebs in Gowland Hopkins laboratory in Cambridge, England, where he had studied the regulation of ketone body production in the liver. He had an encyclopedic memory and kept up with the
latest advances in the U. S. and U. K. I decided to take a year out of my medical training to do
biochemical research and studied the breakdown of pyrimidines. At that time, there was contro-
MAY 30, 2008 VOLUME 283 NUMBER 22

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REFLECTIONS: In Search of the Message
versy about the degradation pathway, but I showed that

the scheme proposed by Robert M. Fink and co-workers
was correct. This involved the intermediate formation of
-ureidopropionic acid from uracil and -ureidoisobutyric acid from thymine (1). There was an initial reduction
of the pyrimidines to dihydropyrimidines, and I set about
to find the dehydrogenase responsible. This was unsuccessful because the only reduced nicotinamide nucleotide
commercially available at that time was NADH, and the
enzyme was later shown to use NADPH. I continued with
my medical studies, and my social life greatly expanded
when I met my future wife, who was a great companion on
our rugged hikes and sailing adventures on the icy waters
of Otago Harbor. She was also a student at the University
of Otago and came from a sheep farming community in
the North Island with the exotic name of Ongaonga. Her
father often employed me on the farm during vacations.
Being a city slicker, I was of doubtful use to him except
during lambing, when my obstetrical skills learned on the
sturdy Scottish women of Dunedin were useful when the
ewes were in difficulties. I suspect he tested me as a suitable son-in-law by sending me out to the endless task of
draining a large swamp by hand shovel. I must have passed
muster because he later gave his daughter in marriage
when I was a final year student.
During my student years at Otago, I supplemented my
scholarship funds by working in a variety of occupations
during the summer breaks. These included working in an
ice cream factory, as a surveyors assistant, in a packing
house, in a wool store, and as a longshoreman. These gave
me the opportunity to interact with a wider socioeconomic group than my middle class family and friends and
encouraged my thinking away from the relentless conservatism of my parents!
At several times during my training, I contemplated
switching to a Ph.D. in Biochemistry, but Norman Edson
advised me strongly to continue in medicine, a course I
have never regretted, particularly because there were
aspects that I really enjoyed, and it gave me fortitude when
I later had to lecture to medical students. After completing
medical studies and an internship in Auckland, I returned
to Dunedin to do a Ph.D. in Biochemistry under Norman
Edson. The thesis topic was the metabolism of isolated rat
liver cells. Michael Berry, an energetic student in the laboratory, had discovered how to prepare these cells, and my
project was to study their carbohydrate and lipid metabolism and to understand why they produced large amounts
of ketone bodies (2, 3). This was traced to a limitation in
the citric acid cycle such that the fatty acids were preferentially oxidized to ketone bodies. At that time, the medJOURNAL OF BIOLOGICAL CHEMISTRY
ical school had only one electron microscope, and when

the cells were examined later, it was found that their
plasma membranes were disrupted and their mitochondria distorted. Nevertheless, the findings were reported in
the Biochemical Journal because studies of isolated mammalian cells were unusual at that time. A visitor from the
U. S. at that time was Harland Wood from Western
Reserve, who came to study pyruvate metabolism. I am
sure an additional attraction for him was the proximity of
great deer hunting to Dunedin. Because of the great fear of
radioactivity in the 1960s, Norman Edson built a small
laboratory for him on the roof of the medical school. Imagine his chagrin when Harland loaded a syringe with
[14C]pyruvate and squirted part of it into the air to expel an
air bubble!
When my Ph.D. was nearing completion, I looked
around for possible postdoctoral positions. The population of New Zealand was then not much more than three
million, and Ph.D. and medical graduates desiring further
training had to look overseas. Britain was the traditional
place, and relatively few went to America. I sought a fellowship to Trinity College Oxford to work on a D.Phil.
under Hans Krebs, although why I needed an additional
degree escapes me now. In case I was not successful in the
fellowship, I looked into several possibilities in the U. S.
The most attractive of these was the Department of Physiology at Vanderbilt University under Charles R. (Rollo)
Park, whose group had just published an impressive series
of papers in the Journal of Biological Chemistry dealing
with the effects of insulin, diabetes, and other hormones
on glucose transport and phosphorylation in heart muscle.
The rigor of their data and their in-depth analysis
impressed me greatly.
I did win a fellowship to Oxford, but when the details
arrived from the British Council, I was frankly outraged.
At that time (1963), my family had grown to two children,
and the Council secretary advised me to leave my family in
New Zealand for two years because the stipend could support only a single person. Furthermore, I could travel only
at the lowest level on the ship to Britain. On the other
hand, a letter came from Rollo Park offering to pay the
travel expenses of the whole family, so I outraged the British by refusing their fellowship to Oxford, which may
never have been done before by a mere colonial and certainly did not please Krebs!
We went by ship to the U. S., stopping at such exotic and
erotic places as Tahiti, Panama City, and Jamaica and disembarking at Port Everglades in Florida. We hired two
Cadillac limousines to take our large pile of baggage to the
train station and then took one of them on a tour of Miami.
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VOLUME 283 NUMBER 22 MAY 30, 2008
It seemed that we were living the American dream a few

hours after arrival. We took a train to Nashville, but it had
been rerouted, and no one on the train knew which station
was Nashville! We discovered for sure only upon alighting
on the platform! Rollo turned out to be as generous as his
correspondence indicated and told me that the scientific
environment of the department had recently been
enhanced by the arrival of Earl Sutherlands group from
Western Reserve. Earl had achieved great recognition as
the discoverer of cyclic AMP, the second messenger of
many hormones.
My project was to look at direct effects of insulin and
other hormones on the liver using the isolated perfused rat
liver preparation. However, the existing perfusion system
left a lot to be desired in that the oxygenation system was
immersed in a water bath for temperature control and
frequently leaked. The temperatures and flow rates of control and experimental livers were often not the same, and
there were other problems. When I arrived, a postdoc was
running the experiments, and he overlooked any perfusion
disasters by ignoring the apparatus while reading Life magazine. When it was discovered that he did not actually have
a Ph.D., he was dispatched back to England posthaste,
where he opened a candy store! The first order of business
was to develop a better perfusion apparatus, and this was
done with the assistance of Howard Morgan and Bailey
Moore, the skilled head of the apparatus shop. Howard
had a very pragmatic attitude and later became Chair of
Physiology at Penn State, President of the American Physiological Society, and President of the American Heart
Association. With the problems of the perfusion system
solved, I began a lengthy series of studies of the hormonal
control of gluconeogenesis and glycogen metabolism in
the isolated liver. In an interesting turn of events, this put
us in competition with the laboratory of Hans Krebs.
Gluconeogenesis occurs principally in the liver and
involves the formation of glucose from non-sugar sources
such as lactate, pyruvate, glycerol, and certain amino acids.
It is essential for life during starvation and recycles lactate
formed during exercise back to glucose (the Cori cycle).
My work with Rollo Park showed that physiological
increases in amino acids and other substrates could alone
increase gluconeogenesis, and measurements of metabolic
intermediates in the pathway revealed that the major ratelimiting step was the substrate cycle between pyruvate and
phosphoenolpyruvate (4, 5). Later, we showed that hormones such as glucagon and epinephrine could directly
stimulate gluconeogenesis in the liver by acting on this
cycle and that their effects were antagonized by insulin.
Work in several other laboratories subsequently showed
that the enzyme affected was pyruvate kinase. Diabetes

also stimulated the process, as did glucocorticoids, which
also exerted a permissive effect on the actions of glucagon
and epinephrine (5, 6). In collaboration with the Sutherland group, we determined that cyclic AMP played a major
role in the regulation of gluconeogenesis at the level of the
pyruvate-phosphoenolpyruvate cycle (7, 8). This work was
conducted with the help of two excellent associates, Leonard (Jim) Jefferson, a graduate student from Kentucky who
drove a Cadillac and ultimately became Chair of Physiology at Penn State Medical School and President of the
American Physiological Society, and Tom Miller, a perpetually good-natured technician who came from a small
town in West Tennessee and became Professor and Dean
of Graduate Studies at the University of Massachusetts
Medical School. These were superb experimentalists,
although I had to avoid scheduling complex experiments
on mornings after they had enjoyed the bars of Nashville.
Another important contributor was Sandy Harper, who
truly qualified for the title of super-tech. Supervising all
this work was the tall aristocratic Rollo Park, with his
impeccable heritage of clerics, generals, and academics.
He provided invaluable insights and conveyed an urbane
relaxed attitude in which issues such as research funding
and priority in publication were not of much concern.
When I complained that one of my papers was taking over
17 months to be submitted because of constant revising,
his response was, In the end, the best paper wins. Rollo
sent me out to look at many departmental chairmanships
despite my relatively young age because he believed that
any candidate older than 40 was over the hill!
A year after my arrival in Nashville, we were joined by
J. G. T. Sneyd, who was also from the Biochemistry
Department at Otago. He was nicknamed Sam after the
golfer Sam Snead, although he never played golf. He was a
brilliant guy but an inveterate prankster. A list of his shenanigans would occupy this entire essay, but I will report
only the episodes when he served rat liver pate to his
mother-in-law and staged a cricket match inside his house.
The ultimate Sneyd story was when his wife put an advertisement in the local newspaper in which she tried to sell
him plus his TV (both in working order) because of his
addiction to football during the holiday season. This was
taken up by countless news sources in the U. S. and
throughout the world and occasioned many TV and radio
interviews in which his wife played it straight!
The presence of Earl Sutherlands group gave a great
boost to my research, but discussions with him frequently
involved mental gymnastics because he would often
change topics mid-sentence. I remember talking about
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glycogen metabolism when he suddenly mentioned TG.

For the life of me I could not see the relevance of triglyceride, but he was referring to transglucosylase, an uncommon name for glycogen synthase! He was quite convinced
that cyclic GMP was the second messenger for insulin, and
this seemed quite logical. However, when Joel Hardman,
subsequently Chair of Pharmacology at Vanderbilt, and I
put this to the test in the perfused liver, the results were
negative (9). Everyone in the department recognized the
fundamental importance of Earls discovery of cyclic AMP
and waited each year for the call from Stockholm. However, when a crew from Swedish TV arrived in late October 1971 to film him and his laboratory, we knew his time
had finally arrived.
In 1968, I was promoted to Associate Professor and also
appointed (anointed) as an Investigator of the Howard
Hughes Medical Institute. At that time, Investigators
received only a stipend and a travel allowance, which
would get me to California and back. Nowadays, it would
get me only a one-way ticket to St. Louis! In 1976, under
pressure from the Internal Revenue Service, which was
perpetually suspicious of the Institute, it was compelled to
disburse more funds, and a cornucopia of equipment, supplies, and support for postdocs and technicians descended
upon my grateful head! The Institute even renovated the
lab using only first-rate materials. At that time, Hughes
Investigators lived in a form of research paradise that was
even better than it is now. Oversight was not very rigorous
and the annual reviews perfunctory. The Medical Advisory Board believed that once Investigators were
approved, they should be left to their own devices. All this
changed in 1978, when the reclusive Howard Hughes died,
and the Institute was restructured. Now, the review process became much more rigorous (terrifying), and they
actually terminated some Investigators. I survived the
review process and retired in 2004 as the longest tenured
Investigator. I felt like a living fossil among all the young
Investigators!
In 1970, I spent a sabbatical in Geneva at the Institut de
Biochimie Clinique, headed by the renowned diabetes
researcher Albert Renold. I worked primarily with his
gifted associate Bernard Jeanrenaud, and I was greatly
impressed by the charming and multilingual Albert and
the handsome and debonair Bernard. I studied the hormonal regulation of glycogenolysis and gluconeogenesis in
mouse liver, working with a superb graduate student,
Francoise Assimacopoulos-Jeannet. I depended on her
skill in adapting our procedures to this small animal. We
rented a nice villa in Geneva and took frequent trips to
France, Italy, and the rest of Switzerland to explore the
countryside and culture. One of my first conferences was

at a Swiss ski resort, where the Nobel Laureate Feodor
Lynen sat at the rear of the room on a large throne-like
chair and made audible derogatory comments about most
of the talks. I approached him with some trepidation after
my talk, but, thankfully, he was complimentary.
While I was in Geneva and after I returned, studies on
the regulation of gluconeogenesis by glucagon and insulin
continued using the perfused liver system. Some of this
work focused on amino acids as gluconeogenic substrates
and was carried out by Larry Mallette, a brilliant M.D./
Ph.D. student, and Michio Ui from Japan, who would later
become famous through his work on a Bacillus pertussis
toxin that inactivates Gi through ADP-ribosylation. Other
work studied the antagonistic action of insulin on the
effects of glucagon in the liver and revealed the primacy of
cyclic AMP changes in this interaction. A key player in this
work was Steve Lewis, an M.D. with enormous energy who
would try any experimental approach including perfusing
the livers backwards or using distilled water as the perfusion medium! It was difficult to restrain him from Geneva.
Soon after my return to Nashville, I switched our experimental system from the liver perfusion procedure to the
use of isolated rat liver parenchymal cells. After years of
persistence by my former New Zealand colleague Michael
Berry, structurally and metabolically intact liver cells
could now be prepared using collagenase. Now the many
perfusion apparatuses were used only to prepare these
hepatocytes. Although the use of hepatocytes greatly
increased the throughput of experiments and permitted
better controls, I was faced with a dilemma as follows.
Whereas all our data obtained using the hepatocytes supported a role for cyclic AMP in the action of glucagon on
gluconeogenesis and glycogen breakdown, this was not
the case for epinephrine. Try as we could, we were unable
to correlate the changes in these processes induced by this
catecholamine with increases in cyclic AMP (10 12). Our
collaborator was Al Robison, a member of the Sutherland
group who was a nocturnal worker and subsequently
Chair of Pharmacology at the University of Texas Medical
School at Houston. We would leave our samples on the
desk, and in the morning, the cyclic AMP measurements
would appear like magic! Our findings caused some consternation in the Sutherland group because the original
work leading up to the discovery of cyclic AMP involved
the effects of epinephrine on glycogen breakdown in dog
liver. This challenge to the dogma was resolved when it
was realized that there was a species difference. In dog
liver, the effects of epinephrine are mediated by -adrenergic receptors acting via cyclic AMP, whereas in rat
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liver, they are mediated by 1-adrenergic receptors acting
via Ca2. The work with epinephrine had a very long heritage starting with Claude Bernard, who found in the 19th
century that stimulation of the brain increased the blood
glucose level (piqure hyperglycemia), and Walter B. Cannon, whose equally classic work (fight or flight response)
showed that increases in blood glucose and other changes
occurred in response to activation of the sympathetic
nervous system. When Mahmoud El Refai, an Egyptian
postdoc, found that the rat liver receptors involved in glycogenolysis were of the 1-type, a competing group tried
to prevent us from publishing because it was contrary to
their incorrect findings! A collaborator in some of this
work was Craig Venter, who had not yet developed his
reputation as the enfant terrible of the sequencing world.
Work by Jim Putney, then at the Medical College of
Virginia, and other investigators utilizing salivary gland,
smooth muscle, and other tissues indicated that the 1-adrenergic mechanism involved a rise in intracellular Ca2,
and we soon confirmed this for rat liver. However, a problem arose because it was then believed that the increase in
Ca2 came from an influx through channels in the plasma
membrane, but our data indicated that it came from an
internal pool (12). A key collaborator in this project was
Peter Blackmore from Australia, who was nicknamed
Quokka after the small Australian marsupial. He was a
remarkably adept experimentalist and is now a Professor
of Physiological Sciences at the East Virginia Medical
School. Also involved were Nancy Hutson, a smart and
very personable graduate student who is now an executive
at Pfizer, and Francoise Assimacopoulos-Jeannet from
Geneva, who is now a Professor Titulaire at the Centre
Medical Universitaire there. The role of Ca2 in the
actions of 1-adrenergic and related agonists was demonstrated unequivocally by the use of Quin-2, a reagent
developed by Roger Tsien that measured free cytosolic
Ca2 (13). The postdoc involved in this work was Bob
Charest. He was a chain smoker, and I worried about the
effects of cigarette ash on the Ca2 measurements!
During this period, we also engaged in some experiments dealing with the effects of epinephrine and insulin
on glycogen metabolism and glucose uptake in skeletal
muscle using the isolated perfused rat hind limb preparation, and also the modulation of these metabolic processes
by adrenalectomy and thyroid status (14, 15). These studies were of value in the interpretation of related in vivo
experiments in man and experimental animals because
they were not confounded by secondary effects. The
importance of studying direct effects of hormones uncomplicated by in vivo changes also applied to our rat liver
perfusion studies. Interestingly, later experiments with in

vivo models largely confirmed our findings in the perfused
liver, thus establishing the validity of our system. JeanLouis Chiasson, an energetic researcher from Montreal,
Melissa Dietz (Lojek), a graduate student and ballet
dancer, and Mike Caldwell, a surgeon, were key to these
experiments.
With the ready availability of hepatocytes, we also conducted many studies examining factors that modulated
hormone effects on the liver. In an extension of our earlier
work indicating a species difference in the adrenergic
receptors involved in epinephrine action in the liver, we
found that age, adrenal cortical status, and gender also
influenced the extent to which epinephrine acted through
- versus -adrenergic receptors (16). Another finding
was that agonists acting through cyclic AMP enhanced
1-adrenergic responses, whereas insulin and phorbol
esters inhibited them (17, 18). These observations not only
cleared up some confusion in the literature, but also
involved some rather colorful postdocs. For example,
there was the energetic, likeable, but budget-busting Chris
Lynch, now a Professor of Physiology at the Hershey Medical School. His philosophy was to order what he might
possibly need rather than what he actually needed. Others
were Bernie Hughes, an Australian with an inexhaustible
store of risque stories, the charming Bernard Bouscarel
from Toulouse, who was notable because his girlfriends
sent him flowers, and Jean-Paul Dehaye from Brussels,
who was nicknamed The Pope after the pope at that
time, Jean Paul II, although he was not as infallible as the
pontiff! These characters were balanced by Tim Chan,
now a Dean at the University of Southern California, and
Noel Morgan, a steadfast Englishman who is now Head of
Biochemistry at a medical school in Cornwall. At this time,
we were visited by Fatima Bosch from Barcelona, whose
energetic pursuit of research and stylish clothing
impressed us greatly.
Studies of the mechanisms involved in the actions of
Ca2-mobilizing agonists reached an exciting phase in
which the hunt was on for the signal that came from the
receptor in the plasma membrane to the internal calcium
pool. Here we became misguided by our findings and
thought that the pool was in the mitochondria, a well
known source of Ca2. Other workers deduced correctly
that the pool was in the endoplasmic reticulum, and I had
to endure some abuse for our sins at several meetings. We
were also skeptical of the emerging phosphoinositide
hypothesis as the basis of signaling to the internal Ca2
store. This posited that the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by certain ago-
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nists generated a second messenger that released the

Ca2. Because we found that an increase in Ca2 stimulated the breakdown of this phospholipid in liver cells and
plasma membranes, we felt that the hypothesis was wrong.
In point of fact, we did not realize that there was a much
earlier phase in the action of the agonists when PIP2 breakdown preceded a rise in Ca2. Our questioning of the
phosphoinositide hypothesis raised the ire of the English
scientists, who were the main proponents. With the recognition of inositol 1,4,5-trisphosphate (IP3) as the intracellular messenger by Robin Irvine, Michael Berridge, and
associates, the controversy went away, except for some
diehards with lingering resentment!
Much evidence was accumulating that a G protein was
involved in the actions of many agonists to stimulate the
breakdown of PIP2 with the resultant mobilization of Ca2
from the endoplasmic reticulum (19, 20). This early work
was done by three American postdocs and one from
Croatia with very different personalities and communication skills: Thom Fitzgerald, who barely said a word, Janet
Atkinson (Colbran), who exuded Southern charm and volubility, Ron Uhing, who was not much more talkative than
Thom, and Vera Prpic, whose accent did not prevent a
marriage with Ron. Because none of the known G proteins
was found to be involved in the breakdown of PIP2, the
hunt was on for a novel G protein. We were successful in
isolating and purifying a G protein that activated the phospholipase C that hydrolyzed PIP2 (PI-PLC), but could not
identify it (21). At the same time, the group of Paul Sternweis at the University of Texas Southwestern Medical
School at Dallas had raised antibodies to two novel G proteins, but could not demonstrate that they activated PIPLC. So we joined forces to demonstrate that the novel G
proteins were in fact activators of the phospholipase and
hence the mediators of hormone action on intracellular
Ca2 mobilization (22). The two proteins were named Gq
and G11, and I am often asked why the Gq. It turns out that
a postdoc involved in the research in Pauls laboratory was
an avid reader of the Gentlemans Quarterly! A key player
in this research was Steve Taylor, a taciturn English postdoc who preferred to fly on British Airways because of the
quality of their gin and tonics. Karen Shaw, a spunky English postdoc, was also involved in this work, as was Iro
Georgoussi from Athens, whose vibrant personality and
social skills livened up the social life of the laboratory. In a
later extension of this work, Gita Venkatakrishnan localized the residues in the Gq -subunit that were specifically
involved in the activation of PI-PLC.
Two interesting side projects arose from this work. The
first was the confirmation by Jonathan Blank, another
English postdoc, of the finding of Peter Gierschik at the

University of Heidelberg that the -components of G
proteins could activate some forms of PI-PLC (23), a finding also reached by Ken Hardens group at the University
of North Carolina. At that time, the idea that -subunits
of G proteins could be involved in cell signaling was an
issue of great controversy, sometimes vicious, and Gierschik had had great difficulty in publishing his findings.
Another side project involved the discovery by Jonathan
Blank and Gabriel Berstein in Elliott Ross laboratory at
UT Southwestern that PI-PLC could stimulate the GTP
hydrolase (GTPase) activity of Gq (24). In other words, the
phospholipase could inactivate its G protein activator,
indicating a novel feedback mechanism. All this and other
work extending to 1999 were supported by the efforts of
Annette Ross, a truly superb technician. Three very competent secretaries, Penny Stelling, Carolyn Sielbeck, and
Judy Childs (Nixon), aided immensely in the preparation
of our publications during my tenure as a Hughes Investigator. After two terms on the Editorial Board of the Journal of Biological Chemistry, I was appointed an Associate
Editor in 1988. I was again lucky to have the superlative
services of two editorial assistants: Carolyn Sielbeck, doing
double duty as my secretary, and Carolyn McDonald,
whose gentle Southern voice calmed many an agitated
author.
The breakdown of PIP2 generates 1,2-diacylglycerol
(DAG) as well as IP3, and if the theory is correct, these
should be produced in equimolar amounts in cells in
which PI-PLC is activated by agonists. Imagine our chagrin when Steve Bocckino, another great postdoc, measured these compounds chemically and found that, in vasopressin-stimulated liver cells, the production of DAG
greatly exceeded that of IP3 and was much more prolonged (25). Now we were at odds with the experts in the
protein kinase C (PKC) area because it was believed that
the DAG that activated this kinase came from the breakdown of PIP2, making a nice story in which second messengers (IP3 and DAG) were simultaneously released in a
bifunctional signaling system. Further work confirmed
that PIP2 was breaking down but that another phospholipid was being hydrolyzed to a greater extent. Measurements of choline release by Helen Irving, an Australian
postdoc, and analyses of the fatty acid composition of the
DAG by Steve Bocckino and Guy Augert, a dashing postdoc and fearless skier from the Haute-Savoie region of
France, indicated that the phospholipid was phosphatidylcholine (PC) (26, 27). This was a surprise because PC is the
major phospholipid of cell membranes, and its breakdown
would be expected to have deleterious effects on the cell.
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However, chemical measurements of PC later showed that

the decrease induced by agonist stimulation was barely
detectable, although the resulting increase in DAG was
sufficient for signaling.
The next surprise came when we set out to determine
the phospholipase responsible for the breakdown of PC.
By analogy with the phosphoinositide signaling system, we
expected the phospholipase to be of the C type. However,
chemical measurements showed that the initial product of
PC breakdown was phosphatidic acid, which was then
converted to DAG. In other words, the phospholipase was
of the D type (PLD) (28). This was somewhat disconcerting because it was generally believed that this enzyme was
present in plants, but not animals! Julian Kanfers group in
Winnipeg had earlier obtained evidence of PLD in mammalian tissues, but their work had been largely ignored.
Once again, these findings were not welcomed by the aficionados of the phosphoinositide hypothesis of cell signaling. However, further work by others, including Claire
Allan, a Scottish postdoc with a delightful accent, showed
that both systems operated, i.e. agonists caused an initial
hydrolysis of PIP2, followed by a larger and more prolonged breakdown of PC.
Reverting back to our studies of PLD, it became clear
that a G protein played a major role in its regulation, but
none of the known heterotrimeric G proteins could be
shown to affect its activity. Then a report from the group
of David Lambeth at Emory appeared showing that
RhoGDI, an inhibitor of the activation of the small G protein Rho, inhibited the activation of PLD by guanosine
5-O-(3-thiotriphosphate) (GTPS) in membranes,
implying a role for members of the Rho family. Ken Malcom, an environmentally conscious postdoc, used recombinant forms of these proteins to show that they could
activate PLD directly (29). An important collaborator in
these studies was Marc Symons, then at Onyx Pharmaceuticals. There were also reports from the Sternweis and
Cockcroft laboratories that another small G protein, ADPribosylation factor (ARF), could also activate partially
purified PLD or when tested in permeabilized neutrophils.
We confirmed this and focused on factors involved in the
action of ARF. This led to the discovery of the novel proteins arfaptins 1 and 2 (30) and arfophilin (31). Interestingly, both arfaptins and arfophilin were found to bind
other families of small G proteins, implying the existence
of novel signaling networks. Two gifted postdocs,
Hiroyuki Kanoh from Japan and Ok-Ho Shin from Korea,
were responsible for this work.
Because Rho was a major regulator of PLD1 in vitro, we
were interested in examining its role in vivo. Steve Plonk,
an M.D./Ph.D. student, showed that introduction of the

C3 exoenzyme of Clostridium botulinum, which inhibits
Rho, inhibited the activation of PLD by lysophosphatidic
acid (LPA) and other G protein-linked agonists, but not
platelet-derived growth factor (PDGF) (32). On the other
hand, Jean Hess, a postdoc now living in rural Vermont,
found that Rac mediated the effect of growth factors. It
was also found that LPA induced the membrane translocation of Rho, but this did not occur with PDGF (33). The
G protein involved in the activation of Rho was shown by
ourselves and others to be G13 and not Gq, illustrating a
new signaling paradigm (34). Because heteromeric G proteins do not link directly to monomeric G proteins, we
next searched for the guanine nucleotide exchange factor
(GEF) involved in agonist activation of Rho family proteins. In collaboration with John Collards group in
Amsterdam, we showed that Tiam1, a GEF for Rac, was
translocated to membranes and phosphorylated on threonine in cells treated with LPA (35, 36). Both PKC and
Ca2/calmodulin-dependent kinase II were shown to
phosphorylate Tiam1, but only the calmodulin-dependent
enzyme induced activation (36). These findings did not
explain how G13 activated Rho (another group showed the
involvement of other GEFs), but pointed to a mechanism
for Rac activation. The postdocs principally involved in
this work were Ian Fleming, not related to the author of the
James Bond series, and Greg Buchanan, the ultimate University of Tennessee fan who wore orange almost
continuously!
Many groups had obtained evidence that PKC could
activate PLD in vivo. This included evidence by Eui-Ju Yeo,
a Korean postdoc in my lab, that PKC mediated the effect
of epidermal growth factor on the enzyme. However, a big
surprise came in in vitro experiments showing that PKC
could directly activate PLD but that this did not involve
phosphorylation, but merely protein-protein interaction
(37). This nontraditional view of how PKC acted was supported by the findings of several other groups, but was not
accepted by others. The lead postdoc in this work was
Kevin Conricode, who was notable in having seven brothers but no sisters. Our studies also showed that the activation of PLD1 by PKC involved the conventional - and
-isozymes, but not the other isozymes. Later work by two
Chinese postdocs, Tianhui Hu and Jun-Song Chen,
showed that PKC could phosphorylate PLD but that this
caused inactivation. This work on PKC led us to examine
the specific PKC isozymes activated by various agonists in
fibroblasts. Surprisingly, Kwon-Soo Ha, an energetic postdoc from Korea, found that -thrombin and PDGF caused
a differential translocation/activation of PKC isozymes in
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these cells. Thrombin caused a rapid membrane association of the - and -isozymes, which correlated with rapid
increases in IP3/Ca2 and DAG due to PIP2 breakdown.
On the other hand, PDGF did not cause a translocation of
PKC, but caused a delayed translocation of PKC that
correlated with an increase in DAG due to PC breakdown
(38). Our general interest in PKC led us to a study of the
atypical PKC isozyme, about which very little was known.
This first had to be isolated and purified before its regulation could be studied. Hiroyuki Nakanishi, a postdoc from
Kobe, examined the effects of various lipids and found the
enzyme to be the first known target of phosphatidylinositol 3,4,5-trisphosphate (39), a lipid now known to be generated by insulin and other growth factors and an important second messenger.
We next devoted a lot of effort to the purification, characterization, and cloning of mammalian PLD. However,
the cloning of the two mammalian (human) PLD1 and
PLD2 isozymes was first accomplished by the group of
Michael Frohman at the State University of New York at
Stony Brook based on their earlier cloning of yeast PLD.
Seung-Kiel Park from Korea, in association with Joe Provost, a colorful ex-Army man, cloned the rat liver enzymes
by an analogous approach (40), and this initiated an extensive program to characterize their structure, kinetics, and
regulation (41). Much of what we found paralleled the
findings of the Frohman group, and in contrast to my earlier research, no controversies arose between us! The
PLD1 isozyme was of particular interest because of its
complex regulation by PIP2, PKC, ARF, and members of
the Rho family of small G proteins (Rho, Rac, and Cdc42).
Furthermore, PKC and the Rho proteins were shown to
mediate the actions of certain agonists on the enzyme in
intact cells. Interestingly, in our early efforts to purify PLD
from rat brain, we inadvertently purified phospholipase A1
to homogeneity, and because little was known about this
enzyme, I encouraged the postdoc to characterize it. This
was Matthew Pete, an ex-Marine who tackled the project
like it was Iwo Jima!
PLD isozymes from most sources are characterized by
the presence of two highly conserved motifs, termed HKD,
which are required for catalytic activity. In PLD1, they are
separately located in the N and C termini. Zhie (Julie) Xie,
a gifted postdoc, ably assisted by Wan-Ting (Tina) Ho,
found that expression of one-half of the enzyme containing one HKD motif produced no catalytic activity, whereas
expression of both halves did (42, 43). The conclusion that
both HKD motifs were required to form a dimeric catalytic
center was borne out by subsequent structural studies.
Julie and Tina later found that a short C-terminal
sequence in mammalian PLDs was absolutely required for

activity. The reason for this remains unknown. Seunghyi
Kook and Do Sik Min, two postdocs from Korea, localized
the binding site of PKC on PLD1 to certain sequences in
the N terminus (44) while Songmin Cai from China identified the binding site for Rho in the C terminus (45). We
also did the reciprocal experiments. Thus, Tianhui Hu
identified a residue in PKC that was critical for PLD1
activation, and Chang Dae Bae from Korea identified residues in the activation loop of RhoA that were specifically
involved in activating the enzyme. Chang was a brilliant
postdoc, but was concerned about his health. He asked me
to move a freezer nearer to his bench because the distance,
not far, was straining his leg muscles! An interesting aspect
of the regulation of PLD1 is the striking synergism
between certain of its activators. In an analysis of this in
collaboration with Alex Brown and Lee Henage, a brilliant
graduate student, it was found that the synergism
occurred only if a catalytic activator such as ARF was combined with a binding activator such as one of the Rho proteins or a mixed activator such as PKC (46).
PLD is a ubiquitous enzyme that is a member of a superfamily that has been conserved through evolution. This
implies that it subserves some important functions. We
and others have identified a variety of cellular functions of
the PLD1 and PLD2 isozymes, but no underlying themes
have emerged, suggesting that the fundamental importance of PLD has yet to be defined. Another area of importance in PLD research is to determine the crystal structure
of the mammalian enzymes. The structures of the enzyme
from Streptomyces antibioticus and of some low molecular
mass members of the PLD superfamily have been defined
and have provided very useful information about the catalytic center and the mechanism of catalysis. However, the
reasons why certain isozymes are critically dependent on
PIP2 for activity and the molecular mechanisms by which
PKC, ARF, and Rho activate PLD1 remain unknown.
Thus, much more work needs to be done to define the
structure, regulation, and functions of these enzymes, the
distribution of which is so widespread.
Address correspondence to: john.exton@vanderbilt.edu.
REFERENCES
1. Batt, R. D., and Exton, J. H. (1956) The catabolism of dihydropyrimidines by rat
tissue preparations. Arch. Biochem. Biophys. 63, 368 375
2. Exton, J. H. (1964) Metabolism of rat-liver cell suspensions. 1. General properties of isolated cells and occurrence of the citric acid cycle. Biochem. J. 92,
457 467
3. Exton, J. H. (1964) Metabolism of rat-liver cell suspensions. 2. Fatty acid oxidation and ketone bodies. Biochem. J. 92, 467 472
4. Exton, J. H., and Park, C. R. (1965) Control of gluconeogenesis in the perfused
liver of normal adrenalectomized rats. J. Biol. Chem. 240, PC955PC957
H106
5. Exton, J. H., and Park, C. R. (1967) Control of gluconeogenesis in liver. I.

General features of gluconeogenesis in the perfused livers of rats. J. Biol. Chem.
242, 26222636
6. Jefferson, L. S., Exton, J. H., Butcher, R. W., Sutherland, E. W., and Park, C. R.
(1968) Role of adenosine 3,5-monophosphate in the effects of insulin and
anti-insulin serum on liver metabolism. J. Biol. Chem. 243, 10311038
7. Exton, J. H., and Park, C. R. (1968) Control of gluconeogenesis in liver. II.
Effects of glucagon, catecholamines, and adenosine 3,5-monophosphate on
gluconeogenesis in the perfused rat liver. J. Biol. Chem. 243, 4189 4196
8. Exton, J. H., and Park, C. R. (1969) Control of gluconeogenesis in liver. III.
Effects of L-lactate, pyruvate, fructose, glucagon, epinephrine, and adenosine
3,5-monophosphate on gluconeogenic intermediates in the perfused rat liver.
J. Biol. Chem. 244, 1424 1433
9. Exton, J. H., Hardman, J. G., Williams, T. F., Sutherland, E. W., and Park, C. R.
(1971) Effects of guanosine 3,5-monophosphate on the perfused rat liver.
J. Biol. Chem. 246, 2658 2664
10. Hutson, N. J., Brumley, F. T., Assimacopoulos, F. D., Harper, S. C., and Exton,
J. H. (1976) Studies on the -adrenergic activation of hepatic glucose output. I.
Studies on the -adrenergic activation of phosphorylase and gluconeogenesis
and inactivation of glycogen synthase in isolated rat liver parenchymal cells.
J. Biol. Chem. 251, 5200 5208
11. Cherrington, A. D., Assimacopoulos, F. D., Harper, S. C., Corbin, J. D., Park,
C. R., and Exton, J. H. (1976) Studies on the -adrenergic activation of hepatic
glucose output. II. Investigation of the roles of adenosine 3:5-monophosphate
and adenosine 3:5-monophosphate-dependent protein kinase in the actions
of phenylephrine in isolated hepatocytes. J. Biol. Chem. 251, 5209 5218
12. Assimacopoulos-Jeannet, F. D., Blackmore, P. F., and Exton, J. H. (1977) Studies
on the -adrenergic activation of hepatic glucose output. III. Studies on the role
of calcium in the -adrenergic activation of phosphorylase. J. Biol. Chem. 252,
26622669
13. Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) Changes in
free cytosolic Ca2 in hepatocytes following 1-adrenergic stimulation. Studies
on Quin-2-loaded hepatocytes. J. Biol. Chem. 258, 8769 8773
14. Dietz, M. R., Chiasson, J.-L., Soderling, T. R., and Exton, J. H. (1980) Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the
perfused rat hindlimb preparation. J. Biol. Chem. 255, 23012307
15. Chiasson, J.-L., Dietz, M. R., Shikama, H., Wootten, M., and Exton, J. H. (1980)
Insulin regulation of skeletal muscle glycogen metabolism. Am. J. Physiol. 239,
E69 E74
16. Blackmore, P. F., Assimacopoulos-Jeannet, F. D., Chan, T. M., and Exton, J. H.
(1979) Studies on -adrenergic activation of hepatic glucose output. Insulin
inhibition of -adrenergic and glucagon actions in normal and calcium-depleted hepatocytes. J. Biol. Chem. 254, 2828 2834
17. Morgan, N. G., Blackmore, P. F., and Exton, J. H. (1983) Age-related changes in
the control of hepatic cAMP levels by 1- and 2-adrenergic receptors in male
rats. J. Biol. Chem. 258, 51035109
18. Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Potentiation of 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.
Proc. Natl. Acad. Sci. U. S. A. 81, 4208 4212
19. Uhing, R. J., Prpic, V., Jiang, H., and Exton, J. H. (1986) Hormone-stimulated
polyphosphoinositide breakdown in rat liver plasma membranes. Roles of guanine nucleotides and calcium. J. Biol. Chem. 261, 2140 2146
20. Taylor, S. J., and Exton, J. H. (1987) Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements. Biochem. J. 248,
791799
21. Taylor, S. J., Smith, J. A., and Exton, J. H. (1990) Purification from bovine liver
membranes of a guanine nucleotide-dependent activator of phosphoinositidespecific phospholipase C. Immunologic identification as a novel G protein
subunit. J. Biol. Chem. 265, 17150 17156
22. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Activation of the 1
isozyme of phospholipase C by purified subunits of the Gq class of G proteins.
Nature 350, 516 518
23. Blank, J. L., Shaw, K., Ross, A. H., and Exton, J. H. (1993) Purification of a
110-kDa phosphoinositide phospholipase that is activated by G-protein subunits. J. Biol. Chem. 268, 25184 25191
24. Berstein, G., Blank, J. L., Jhon, D.-Y., Exton, J. H., Rhee, S. G., and Ross, E. M.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
H107
(1992) Phospholipase C-1 is a GTPase-activating protein for Gq/11, its physiological regulator. Cell 70, 411 418
Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) Stimulation of 1,2diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and
angiotensin II. J. Biol. Chem. 260, 1420114207
Irving, H., and Exton, J. H. (1987) Phosphatidylcholine breakdown in rat liver
plasma membranes. Roles of guanine nucleotides and P2-purinergic agonists.
J. Biol. Chem. 262, 3440 3443
Augert, G., Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1989) Hormonal
stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown. J. Biol. Chem. 264, 21689 21698
Bocckino, S. B., Blackmore, P. F., Wilson, P.B., and Exton, J. H. (1987) Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D
mechanism. J. Biol. Chem. 262, 15309 15315
Malcolm, K. C., Ross, A. H., Qiu, R.-G., Symons, M., and Exton, J. H. (1994)
Activation of rat liver phospholipase D by the small GTP-binding protein
RhoA. J. Biol. Chem. 269, 2595125954
Kanoh, H., and Exton, J. H. (1997) Arfaptin 1, a putative cytosolic target protein
of ADP-ribosylation factor, is recruited to Golgi membranes. J. Biol. Chem. 272,
54215429
Shin, O.-K., Ross, A. H., Mihai, I., and Exton, J. H. (1999) Identification of
arfophilin, a target protein for GTP-bound class II ADP-ribosylation factors.
J. Biol. Chem. 274, 36609 36615
Malcolm, K. C., Elliott, C. M., and Exton, J. H. (1996) Evidence for Rho-mediated agonist stimulation of phospholipase D in Rat1 fibroblasts. Effects of Clostridium botulinum C3 exoenzyme. J. Biol. Chem. 271, 1313513139
Fleming, I. N., Elliott, C. M., and Exton, J. H. (1996) Differential translocation of
Rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J. Biol. Chem. 271, 3306733073
Plonk, S. G., Park, S.-K., and Exton, J. H. (1998) The -subunit of the heterotrimeric G protein G13 activates a phospholipase D isozyme by a pathway requiring Rho family GTPases. J. Biol. Chem. 273, 4823 4826
Buchanan, F. G., Elliot, C. M., Gibbs, M., and Exton, J. H. (2000) Translocation
of the Rac1 guanine nucleotide exchange factor Tiam1 induced by plateletderived growth factor and lysophosphatidic acid. J. Biol. Chem. 275, 97429748
Fleming, I. N., Elliott, C. M., Buchanan, F. G., and Exton, J. H. (1998) Ca2/
calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein
phosphorylation. J. Biol. Chem. 274, 1275312758
Conricode, K. M., and Exton, J. H. (1992) Activation of phospholipase D by
protein kinase C. Evidence for a phosphorylation-independent mechanism.
J. Biol. Chem. 267, 7199 7202
Ha, K.-S., and Exton, J. H. (1993) Differential translocation of protein kinase C
isozymes by thrombin and platelet-derived growth factor. J. Biol. Chem. 268,
10534 10539
Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) Activation of the isoform
of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem.
268, 1316
Park, S.-K., Provost, J. J., Bae, C. D., Ho, W.-T., and Exton, J. H. (1997) Cloning
and characterization of phospholipase D from rat brain. J. Biol. Chem. 272,
2926329271
Min, D. S., Park, S.-K., and Exton, J. H. (1998) Characterization of a rat brain
phospholipase D isozyme. J. Biol. Chem. 273, 7044 7051
Xie, Z., Ho, W.-T., and Exton, J. H. (1998) Association of N- and C-terminal
domains of phospholipase D is required for catalytic activity. J. Biol. Chem. 273,
34679 34682
Xie, Z., Ho, W.-T., and Exton, J. H. (2000) Association of the N- and C-terminal
domains of phospholipase D. Contribution of the conserved HKD motifs to the
interaction and the requirement of the association for Ser/Thr phosphorylation
of the enzyme. J. Biol. Chem. 275, 2496224969
Park, S.-K., Min, D. S., and Exton, J. H. (1998) Definition of the protein kinase C
interaction site of phospholipase D. Biochem. Biophys. Res. Commun. 244,
364 367
Bae, C. D., Min, D. S., Fleming, I. N., and Exton, J. H. (1998) Determination of
interaction sites on the small G protein RhoA for phospholipase D. J. Biol.
Chem. 273, 11596 11604
Henage, L. G., Exton, J. H., and Brown, H. A. (2006) Kinetic analysis of phospholipase D. J. Biol. Chem. 281, 3408 3413
REFLECTIONS

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 50, pp. 34469 34478, December 11, 2009
Authors Choice
From the -Glutamyl Cycle to

the Glycan Cycle: A Road
with Many Turns and
Pleasant Surprises
Published, JBC Papers in Press, October 19, 2009, DOI 10.1074/jbc.X109.023150
Naoyuki Taniguchi
From the Department of Disease Glycomics, Institute of Scientific and Industrial Research, Osaka
University, Ibaraki, Osaka 567-0047 and the Systems Glycobiology Research Group, Chemical
Biology Department, Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
s I have written previously about my personal history and research journey since
graduating from medical school (1), in this article, I wish to focus on the biochemistry
of glutathione and glycosyltransferases, which, on the surface, would appear to be
quite different subjects.
Early Training in Biochemistry and Studies of Glutathione Metabolism during

Hepatocarcinogenesis
After obtaining an M.D. and finishing an internship at the university hospital in 1965, my
early training in research was obtained in the Department of Biochemistry, Hokkaido University Graduate School of Medicine, where I received a Ph.D. degree in 1972. At that time, the
department was chaired by Professor Hidematsu Hirai, and the focus of the departmental
research effort was primarily on the biological and clinical significance of -fetoprotein, an
oncofetal protein that is synthesized by embryonic tissues and primary hepatomas but not by
normal tissues (2). I had already developed an interest in enzymology, and I also became
interested in an area that was very different from the mainstream program in the department.
I decided to focus on the enzymes involved in glutathione metabolism during experimental
hepatocarcinogenesis of rats that had been fed 0.06% 3-methyl-4-dimethylaminoazobenzene
under the guidance of Dr. Yutaka Tsukada. In these studies, rats were killed at weekly intervals, and the glutathione levels and glutathione-related enzymes were assayed spectrophotometrically. The experiments started early in the morning and usually ended at around midnight. I found that the activity of -glutamyl transpeptidase, one of the glutathione-degrading
enzymes, in rats the fed azo dye underwent two biphasic changes, i.e. the activity was
increased at an early stage of hepatocarcinogenesis, then decreased, and finally increased
again at the late stage. Similar changes in the pattern for -fetoprotein had been reported by
a group in the same department. The actual level of glutathione decreased at the early stage,
then increased, and again decreased at the late stage, mirroring the pattern observed for
-glutamyl transpeptidase (3).
I hypothesized that -glutamyl transpeptidase might also be an oncofetal protein, analogous to
-fetoprotein. In fact, -glutamyl transpeptidase activity is high in fetal tissues, but the enzyme is
also ubiquitously distributed and abundant in the kidney.
After receiving my Ph.D., I spent one year as a research fellow in the same laboratory and then
moved to the Department of Hygiene and Preventive Medicine.
DECEMBER 11, 2009 VOLUME 284 NUMBER 50

H108
REFLECTIONS: From the -Glutamyl Cycle to the Glycan Cycle
FIGURE 1. -Glutamyl cycle proposed by Alton Meister (12). The cycle is involved primarily in the turnover of glutathione, i.e. glutathione
synthesis and degradation. AA, amino acid.
-Glutamyl Cycle and Alton Meisters Laboratory

in New York
In 1974, Alton Meister, Professor and Chair of the
Department of Biochemistry at the Cornell Medical
School, presented a plenary lecture on the -glutamyl
cycle at the annual meeting of the Japan Biochemical Society held in Okayama, Japan. He was agreeable to my working with him in New York, and I joined his group as a
visiting associate professor on September 1, 1976. At that
time, there were many postdoctoral fellows and faculty
members in Meisters laboratory, and they published
numerous excellent papers on glutathione metabolism,
including the chemical synthesis of buthionine sulfoximine, a specific inhibitor of -glutamylcysteine synthetase; the chemical synthesis of glutathione esters that are
able to enter the cell; the feedback inhibition of -glutamylcysteine synthetase; and the characterization of metabolic disorders of glutathione via the -glutamyl cycle,
first thought to play a role in amino acid transport but later
shown to be largely involved in the turnover of glutathione
(Fig. 1) (4, 5). He was a heavy smoker, so I could soon
recognize him by the smell of his cigars and pipes when he
showed up in the lab and when I visited him to discuss
research progress in his office on the first floor.
Glutathione is synthesized via two enzymes, glutathione synthetase and -glutamylcysteine synthetase, both of
which require ATP. Likewise, the degradation of glutathione is catalyzed by two enzymes, -glutamyl transpeptidase and -glutamylcyclotransferase. I was involved in the
purification of -glutamylcyclotransferase from rat kidney, which was highly heterogeneous because of sulfhydryl
modifications of the enzyme.
Hokkaido University and Cancer-associated Changes

in Glycoproteins: A Saints Maid Quotes Latin
I was supposed to become an associate professor in a
newly launched department, i.e. the Graduate School of
Environmental Science at Hokkaido University. I was
obliged to return to Japan, but the research situation was
not well organized, and I became interested in moving to
another laboratory. Half a year later, Professor Akira
Makita kindly offered me a position of associate professor
in the Cancer Institute of the same university. His group
discovered various cancer-associated changes in various
enzymes such as arylsulfatases (6), -glucuronidase, hexokinases, cathepsin D, and galactosyltransferase-1 and glycolipids such as sulfatides. Some of the work on sulfatides
was subsequently carried out by Koichi Honke, who purified sulfatide synthase, cloned the cDNA and gene, pro-
H109
VOLUME 284 NUMBER 50 DECEMBER 11, 2009
duced null mice, and discovered many interesting phenotypes (7). He later joined our group as an associate
professor and was a valuable member of our department.
When I first joined the Makita laboratory, I was not familiar with glycoprotein or glycolipid research, and I was just
like a saints maid quoting Latin. Finally, however, I
learned many techniques that subsequently proved to be
useful in the area of glycobiology and published several articles under his guidance. I enjoyed my tenure as an associate
professor in his group very much, as well as my interactions
with the various graduate students in the group. I also published several papers on non-enzymatic glycosylation (glycation), the Maillard reaction, in relation to free radical research
or diabetes, which subsequently became the other project of
my research journey when I moved to Osaka University (8).
While there, I was able to restart experiments on -glutamyl transpeptidase. At that time, several glycolytic
enzymes such as aldolase C, hexokinase, and pyruvate
kinase had been reported to be activated in cancer and
fetal and muscle tissues, and isozymic forms such as the
muscle type were present in hepatoma tissues. Even
though my preliminary experiments on -glutamyl
transpeptidase suggested that isozymic forms were not
present, I nevertheless continued to wonder if a specific
isozymic form of -glutamyl transpeptidase was also activated in hepatomas. -Glutamyl transpeptidase is a glycoprotein and was first purified from bovine kidney by Meisters group. Therefore, I was interested in purifying the
enzymes again from various rat tissues such as fetal, kidney, normal liver, and tumor tissues with the objective of
comparing their biochemical properties as judged by
kinetic properties, isoelectric focusing patterns, antigenicity, and amino acid analysis. The only difference we found
was in the sugar composition, i.e. the levels of neutral sugars and sialic acid, as the isoelectric focusing patterns varied before and after treatment with sialidase (9, 10). Since
then, I decided to focus on the sugar chains of the enzyme.
In collaboration with Professor Akira Kobata (Kobe
University), an eminent glycobiology expert, we found
that the enzyme purified from ascites hepatoma AH-66
cells contained bisecting GlcNAc, whereas the enzyme
purified from normal liver did not (11).
Osaka University Medical School, Department of

Biochemistry: From Protein Chemistry to Glycobiology (Purification of Glycosyltransferases) and from
Gene Cloning to Functional Glycomics
In February 6, 1986, I received an unexpected phone call
from the dean of the Osaka University Medical School,
who told me that I was a candidate for the position of
Professor and Chair of the Department of Biochemistry,
one of the oldest and most prestigious biochemistry

departments in Japan. The First Chair of the department
was Professor Yashiro Kotake, who elucidated the chemistry of kynurenine and was an eminent biochemist and an
expert in tryptophan metabolism. The Third Chair was
Professor Osamu Hayaishi, a world-renowned and eminent biochemist famous for his pioneering work on oxygenase and prostaglandins. I was very happy and honored
to accept this position and subsequently moved to Osaka
on April 1, 1986. I decided to change my project from
protein chemistry to glycobiology in this new department.
Purification of Glycosyltransferases and Gene Cloning
As 1988 was the 100th anniversary of the discovery of
glutathione, I co-organized a meeting on the Glutathione
Centennial: Molecular Perspectives and Clinical Implications with Dr. Yukiya Sakamoto and his group. Alton
Meister also attended the conference as an organizer. At
the meeting, he comprehensively reviewed the history of
glutathione and its metabolism, designated as the -glutamyl cycle (12), as shown in Fig. 1. His group had been
involved in studies in this area for the last 10 years or so.
We reported on the purification and cDNA cloning of
human -glutamyl transpeptidase and also reported on
the significance of highly activated UDP-N-acetylglucosamine:-D-mannoside 1,4-N-acetylglucosaminyltransferase III (GnT-III), which may modify -glutamyl
transpeptidase in hepatoma tissues and hyperplastic nodules. In 1988, we also reported on a marked activation of
GnT-III activity in primary hepatoma and ascites hepatoma, which could explain the unique changes in the sugar
units attached to -glutamyl transpeptidase, i.e. the addition of a bisecting GlcNAc, a product of GnT-III.
In 1992, we organized the Sapporo Cancer Seminar,
which was founded by Dr. Hiroshi Kobayashi, Professor
Emeritus of Hokkaido University; this seminar is a Gordon
Conference-like meeting that focuses on cancer research.
I invited Alton Meister, Owen Griffith, Susumu Nishimura, Cecil Pickets, Harold Deutsch, and other distinguished speakers who were actively involved in glutathione research, redox regulation, and nitric oxide and
reactive oxygen species (Fig. 2). Immediately after the
meeting, we celebrated Altons 70th birthday in Osaka,
and many Japanese scientists, good friends of his or former
colleagues, attended. Since Yoshitaka Ikeda in our group
joined Altons laboratory in 1993, we continued the work
on the catalytic properties of -glutamyl transpeptidase in
a collaborative effort, which focused mainly on studies of
the catalytic mechanism of the enzyme by site-directed
mutagenesis. Two years later, in 1995, Alton suffered a
stroke during a trip to New Zealand, and after returning to
H110
FIGURE 2. 12th Sapporo Cancer Seminar in 1992. Shown are Alton Meister, to the left of me (front row, fourth from the right), and Hiroshi Kobayashi,
to the left of Meister.
New York, he passed away in December at the age of 72.

The final paper that Alton actually handled was ours,
which appeared in the Journal of Biological Chemistry in
September 1995 (13); this article was the culmination of
his work. Altons death was a great loss not only for glutathione metabolism but also for biochemistry in the world.
His enormous contributions to our understanding of glutathione metabolism through the concept of the -glutamyl cycle (Fig. 1) and amino acid metabolism will remain
forever as his legacy. Our data on the catalytic mechanism
of -glutamyl transpeptidase, including the work with
Alton, were published (14).
Starting in 1986, we began to purify the glycosyltransferase, which catalyzes the biosynthesis of the bisecting
GlcNAc found in -glutamyl transpeptidase purified from
ascites hepatoma AH-66 cells. At that time, the method
used in assaying glycosyltransferases involved the incorporation of 14C- or 3H-labeled nucleotide sugars into the
acceptor substrate, but this was a time-consuming and
rather expensive method. Therefore, we attempted to
develop a simpler, less costly method of analysis. The
groups of Drs. Hase and Ikenaka at Osaka University

developed a method involving the pyridylamination of the
nonreducing end of glycans to analyze sugar chains.
Atsushi Nishikawa and co-workers (15) successfully employed this technique to assay the activity of various glycosyltransferases such as GnT-III, -IV, -V, and -VI. Using
this assay method, we measured the GnT-III activity of
various hepatoma cells and azo dye-induced hepatomas
and found that the enzyme activity was substantially elevated in most hepatomas. In some cases, the activity was
100-fold greater than that in a normal rat liver.
To obtain a cDNA clone, we attempted to purify GnTIII by affinity chromatography using biantennary sugar
chains, the substrate for GnT-III. We first prepared a large
amount of bisected biantennary sugar chains from human
serum transferrin. Because GnT-III activity is very high in
rat kidney, we collected a large number of kidneys from
rats that had been killed during physiological experiments
in other laboratories. We ultimately collected 10 kg of rat
kidneys. The enzyme was purified 153,000-fold in 1.5%
yield from a Triton X-100 extract of rat kidneys by frac-
H111
FIGURE 4. Glycosyltransferases involved in N-glycan branching

characterized by our group. The enzymes in boxes indicate the names
of glycosyltransferases purified and their cDNAs cloned by our group.
GnT-IX was cloned based on the genome sequence.
FIGURE 3. Dr. Schachters laboratory in Toronto (from left to right):

Jim Dennis, Harry Schachter, Naoyuki Taniguchi, and Jeremy P.
Carver.
tionation procedures utilizing QAE-Sepharose, Cu2chelating Sepharose, and affinity chromatography on

UDP-hexanolamine and substrate-conjugated Sepharose,
and finally, the cDNA encoding GnT-III was successfully
cloned (16). The enzyme is responsible for the formation
of a bisecting glycan, as first published by Dr. Harry
Schachter, who reported the presence of numerous
N-acetylglucosaminyltransferases such as GnT-I, -II, -III,
-IV, -V, and -VI. His group purified GnT-I and GnT-II for
the first time and then cloned their genes (17). His contribution in this field has been enormous, and I met Harry for
the first time in 1989, when Dr. Makita organized the Second Sapporo Cancer Seminar at a time when I was a new
comer in this field. Since then, every time I met Harry, he
gave me valuable suggestions and encouragement in our
work on glycosyltransferases even though his lab has been
our major competitor in this area because very few groups
have attempted to purify and biochemically characterize
the enzyme and then clone the gene (Fig. 3).
We further purified various glycosyltransferases involved in N-glycan branch biosynthesis one by one and
cloned their genes; these included GnT-V (18) and GnTIV, GnT-VI, synthetic enzyme for poly-N-acetyllactosamine, and 1,6-fucosyltransferase (FUT8) (19, 20).
Our group also cloned other glycosyltransferases using
molecular cloning techniques as depicted in Fig. 4. In 1989,
I organized the first glycosyltransferase meeting in Osaka,
and numerous eminent scientists in this field, including
Robert Hill, Pamela Stanley, Jamey Marth, Gerald Hart,
Ajit Varki, Jurgen Roth, Minoru Fukuda, Laurence Tabak,
Andre Verbert, John Lowe, and many renowned Japanese
glycobiologists, attended, and the significance of glycosyltransferases was discussed. Last year, the sixth meeting
(now called Glyco T) was organized by Richard Cummings
and Michael Pierce and held in Atlanta.
Functional Glycomics Approach and Lessons from GnTIII TransfectantsUsing the cloned glycosyltransferase
genes, we next initiated studies related to functional glycomics using sugar-remodeling techniques by gene transfections, knock-out and/or transgenic mice, and small
interfering RNA. We were able to show various functional
changes in glycans as the result of aberrant glycosylation
(2124) using the cloned glycosyltransferases. Moreover,
to utilize a functional glycomics approach, we emphasized
the importance of identifying the target protein(s) of the
glycosyltransferase genes. At that time, some researchers
asked, What would happen if you were able to identify
one or two target proteins among the numerous glycosylated proteins that could undergo glycosylation? This is
analogous to pointing out one star among the universe.
However, we now all realize that, even though the knockout of a certain glycosyltransferase gene may lead to various phenotypic changes due to a lack of glycan in various
glycoproteins, in fact, phenotypic changes accompanied
by functional changes in glycans are very limited. This
indicates that the identification of target protein(s) is very
important in characterizing glycan functions. In 2002, I
co-edited and published a handbook in which most of the
biochemical and molecular properties of glycosyltransferases and related genes are described (25). Our major
glycosyltransferases of interest are depicted in Fig. 4.
As we observed previously, -glutamyl transpeptidase
was one of the target proteins for GnT-III. To explore the
other functions of the GnT-III gene and its target proteins,
our group conducted experiments using cells that had
been transfected by the GnT-III gene and found different
types of likely target proteins as follows (2124). K562 cells
are usually killed by the immune systems in the spleen, but
GnT-III-transfected K562 cells are not. This is due to a
decreased sensitivity to natural killer cell cytotoxicity,
which leads to increased spleen colonization by these cells
in athymic mice. We identified target proteins that recognize bisecting GlcNAc, a product of GnT-III, as annexin V
and apoprotein B.
H112
FIGURE 5. Competitive reaction between GnT-III and GnT-V. Because GnT-III and GnT-V act on the same substrate but GnT-III acts on the substrate
first and produces a bisecting GlcNAc structure, GnT-V is not able to act further. This leads to a suppression of the metastatic potential in vivo.
Computer modeling of these reactions provided support for the this hypothesis.
Epidermal growth factor receptors (EGFRs) also contain N-glycans, and several researchers have proposed
that they play a role in growth factor signaling. The
deletion of Asn241 from the EGFR permits the molecule
to dimerize spontaneously without phosphorylation
(26). Erb-2 glycans play a key role in growth factor signaling because, without glycans, Erb-2 dimerizes without phosphorylation and enhances tumor development
in vitro (27). In GnT-III-transfected glioma cells, EGF
binding was blocked, and autophosphorylation of the
EGFR occurred. In HeLa cells, GnT-III transfection inhibited the low affinity binding of EGF to the EGFR but
enhanced the high affinity binding and the internalization
rate of the EGFR. The transfection of GnT-III further
resulted in 1) the inhibition of the growth factor response
of tyrosine phosphorylation of the Trk/nerve growth factor receptor following the addition of nerve growth factor,
2) the inhibition of neurite growth induced by co-stimulation of EGF and integrins, and 3) the inhibition of EGFRmediated extracellular signal-regulated kinase activation.
The target proteins of GnT-III responsible for these effects
were identified as the EGFR and integrin 31. CD44
obtained from GnT-III-transfected mouse melanoma
cells exhibited increased adhesion to hyaluronate, which
enhanced CD44-mediated tumor growth and metastasis
in the spleen after subcutaneous inoculation into mice.
The 13Gal epitope is a xenotransplantation antigen
absent in humans. Thus, in the case of xenotransplantation from pigs to apes or humans, subacute rejection will
occur because of the presence of an antibody against the
Gal epitope in humans and apes. Interestingly, the transfection of GnT-III into pig cells or the generation of GnTJOURNAL OF BIOLOGICAL CHEMISTRY
III transgenic pigs markedly reduced the Gal epitope by

inhibiting 13Gal-transferase action, thus reducing its
antigenicity.
GnT-III and GnT-V Have Opposite Effects on Cancer
Metastasis and Cell AdhesionGnT-III serves as a STOP
signal for other glycosyltransferases such as GnT-II, -IV,
and -V, and once a bisecting GlcNAc is incorporated,
those enzymes no longer act. Therefore, a high expression
of GnT-III would be expected to inhibit the synthesis of
multiantennary complex carbohydrates and to evoke various phenotypic alterations reflecting abolished glycan
functions. Gu et al. (18) confirmed this substrate specificity using purified GnT-V and demonstrated that GnT-V
does not act on a bisected biantennary chain. This catalytic
function was originally reported by Schachter et al. (17)
using a crude enzyme preparation. B16-hm cells, in which
GnT-V is highly expressed, therefore leading to increased
1,6 branching, have a high metastatic potential. In contrast, lung metastasis in syngeneic and athymic mice after
an intravenous injection of B16-hm cells transfected with
the gene encoding GnT-III was dramatically suppressed
(28). GnT-V catalyzes the addition of a 1 6 linkage to
the Man1 6 residue of GlcNAc12Man1 6Man
between the arm of the N-glycan core. In the GnT-III
transfectants, the level of 1,6 branches is decreased due
to competition for the substrate by intrinsic GnT-V and
ectopically expressed GnT-III (Fig. 5). The underlying
mechanism by which lung metastasis is suppressed is due
to elevated E-cadherin levels on cell-cell borders. Upon
overexpression of GnT-III in melanoma cells, E-cadherin
remains on the cell-cell border and enhances the attachment of cancer cells to protect cancer metastasis. As
H113

E-cadherin-catenin complexes are quite stable, the
increased E-cadherin levels result in the complete downregulation of the tyrosine phosphorylation of -catenin
after EGF treatment (23, 24).
Conversely, the disruption of E-cadherin-mediated cell
adhesion appears to be a central event in the transition
from noninvasive to invasive carcinomas. Interestingly, we
recently found that E-cadherin-mediated cell-cell interaction up-regulated GnT-III expression, suggesting that the
regulation of GnT-III and E-cadherin expression may represent a positive feedback loop and may be implicated
in endothelial-mesenchymal transition (29). E-cadherin
contains four N-glycan sites, and the role of glycans in
terms of cell adhesion is now well recognized.
The increased GnT-V levels were attributed to the upregulation of the Ets family of transcriptional activators,
which increase GnT-V expression. Knock-out mice of
Mgat5, encoding GnT-V, develop into apparently normal
adults, but the incidences of polyoma virus middle T oncogene-induced mammary tumor growth and metastases
are considerably lower in knock-out mice than in transgenic littermates. N-Glycans bearing the GnT-V branch
amplify oncogene signaling of tumor growth in vivo (30).
Integrins, crucial molecules for cell adhesion, have been
shown to be the major N-glycan-containing molecules on
cell membranes. For example, integrin 51 contains 14
and 12 putative N-glycosylation sites on the 5 and 1
subunits, respectively. Introduction of a bisecting GlcNAc
to N-glycans of integrin 5 by the overexpression of GnTIII diminished ligand binding, cell adhesion, and cell
migration. Contrary to the functions of GnT-III, the overexpression of GnT-V promoted integrin 51-mediated
cell migration (31). Indeed, the opposing effects of GnT-III
and GnT-V have been observed for the same target protein, integrin 31. N-Glycosylation of the -propeller
domain on the 5 subunit (32) and the I-like domain on
the 1 subunit is essential to both the heterodimerization
and biological functions of the subunits. In addition,
among many N-glycosylation sites of the 5 subunit, only
N-glycosylation site-4 on the -propeller was specifically
modified by GnT-III, thereby regulating integrin 51mediated cell spreading and cell migration.
Our group identified matriptase as a target protein for
GnT-V and showed that the overexpression of GnT-V
results in the increased expression of matriptase due to the
resistance of matriptase to autodegradation. Matriptase is
known to be associated with cancer invasion and metastasis, and its expression is enhanced in some types of tumor
cells. Moreover, GnT-V may function as an inducer of
angiogenesis, thereby promoting metastasis (2224).
TIMP-1 was identified as a target protein for GnT-V. The

poly-N-acetyllactosamine chains on GnT-V-dependent
N-glycan antennae serve as ligands for galectin-3, and it
has been suggested that GnT-V strengthens the galectin3/glycoprotein lattice on the T-cell receptor, thereby
restricting the recruitment of T-cell receptors to the site of
antigen presentation and impeding antigen-dependent
receptor clustering and signal transduction (33).
Core Fucosylation of Glycoproteins Is Implicated in
Growth, Lung Emphysema, Antibody Therapy, and a Cancer BiomarkerL-Fuc:N-acetyl--D-glucosaminide 1,6fucosyltransferase (FUT8) catalyzes the transfer of a
fucose residue from GDP-Fuc to position 6 of the innermost GlcNAc residue (core fucose residue) of an N-linked
sugar hybrid and complex types of N-linked oligosaccharides on glycoproteins. The enzyme was first purified to
homogeneity and cloned by our group from porcine brain
as well as from human gastric cancer cells (19, 20). Deletion of the Fut8 gene resulted in marked phenotypic
changes in mice, namely 60% of the mice died during the
neonatal period, and the remainder died within 3 weeks.
Using Fut8 knock-out mice, we found that the mRNA levels of MMP-1, -9, and -12 were specifically up-regulated
among the various matrix metalloproteinases (MMPs)
examined. MMP gene expression occurs specifically due
to the deletion of the core fucose in the transforming
growth factor- (TGF-) receptor, which usually suppresses MMP expression.
The up-regulation of MMPs results in the degradation
of extracellular matrix proteins such as laminin and type
IV collagen, which may facilitate the development of lung
emphysema in mice and humans. In Fut8 knock-out mice,
the phosphorylation of SMAD proteins was markedly
down-regulated, suggesting an impairment in the TGF-
cascade. The core fucosylation of the TGF- receptor was
completely abolished in Fut8 knock-out mice, indicating
that the binding affinity for the ligand, TGF-, was
impaired, and therefore, the negative regulation of TGF-
signaling was also suppressed (34). The Fut8 null mutation
in mice also leads to the impairment of LRP-1 (low density
lipoprotein receptor-related protein-1) (35) and an
impaired pre-B-cell re-population due to dysfunction of
VECAM-2 (vascular endothelial cell adhesion molecule-1) and integrin 41 (36).
Some natural killer cells contain receptors for the Fc
domain of IgG and bind to the Fc portion of the IgG antibody on the surface of a target cell, such as tumor cells, and
release cytolytic components that kill the target cell. This
killing mechanism is referred to as antibody-dependent
cell-mediated cytotoxicity.
H114
FIGURE 6. Schematic drawing of the glycan cycle (fucose cycle). Glycosylation is affected by various factors as described in text, and the pro-form
is converted to the mature form of glycoproteins by glycosyltransferases and then localized on the cell surface, such as growth factor receptors by
binding to lectins. The similar cycles such as the GlcNAc cycle, sialic acid cycle, etc., will be present. TGF-R; TGF- receptor; GMD, GDP-mannose
4,6-dehydratase; FX, GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase; ER, endoplasmic reticulum.
It is well known that mouse monoclonal antibodies

and humanized antibodies, including clinically effective
agents such as trastuzumab (Herceptin) and rituximab
(Rituxan), require both activation via FcRIII and
inhibition via FcRIIB antibody receptors. The expression of antibodies with altered glycoforms, especially
the addition of bisecting GlcNAc, leads to an increase
in antibody-dependent cell-mediated cytotoxicity
through a higher affinity for FCRIII by up to 10 20fold. Similar to the line of evidence described above, two
groups reported that binding of fucose-deficient IgG1
to human FcRIIIA was improved by up to 50 100-fold
(37, 38). Core fucosylation also plays important roles in
the adhesion of tumor cells via integrins (39). These
findings strongly suggest that the 1,6-fucosylation of
N-glycans modifies the function of the glycoproteins.
The Glycomics Approach Is One of the More Promising
Approaches for the Discovery of Cancer BiomarkersIn
2003, the International Union of Biochemistry and Molecular Biology (IUBMB) Congress in Toronto was cancelled
due to the SARS problem, and a joint meeting of the
Human Proteome Organization (HUPO) with IUBMB
was held in Montreal. I attended the council meeting of
HUPO and was asked by several participants to launch a
glycomics initiative because there was no official initiative
on glycomics under HUPO. We finally launched the
Human Disease Glycomics/Proteome Initiative (HGPI),
with myself as the chair, along with many glycobiologists
and biochemists who agreed to collaborate. Many experts,
especially experts in mass spectrometry, joined this initiative and participated in the steering committee meeting.
Among them was the Nobel Laureate Koichi Tanaka and
many others. Since the launch of HGPI, two pilot studies
of N-glycan (40) and O-glycan analyses have been performed with 26 participating laboratories. In 2006, with
Jim Paulson, Sudhir Srivastava, Pamela Marino, and Ram
Sasisekharan, I organized a joint meeting at the National
Institutes of Health entitled Frontiers in Glycomics:
Bioinformatics and Biomarkers in Disease, and on this
occasion, we proposed a white paper emphasizing the
importance of glycomics (41). In terms of identifying cancer biomarkers using glycomics, Dr. Sen-ichiro Hakomori
has made many pioneering discoveries. The fucosylation
of -fetoprotein is markedly increased in patients with a
primary hepatoma, and affinoelectrophoresis using lectin
and antibody can be used to distinguish between patients
with hepatocellular carcinoma and those with chronic
hepatitis and liver cirrhosis as reported by Taketa et al.
(42). In 2006, the Food and Drug Administration approved
the use of fucosylated -fetoprotein for the differential
diagnosis of patients with liver cirrhosis and primary hepatoma. Glycan patterns of the -fetoprotein L3 fraction
and its enzymatic basis were reported by our group (43).
Using a similar approach, fucosylated haptoglobin was
identified as a possible marker for pancreatic cancer. Most
fucosylated proteins are found in the bile duct under normal conditions, but in the case of cancer, polarity changes
occur, which are possibly due to the expression of carrier
H115
protein(s) or the disorganization of tumor cells (44). The

levels of GDP-fucose, as well as synthetic enzymes such as
GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase
and GDP-mannose 4,6-dehydratase, play an important
role in core fucosylation.
Glycan Cycles as Functional Units of Glycan Functions
for Understanding the Systemic Approach: To Make Movies Rather Than to Take a SnapshotHere, I propose the
concept of the glycan cycle as a functional unit of glycans
that will permit the integration of glycan functions in analogy with the -glutamyl cycle for understanding glutathione metabolism (Fig. 1). Jim Dennis reported that Mgat5,
a gene that encodes GnT-V, plays a key role in growth and
arrest via the glycan structure of growth factor receptors
and GLUT1 by elucidating the binding of galectin-3 (45).
However, glycosylation is affected not only by a single glycosyltransferase such as GnT-V but also by various components such as the levels of mono-oligosaccharides,
nucleotide sugars, nucleotide transporters, the localization of glycosyltransferases, nucleotide sugar levels in
the Golgi, transcription factors, the pro-form and
mature form of glycoproteins, and the structure of cellsurface glycoproteins (46). All of these components
should be considered in detecting changes in more
dynamic ways; namely, it is necessary not only to take a
snapshot but also to make movies of the dynamic
changes in glycan metabolism.
For example, glucose enters the cell via a glucose transporter and is converted to UDP-GlcNAc via the hexosamine pathway. UDP-GlcNAc plays a key role in the cytosol
and is then incorporated into the Golgi via the UDPGlcNAc transporter and serves as a donor substrate for
various GlcNAc-transferases from GnT-I to GnT-VI.
UDP-GlcNAc also serves as a donor for O-GlcNAc-transferase in the cytosol. These enzymes function to modify
target proteins such as TGF-, the EGFR, and the glucose
transporter. These receptors are localized on the cell surface and bind to lectins such as galectin-3. When the affinity of lectin binding is decreased due to changes in receptor glycosylation, the receptors are endocytosed, and the
free monosaccharide is probably recycled. This conceptual functional unit of the glycan cycle, such as just
described for GlcNAc, is intended to help develop an
understanding of the integrative and dynamic analysis of
glycan functions. The same is true for the fucose cycle,
in which GDP-fucose, GDP-mannose 4,6-dehydratase,
GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase,
the GDP-fucose transporter, and fucosyltransferases are
key components for core fucosylation as depicted in Fig. 6.
Similarly, the galactose cycle and sialic acid cycle, etc.,

would be present.
Among the components of the glycan cycle, nucleotide
sugars play key roles in the function of glycans. Very
recently, our group developed a method for the simultaneous assay of nucleotide sugars using ion-pair reversedphase high pressure liquid chromatography and observed
dramatic changes in nucleotide sugar levels in cells cultured under dense or sparse conditions (K. Nakajima, S.
Kitazume, R. Fujinawa, E. Miyoshi, and N. Taniguchi,
unpublished data). This indicates that nucleotide sugars
may control glycan structures and function via the glycan
cycle (Fig. 6). To clarify glycan functions in a more
dynamic way, after I retired from the Osaka University
Medical School in 2006 after reaching retirement age, I
became an Endowed Chair Professor of the Department of
Disease Glycomics in the same university, with support
from Seikagaku Corp., and we subsequently launched a
Systems Glycobiology Research Group at RIKEN (Wako,
Japan). We expect that our understanding of glycan cycles
will be enhanced considerably in the near future.
AcknowledgmentsI thank my many colleagues who collaborated with
me over three decades, many whose names also appear in the list of
literature citations. During my stay at Osaka University, many eminent
scientists came to visit as visiting professors and contributed to our
research and education in our M.D./Ph.D. program as well. Among them,
I thank Professors Dirk van den Eijnden, John Gutteridge, Kunihiko
Suzuki, Harold F. Deutsch, Kosaku Uyeda, Takashi Yonetani, Peng
George Wong, Mary Anderson, Owen W. Griffith, and William Lennarz,
who spent from three weeks to one year as visiting professors and supported our research and the M.D./Ph.D. program in our faculty. I thank
Drs. Vincent C. Hascall, Etorre Appella, Milton Feather, Jianguo Gu, and
Eiji Miyoshi and Lisa Jenkins for reading this manuscript prior to submission and for valuable suggestions. I thank Dr. Kazuki Nakajima and
Fumi Ota for help in preparing this manuscript. I acknowledge continued
grant support from the Japan Society for the Promotion of Science and the
Ministry of Education, Sports, Culture, Science, and Technology, Japan
(from 1973 to the present).
Address correspondence to: tani@sanken.osaka-u.ac.jp.
Authors ChoiceFinal version full access.
REFERENCES
1. Taniguchi, N. (2008) IUBMB Life 60, 703705
2. Hirai, H., Nishi, S., Watabe, H., and Tsukada, Y. (1973) Gann Monogr. 14,
19 34
3. Taniguchi, N., Tsukada, Y., Mukuo, K., and Hirai, H. (1974) Gann 65, 381387
4. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 711760
5. Meister, A. (1986) in Molecular Perspectives and Clinical Implications (Taniguchi, N., Higashi, T., Sakamoto, Y., and Meister, A., eds) pp. 321, Academic
Press, New York
6. Gasa, S., Balbaa, M., Nakamura, M., Yonemori, H., and Makita, A. (1987) J. Biol.
Chem. 262, 1230 1238
7. Honke, K., Hirahara, Y., Dupree, J., Suzuki, K., Popko, B., Fukushima, K., Fukushima, J., Nagasawa, T., Yoshida, N., Wada, Y., and Taniguchi, N. (2002) Proc.
Natl. Acad. Sci. U.S.A. 99, 4227 4232
8. Taniguchi, N. (1992) Adv. Clin. Chem. 29, 159
9. Yokosawa, N., Taniguchi, N., Tsukada, Y., and Makita, A. (1981) Oncodev. Biol.
H116
Med. 2, 165177
10. Taniguchi, N., Yokosawa, N., Iizuka, S., Tukada, Y., Sako, F., and Miyazawa, N.
(1983) Gann Monogr. 29, 263272
11. Yamashita, K., Hitoi, A., Taniguchi, N., Yokosawa, N., Tsukada, Y., and Kobata,
A. (1983) Cancer Res. 43, 5059 5063
12. Meister, A. (1998) in Glutathione Centennial (Taniguchi, N., Higashi, T., Sakamoto, Y., and Meister, A., eds) pp. 322, Academic Press, New York
13. Ikeda, Y., Fujii, J., Anderson, M. E., Taniguchi, N., and Meister, A. (1995) J. Biol.
Chem. 270, 2222322228
14. Taniguchi, N., and Ikeda, Y. (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72,
239 278
15. Taniguchi, N., Nishikawa, A., Fujii, S., and Gu, J. G. (1989) Methods Enzymol.
179, 397 408
16. Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K., and Taniguchi, N.
(1992) J. Biol. Chem. 267, 18199 18204
17. Schachter, H., Narasimhan, S., Gleeson, P., and Vella, G. (1983) Can. J. Biochem. Cell Biol. 61, 1049 1066
18. Gu, J., Nishikawa, A., Tsuruoka, N., Ohno, M., Yamaguchi, N., Kangawa, K.,
and Taniguchi, N. (1993) J. Biochem. 113, 614 619
19. Uozumi, N., Yanagidani, S., Miyoshi, E., Ihara, Y., Sakuma, T., Gao, C. X.,
Teshima, T., Fujii, S., Shiba, T., and Taniguchi, N. (1996) J. Biol. Chem. 271,
27810 27817
20. Yanagidani, S., Uozumi, N., Ihara, Y., Miyoshi, E., Yamaguchi, N., and Taniguchi, N. (1997) J. Biochem. 121, 626 632
21. Taniguchi, N., Yoshimura, M., Miyoshi, E., Ihara, Y., Nishikawa, A., and Fujii, S.
(1996) Glycobiology 6, 691 694
22. Taniguchi, N., Ihara, S., Saito, T., Miyoshi, E., Ikeda, Y., and Honke, K. (2001)
Glycoconj. J. 18, 859 865
23. Taniguchi, N., Gu, J., Takahashi, M., and Miyoshi, E. (2004) Proc. Jpn. Acad. Ser.
B Phys. Biol. Sci. 80, 8291
24. Taniguchi, N., Miyoshi, E., Gu, J., Jianguo, G., Honke, K., and Matsumoto, A.
(2006) Curr. Opin. Struct. Biol. 16, 561566
25. Taniguchi, N., Honke, K., and Fukuda, M. (eds) (2002) Handbook of Glycosyltransferases and Related Genes, Springer, Tokyo
26. Tsuda, T., Ikeda, Y., and Taniguchi, N. (2000) J. Biol. Chem. 275, 21988 21994
27. Takahashi, M., Yokoe, S., Asahi, M., Lee, S. H., Li, W., Osumi, D., Miyoshi, E.,
and Taniguchi, N. (2008) Biochim. Biophys. Acta 1780, 520 524
28. Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S., and Taniguchi, N.
(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 8754 8758
29. Akama, R., Sato, Y., Kariya, Y., Isaji, T., Fukuda, T., Lu, L., Taniguchi, N.,
Ozawa, M., and Gu, J. (2008) Proteomics 8, 32213228
30. Granovsky, M., Fata, J., Pawling, J., Muller, W. J., Khokha, R., and Dennis, J. W.
(2000) Nat. Med. 6, 306 312
31. Guo, H. B., Lee, I., Kamar, M., Akiyama, S. K., and Pierce, M. (2002) Cancer Res.
62, 6837 6845

32. Zhao, Y., Nakagawa, T., Itoh, S., Inamori, K., Isaji, T., Kariya, Y., Kondo, A.,
Miyoshi, E., Miyazaki, K., Kawasaki, N., Taniguchi, N., and Gu, J. (2006) J. Biol.
Chem. 281, 3212232130
33. Lagana, A., Goetz, J. G., Cheung, P., Raz, A., Dennis, J. W., and Nabi, I. R. (2006)
Mol. Cell. Biol. 26, 31813193
34. Wang, X., Inoue, S., Gu, J., Miyoshi, E., Noda, K., Li, W., Mizuno-Horikawa, Y.,
Nakano, M., Asahi, M., Takahashi, M., Uozumi, N., Ihara, S., Lee, S. H., Ikeda,
Y., Yamaguchi, Y., Aze, Y., Tomiyama, Y., Fujii, J., Suzuki, K., Kondo, A., Shapiro, S. D., Lopez-Otin, C., Kuwaki, T., Okabe, M., Honke, K., and Taniguchi, N.
(2005) Proc. Natl. Acad. Sci. U.S.A. 102, 1579115796
35. Lee, S. H., Takahashi, M., Honke, K., Miyoshi, E., Osumi, D., Sakiyama, H.,
Ekuni, A., Wang, X., Inoue, S., Gu, J., Kadomatsu, K., and Taniguchi, N. (2006)
J. Biochem. 139, 391398
36. Li-Nakagawa, T., Miyoshi, E., Yakushijin, T., Hiramatsu, N., Igura, T., Hayashi,
N., Taniguchi, N., and Kondo, A. (2008) J. Proteome Res. 7, 22222233
37. Shields, R. L., Lai, J., Keck, R., OConnell, L. Y., Hong, K., Meng, Y. G., Weikert,
S. H., and Presta, L. G. (2002) J. Biol. Chem. 277, 2673326740
38. Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y.,
Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M., Hanai, N., and
Shitara, K. (2003) J. Biol. Chem. 278, 3466 3473
39. Zhao, Y., Itoh, S., Wang, X., Isaji, T., Miyoshi, E., Kariya, Y., Miyazaki, K.,
Kawasaki, N., Taniguchi, N., and Gu, J. (2006) J. Biol. Chem. 281, 3834338350
40. Wada, Y., Azadi, P., Costello, C. E., Dell, A., Dwek, R. A., Geyer, H., Geyer, R.,
Kakehi, K., Karlsson, N. G., Kato, K., Kawasaki, N., Khoo, K. H., Kim, S., Kondo,
A., Lattova, E., Mechref, Y., Miyoshi, E., Nakamura, K., Narimatsu, H., Novotny, M. V., Packer, N. H., Perreault, H., Peter-Katalinic, J., Pohlentz, G., Reinhold, V. N., Rudd, P. M., Suzuki, A., and Taniguchi, N. (2007) Glycobiology 17,
411 422
41. Packer, N. H., von der Lieth, C. W., Aoki-Kinoshita, K. F., Lebrilla, C. B., Paulson, J. C., Raman, R., Rudd, P., Sasisekharan, R., Taniguchi, N., and York, W. S.
(2008) Proteomics 8, 8 20
42. Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., Koji, T., Taga, H., Satomura, S.,
Matsuura, S., Kawai, T., and Hirai, H. (1993) Cancer Res. 53, 5419 5423
43. Ohno, M., Nishikawa, A., Koketsu, M., Taga, H., Endo, Y., Hada, T., Higashino,
K., and Taniguchi, N. (1992) Int. J. Cancer 51, 315317
44. Nakagawa, T., Uozumi, N., Nakano, M., Mizuno-Horikawa, Y., Okuyama, N.,
Taguchi, T., Gu, J., Kondo, A., Taniguchi, N., and Miyoshi, E. (2006) J. Biol.
Chem. 281, 2979729806
45. Partridge, E. A., Le Roy, C., Di Guglielmo, G. M., Pawling, J., Cheung, P.,
Granovsky, M., Nabi, I. R., Wrana, J. L., and Dennis, J. W. (2004) Science 306,
120 124
46. Taniguchi, N. (2007) Nat. Chem. Biol. 3, 307309
H117
ADDITIONS AND CORRECTIONS

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 5, p. 3524, January 29, 2010
VOLUME 284 (2009) PAGES 34469 34478

DOI 10.1074/jbc.A109.023150
From the -glutamyl cycle to the glycan cycle: a road

with many turns and pleasant surprises.
Naoyuki Taniguchi
On page 34472, right column, second paragraph, line 3, the sentence

should read as follows: We first prepared a large amount of non-bisected biantennary sugar chains from human serum transferrin.
We suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original
article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry
notice of these corrections as prominently as they carried the original abstracts.
H118
VOLUME 285 NUMBER 5 JANUARY 29, 2010
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JBC Hist Persp Glycobiology

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HISTORICAL PERSPECTIVES

... but true! MARS - The easiest Data Analysis

Find our microplate readers on www.bmglabtech.com

HTRF is a registered trademark of Cisbio International.

Overlay plot of esterase catalysed pNPA

The Journal of Biological Chemistry

H34 Proteoglycans and Orchids: the Work of Vincent Hascall

H1 JBC Historical Perspectives: Glycobiology and Carbohydrates.

H37 Lactose Synthesis in the Mammary Gland: Lactose Synthase

Nicole Kresge, Robert D. Simoni, and Robert L. Hill

and the Work of Robert L. Hill

the Clinical Chemistry Work of Stanley R. Benedict

Work of Michael Doudoroff

Catabolism: the Work of Gilbert G. Ashwell

Work of Stuart A. Kornfeld

H17 Albert Dorfman and the Biosynthesis of Hyaluronic Acid

Phosphotransferase System: Saul Rosemans Contributions to

of William Zev Hassid

Recognition Molecules. Nathan Sharon

Many Turns and Pleasant Surprises. Naoyuki Taniguchi

JOURNAL OF BIOLOGICAL CHEMISTRY

This paper is available online at www.jbc.org

Printed in the U.S.A.

JBC Historical Perspectives: Glycobiology and Carbohydrates*

Defined in the broadest sense, glycobiology is the study of

nucleotide sugars uridine diphosphate glucose (UDPG),

PROLOGUE: Glycobiology and Carbohydrates

JOURNAL OF BIOLOGICAL CHEMISTRY

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Vol. 277, No. 16, Issue of April 19, p. e5, 2002

Benedicts Solution, a Reagent for Measuring Reducing

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Stanley R. Benedict. Photo courtesy of the National Library of Medicine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 277, No. 39, Issue of September 27, p. e27, 2002

The Discovery of Hyaluronan by Karl Meyer

Karl Meyer. Photo courtesy of the National Library of Medicine.

Meyers scientific contributions were not limited to the discovery of hyaluronan. He is

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Vol. 280, No. 4, Issue of January 28, p. e3, 2005

Otto Fritz Meyerhof and the Elucidation of the Glycolytic

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Otto F. Meyerhof. Photo courtesy of the National Library of Medicine.

Warburg, O., and Christian, W. (1939) Biochem. Z. 301, 201

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Vol. 280, No. 19, Issue of May 13, p. e16, 2005

Luis F. Leloir and the Biosynthesis of Saccharides

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Luis F. Leloir. Photo courtesy of the National Library of Medicine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 280, No. 29, Issue of July 22, p. e26, 2005

Bernard L. Horeckers Contributions to Elucidating the

This paper is available on line at http://www.jbc.org

All biographical information on Bernard L. Horecker was taken from Ref. 3.

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Vol. 280, No. 27, Issue of July 8, p. e24, 2005

The Entner-Doudoroff Pathway for Glucose Degradation:

This paper is available on line at http://www.jbc.org

All biographical information on Michael Doudoroff was taken from Ref. 4.

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 280, No. 31, Issue of August 5, p. e28, 2005

Albert Dorfman and the Biosynthesis of Hyaluronic Acid

This paper is available on line at http://www.jbc.org