Thalassas, 2004, 20 (1): 15-24
An International Journal of Marine Sciences
MACROMOLECULES IN BIOMINERALS
OF AQUATIC ORGANISMS
HIROMICHI NAGASAWA(1)
Keywords: biomineralization; calcium carbonate; cDNA cloning; crystal growth; molecular biology; organic matrix
ABSTRACT
Macromolecules in biominerals of aquatic animals
and plants play important roles in the regulation of
mineralization. In the past several years, most
macromolecules including proteins and
polysaccharides have been characterized mainly by
their amino acid sequences and chemical structures,
respectively. Molecular biological techniques have
been useful not only for determination of the entire
amino acid sequences but also for analysis of
spatiotemporal expression of their genes.
Immunohistochemistry has clarified distribution of
matrix proteins. The in vitro anti-calcification assay
permitted us to purify minor organic components from
biominerals such as crayfish exoskeleton and coccolith
of marine unicellular algae. Matrix proteins thus far
identified have characteristic structural features. They
do not have sequence homology to one another except
for a few proteins that originate from taxonomically
close species. Many matrix proteins carry repeats with
(1) Department of Applied Biological Chemistry,
Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657,
Japan.
Correspondence: Hiromichi Nagasawa
Tel: +81-3-5841-5132; Fax: +81-3-5841-8022
e-mail: anagahi@mail.ecc.u-tokyo.ac.jp
varying sequences and peptide lengths. Based on these
findings, matrix proteins are not likely to have
divergently evolved from an ancestral protein.
Development of assay methods for the functions of
matrix proteins is urgently needed. Protein chemistry
including synthesis of proteins and peptides, structure-
function relationship studies and three-dimensional
structure analysis will also contribute to clarification of
the mechanism of biomineralization.
INTRODUCTION
Biomineralization is a process strictly controlled
by organisms ranging from higher vertebrates and
plants to microorganisms. Representative examples
of biominerals are bones and teeth in vertebrates,
shell in molluscs, exoskeleton in crustaceans,
coccoliths in marine algae, plant opal in higher plants
and magnetite in bacteria. They play important roles
in each organism such as support of the body, storage
of calcium, defence from enemies, and detoxification
of some toxic elements. The mechanism of
biomineralization has been a target area of many
research such as paleontology, evolutionary biology,
physiology and materials science. Although
enormous data have been accumulated so far, the
mechanism of biomineralization is not yet well
understood.
15
Hiromichi Nagasawa
Biomineralization is distinct from non-biological
mineralization with respect to slower crystallization,
controlled crystalline morphology, selected polymorph
and enhanced mechanical strength of crystals. These
characteristics of biominerals are thought to be
attributed partially to the involvement of matrix
macromolecules such as proteins and polysaccharides
in biominerals, although they are present in minute
amounts. In aquatic animals and plants, the chemical
structures of such macromolecules had never been
characterized until around 1995 except for coccolith
matrix polysaccharide, PS-2, (Marsh et al., 1992) and
spicule matrix proteins, SM30 (George et al., 1991)
and SM50 (Sucov et al., 1987; Katoh-Fukui et al.,
1991), in the sea urchin. However, spicule formation
was first regarded as a developmental event during
embryogenesis rather than biomineralization. Most of
the works on macromolecules in biominerals of
aquatic organisms before 1995 had included
experiments using crude extracts or partially purified
preparations of matrix proteins, and only their amino
acid compositions or protein profiles on SDS-PAGE
had been reported. Although some of the data showed
characteristic compositions, suggesting the existence
of a unique protein as a major component, the amino
acid composition of a mixture of proteins is
meaningless. Indeed, this speculation was later proved
to be correct by amino acid sequence analysis of a
purified protein from the mixture. In the past several
years, molecular biological techniques were applied to
the characterization of individual matrix proteins or
peptides. In this review, some of the examples are
described together with the introduction of a
methodology for bioactive compound chemistry to
isolate functionally important organic matrices. In
addition, the chemical characteristics of matrix
proteins identified thus far will also be discussed.
1. Application of Molecular Biological Techniques
to Organic Matrix Research
1-1. Structure determination of organic matrices in
biominerals
Matrix proteins have recently been extracted and
purified to homogeneity from the extract of
biominerals of many aquatic animals and algae. Table
1 summarizes the organic matrices thus far
characterized. The method adopted to obtain the
16
primary sequences of matrix proteins are orthodox in
almost all cases. In those cases, the amino-terminal
and internal amino acid sequences of a certain matrix
protein are first analyzed. Next, based on the
sequences a set of primers are designed, and a
fragment of a cDNA encoding the protein is amplified
by RT-PCR using the primers. Finally, a whole cDNA
is obtained by 5' and 3' RACE or by screening a cDNA
library using the whole or a part of the fragment as a
probe. Another method for cloning a cDNA is to use
an antibody raised against a matrix protein and an
expression library. From the nucleotide sequence of an
open reading frame of the cDNA thus cloned, the
amino acid sequence of a precursor of the matrix
protein can be deduced. The precursor usually has a
signal peptide at the amino-terminus for secretion
from the producing cells, because the mature protein
is found in an extracellular matrix. Thus, the primary
structure of a protein can be deduced, and many
matrix proteins have been characterized by this
method combining protein chemistry with molecular
biology.
Post-translational modifications other than the
removal of a signal peptide may occur in other
extracellular matrix proteins. Some matrix proteins
were found to be glycosylated at Asn residues, which
was predicted by the presence of a consensus
sequence, Asn-X-Ser/Thr, and verified by the decrease
in molecular mass on an SDS-PAGE gel after
treatment with endoglycosidase to cleave the
carbohydrate chain (Murayama et al., 2000 and 2002).
Chemical deglycosylation can also be performed to
demonstrate the existence of a carbohydrate moiety
(Marin et al., 2000). However, the structure of the
carbohydrate chain moiety has never been analyzed in
any matrix glycoproteins. Moreover, the functions of
the carbohydrate moiety is unclear. Phosphorylation,
which causes a protein to be more acidic, was observed
at a specific Ser residue of CAP-1, a crayfish cuticle
peptide, and might contribute to the regulation of
mineralization (Inoue et al., 2001). Gla proteins, γ-
carboxyglutamic acid residue-containing proteins first
characterized in terrestrial mammals, were also
isolated from the bone and/or scale of the seabream,
Sparus aurata, (Pinto et al., 2001) and the freshwater
sunfish, Lepomis macrochirus, (Nishimoto et al.,
1992). These proteins have not been found in
elasmobranchs, and therefore seems to be present in
 Macromolecules in Biominerals of Aquatic Organisms

Table 1.
Organic matrices in biominerals of aquatic animals and plants

vertebrates higher than teleost fish in the phylogenic
tree and to be required for adequate maturation of
hydroxyapatite crystals. The γ-carboxyglutamic acid
residues may participate in this function.
Another approach to obtain matrix proteins
associated with calcification is differential display
analysis, which was applied to search for proteins in
crustacean calcified exoskeleton (Watanabe et al.,
2000; Endo et al., 2000; Ikeya et al., 2001). In
crustaceans, calcification in the exoskeleton occurs
only during the post-molt stage. Differential display
analysis was performed with RNA's isolated from the
tail fan blade of the kuruma prawn Penaeus japonicus
at several molting stages (from the postmolt to the late
premolt stages); and four novel cDNA fragments
specifically expressed only during the post-molt stage
were identified. The full-length cDNAs were obtained,
and the putative proteins were named DD4, DD5,
DD9A and DD9B. DD4, now renamed crustocalcin, is
a Ca2+-binding protein, and the other three are members
of a family of arthropod cuticular proteins which
contain a Rebers-Riddiford consensus sequence
(Rebers & Riddiford, 1988) that is possibly
responsible for chitin-binding. This approach is useful
when calcification occurs temporally.

17
Hiromichi Nagasawa
In coccolithophorid algae, acidic polysaccharides
were purified from coccoliths as major organic
matrices (Marsh et al., 1992; Ozaki et al., 2001). PS-
2 was isolated from the EDTA-soluble fraction of
coccoliths of a coccolithophorid alga Pleurochrysis
carterae. It is an acidic polysaccharide with the
unique polymeric structure of a disaccharide unit,
consisting of glucuronic acid and its oxidative
degradation product at the C2-C3 bond (meso-tartaric
acid and glyoxylic acid) (Marsh et al., 1992). Though
the structures of matrix proteins and polysaccharides
are completely different chemically, they share acidic
nature and may contribute to the regulation of
calcification by a similar mechanism; for example,
serving as a nucleation site, supplying a calcium ion
for crystal growth or, in contrast, inhibiting crystal
growth. Given that the acidic polysaccharides play
important roles in coccolith formation, the
carbohydrate moiety of glycoproteins obtained from
other calcified tissues may also have roles in
calcification. Though molecular biological
techniques can not be applied directly to the structure
determination of carbohydrates, characterization of
enzymes associated with biosynthesis of
carbohydrates may be possible using molecular
biological techniques.
1-2. Spatiotemporal synthesis and distribution of
matrix proteins
Once a cDNA encoding a matrix protein has been
identified, the expression of the corresponding gene
can be analyzed by in situ hybridization. This method
revealed that the SM30 mRNA was expressed
specifically in the spicule-producing primary
mesenchyme cells of the sea urchin embryo (George et
al., 1991). The mRNA for MSI60 was expressed in the
inner epithelial region of the mantle of the oyster,
Pinctada fucata, while MSI31 was seen in the outer
region (Sudo et al., 1997). Since the inner and outer
regions of the mantle face the nacreous (aragonite) and
prismatic layers (calcite), MSI60 and MSI31 might be
associated with the regulation of crystalline
polymorphism. DD4 and DD5, putative exoskeletal
proteins of the kuruma prawn, Penaeus japonicus,
were found to be expressed in the epidermal cells of
that organism (Endo et al., 2000; Ikeya et al., 2001).
Orchestin transcripts were detected only in the caecal
epithelium mainly during the late premolt period
18
(Testeniere et al., 2002). The bone Gla protein as
mentioned above was found to be expressed only in
jaw and verterba of the seabream, S. Aurata, (Pinto et
al., 2001), indicating the involvement of this protein in
calcification.
Northern blot analysis and RT-PCR can also be
used to study the spatiotemporal expression of matrix
proteins. GAMP originally isolated from gastroliths
of the crayfish was found to be produced not only at
the gastrolith disk in the stomach during the premolt
stage, but also in the exoskeleton during the early
intermolt stage by Northern blot analysis (Tsutsui et
al., 1999; Takagi et al., 2000). The site and
developmental stage of GAMP expression coincided
well with the site and timing of calcification,
suggesting the association of GAMP with
calcification in both gastrolith and exoskeleton. The
expression of the GAMP gene was found to be
induced by molting hormone, 20-hydroxyecdysone,
in in vitro culture of the gastrolith disk, a part of
stomach epithelia with the ability to produce GAMP.
Such an experiment is possible because of the
periodic formation of gastolith occurs synchronously
and the molting is regulated endocrinologically in
crustaceans. Similar experiments were conducted
with orchestin, a matrix protein from the amphipod,
Orchestia cavimana, whose expression was also
induced by molting hormone (Testeniere et al., 2002).
The endocrine control of the expression of a matrix
protein of the calcified tissue was observed in fish
scale. The expression of mRNA encoding
Osteonectin, a collagen-binding protein, was found to
be regulated by estrogen in the female goldfish,
Carassius auratus, during vitellogenesis (Lehane et
al., 1999). This experiment was done using a goldfish
scale cell line. The expression of OMP-1, a matrix
glycoprotein in the otolith of the rainbow trout, was
expected to be regulated by a circadian rhythm,
because daily rings are formed by alternate deposition
of CaCO3 and organic matrices. This is the basis for
estimation of age in this organism. However, no clear
rhythmic change in the OMP-1 mRNA levels was
observed (Murayama et al., 2000). Therefore, a
different type of regulation for diurnal changes in
deposition of organic matrices might exist (e.g.
regulation at the level of translation and /or secretion
levels). Some endocrine regulation might be involved
in this daily ring formation.

Table 2.
Proteins having amino acid sequence similarities with mineralized tissue matrix proteins
Antibodies against matrix proteins can be raised
using natural or recombinant proteins, the latter of
which are synthesized using a cDNA encoding a
matrix protein and an expression system. Using such
antibodies, the distribution of matrix proteins can be
examined immunohistochemically. Mucopearlin, a
mucin from the nacreous layer of the fan mussel Pinna
nobilis was detected immunohistochemically using an
antibody raised against recombinant mucoperlin. The
result demonstrated that it is only present in the
nacreous aragonitic layer but not in the prismatic
calcitic layer (Marin et al., 2000).
Immunohistochemical studies using antibodies against
purified PS-1 and PS-2 demonstrated that they were
localized in the crystal coats of coccoliths (Marsh,
1994). These polysaccharides were found to be
synthesized in medial Golgi cisternae and co-
aggregate with calcium ions. GAMP, a matrix protein
of gastrolith, was found to be localized in the gastrolith
disk of the stomach and distributed uniformally
throughout the gastrolith (Takagi et al., 2000).
Unexpectedly, immunoreactivity was also observed in
the cuticle, especially at the epidermal cells,
endocuticle and exocuticle. The latter two were other
sites of calcification. Therefore, GAMP are produced
in the epidermal cells, and secreted into the cuticle and
might be associated with calcification of the cuticle
(Takagi et al., 2000).
Macromolecules in Biominerals of Aquatic Organisms
2. Application of Bioactive Compound Chemistry
to Organic Matrix Research
Though organic matrices are considered to play
important roles in biomineralization, the precise role is
still unclear. Until now, almost all work have been
done on major components of biominerals, irrespective
of their function, because there has not been an
appropriate assay method for examining the function
of organic matrices. A simple in vitro CaCO3 inhibition
assay method was developed by Wheeler et al. (1981).
This method consists of two steps of procedure; first,
mixing 20 mM CaCl2 and 20 mM NaHCO3 containing
a test sample to make a supersaturated solution of
CaCO3, followed by examining the effect of the sample
on the inhibition of CaCO3 precipitation (anti-
calcification activity). This method was first applied to
the purification of CMAP from EDTA-soluble extracts
of coccoliths of P. carterae (Ozaki et al., 2001).
CMAP, an acidic polysaccharide, was purified by
assaying anti-calcification activity in each fraction at
every step of purification (Ozaki et al., 2001). But the
final preparation still contained a small amount of PS-
2, another acidic polysaccharide, which was found to
be far less active. This method was next applied to the
purification of CAP-1 from SDS extracts of the
exoskeleton of the crayfish, Procambarus clarkii.
(Inoue et al., 2001). CAP-1, a 78-amino acid residue
19
Hiromichi Nagasawa
peptide, was a minor component but most active
among many components in the extract. The anti-
calcification assay enabled the purification of CAP-1,
and the yield of CAP-1 was only 7.5 µg from 1 g of
dried exoskeleton.
A functional assay was also performed to search
for a compound responsible for inducing
crystallization of aragonite, a specific polymorph of
CaCO3 which is less stable than calcite. It was
demonstrated that a macromolecule responsible for
inducing aragonite formation was present in the pearl
layer of shell, which consists of aragonite crystals
(Watabe & Wilbur, 1960). Later, this aragonite-
inducing compound was partially characterized by two
research groups (Falini et al., 1996; Belcher et al.,
1996), but has not yet been isolated. Once this
substance is successfully isolated, its function could be
determined straightforwardly.
There are many reports describing isolated
macromolecules that have been tested for anti-
calcification activity or aragonite inducing ability:
nacrein (Miyamoto et al., 1996), mucoperlin (Marin et
al., 2000), N16 (Samata et al., 1999), N66 and N14
(Kono et al., 2000), and GAMP (Tsutsui et al., 1999).
3. Molecular Characteristics of Matrix Proteins in
Mineralized Tissues
Many matrix proteins thus far characterized have
some structural characteristics such as sequence
similarity to known proteins and the presence of
repeated sequences. Table 2 lists matrix proteins which
have sequence similarity to known proteins. Similar
proteins are very diverse; structural proteins, enzymes,
an ion-transport protein, an ion-binding protein and
cuticular proteins, which have no structural or
functional relationship to one another. Overall, matrix
proteins thus far characterized in aquatic animals have
no sequence similarity to one another, although there
are a few exceptions. Proteins obtained from
taxonomically close species are similar; for example,
nacrein (Miyamoto et al., 1996) and N66 (Kono et al.,
2000), and pearlin (Miyashita et al., 2000; N16, an
almost identical protein to pearlin, Samata et al., 1999)
and N14 (Kono et al., 2000). Nacrein, pearlin and N16
are from the pearl oyster, Punctada fucata, while N66
and N14 are from the same genus P. maxima. A
20
homolog of OMP-1 obtained from otoliths of the chum
salmon Oncorhynchus keta was found in otoliths of the
rainbow trout, Oncorhynchus mykiss, belonging to the
same genus (Murayama et al., 2000). Osteonectin and
the Gla protein, originally discovered in higher
terrestrial vertebrates, were also found in fish and each
molecule share the respective sequence characteristics
(Lehane et al., 1999; Nishimoto et al., 1992; Pinto et
al., 2001). These examples do not imply that there was
a universally common ancestral matrix protein
molecule which has evolved divergently. Instead,
matrix proteins might have evolved separately from
multiple distinct ancestral molecules. From the limited
information obtained thus far about the structures of
matrix proteins, divergent evolution of matrix proteins
is not likely, because their structures are very different
from each other.
As mentioned previously, in most cases only major
matrix components have been characterized thus far,
while minor components have remained neglected.
Therefore, the number of different matrix components
in biominerals is unclear. More than 40 proteins were
found in the sea urchin embryonic spicule by two-
dimensional gel electrophoresis (Killian & Wilt,
1996). But, only two of them, SM30 and SM50, have
been characterized, although their functions are
unclear. Until now, only a few major matrix proteins at
most have been characterized from each mineralized
tissue. Therefore, our knowledge about organic
matrices is so limited that we can not give an answer to
the question how matrix proteins have evolved.
Many matrix proteins have various types of
repeated sequences, which are shown in Table 3.
Peptide chain length and repeated sequence vary from
protein to protein. Some proteins such as GAMP,
MSP-1 and lustrin A have two distinct types of
repeated sequences in one molecule. In the repeated
sequence, Gly, Ser and Asn are most often observed,
but they never form a homologous sequence. The
possible significance of the repeated sequence can only
be estimated for a few examples. Otolin-1 from fish
otoliths, has a repeated sequence typical of collagen,
Gly-Xaa-Yaa, and the overall sequence is most similar
to type VIII and X collagens that form a meshwork
(Murayama et al., 2002). Therefore, otolin-1 may form
a meshwork that offers appropriate sites for crystal
nucleation directly or indirectly. GAMP, a matrix

Table 3.
Repeated sequences present in matrix proteins
protein in the gastrolith of the crayfish, has 17 tandem
repeats of a 10-residue sequence (Tsutsui et al., 1999),
which is similar to that of human involucrin produced
in the keratinocyte of the skin; however, the role of
involucrin is not clear. Although both proteins share
common features, they are produced in the epidermal
cells and have a similar repeated sequence. It is
unlikely that this repeated sequence contributes to
calcification directly, because human skin is not
mineralized.
All other repeated sequences are unique and have
no homology with known sequences. Therefore, at
present, it is difficult to estimate the significance of the
repeated sequences. The repetition of amino acid
sequences might correlate with the regular crystalline
structure of CaCO3. Alternatively, the repeated
sequence in each protein might form a specific
structure that binds to the crystal surface. Acidic amino
acid residues such as Asp and Glu are thought to
Macromolecules in Biominerals of Aquatic Organisms
participate in the interaction with an calcium ion or
calcium in CaCO3 crystals (Addadi & Weiner, 1985).
Some matrix proteins such as MSP-1 (Sarashina &
Endo, 1998, 2001) and CAP-1 (Inoue et al., 2001)
contain a high proportion of acidic amino acid
residues; however, acidic amino acid residues are not
always involved in most matrix proteins of
biominerals. In order to clarify the meaning of
repeated sequences, it will be necessary to determine
the tertiary structure of matrix proteins, which may
permit us to assume exact interactions between matrix
proteins and the surface of CaCO3 crystals.
Matrix proteins are generally categorized into two
groups in terms of solubility in EDTA, "soluble" and
"insoluble" proteins. When CaCO3 biominerals are
decalcified with EDTA or acidic solution, the
solubilized proteins are called "soluble" proteins, and
the proteins remaining in the residual material are
called "insoluble" proteins. Some "insoluble" proteins
21
Hiromichi Nagasawa
can be extracted and dissolved in a buffer solution
containing a detergent such as SDS and Chaps. Even
after they are extracted, they sometimes become
soluble in a buffer solution without a detergent. Thus,
"insoluble" only refers to their state after extraction
from biominerals but not necessarily to their intrinsic
chemical property, and therefore this classification is
arbitrary. Such proteins may be bound tightly to
insoluble materials such as chitin and collagen in the
natural state. For example, GAMP in the gastrolith of
the crayfish and CAP-1 in the exoskeleton could not be
extracted with EDTA or acidic solution due to tight
association with chitin fibril, but after extraction with
SDS-containing solution, they became "soluble" (Ishii
et al., 1996 and 1998; Inoue et al., 2001). Therefore,
different extraction methods will not always maintain
the chemical properties of matrix proteins in terms of
solubility. It is possible that the tertiary structures of
such proteins may change after extraction.
4. Prospects for the Future Study
Highly developed techniques for purification and
characterization of macromolecules and molecular
biological techniques have recently enabled us to
characterize macromolecules much more easily than
before. Thus, the primary structures of many matrix
proteins have been deduced from the nucleotide
sequences of their cDNAs. In the future, the number of
identified matrix proteins will increase faster, and
information on their primary structure will accumulate.
However, this may not reveal functionally important
structure determinants necessary for mineralization,
since we have not found any consensus sequences
among known matrix proteins, with a few exceptions.
In the future, investigations toward at least two
directions shall be developed. One is to develop
methods to identify the role of each macromolecule in
biomineralization. It is presumed that some of the
macromolecules contribute to the regulation of crystal
nucleation, some to the control of crystal growth, some
to the selection of crystal polymorph such as calcite,
aragonite and vaterite in calcium carbonate crystals,
and the others to the regulation of the morphology of
the whole biomineral. There might be other functions
of macromolecules which have barely been
considered; for example, self-organization or self-
assembling of macromolecules, or interaction among
22
macromolecules. Although many aspects of matrix
macromolecules have been studied thus far, the role of
even a single macromolecule has not been definitely
identified. Atomic force microscopy is the powerful
tool that can be used to observe the effects of
macromolecules on crystal growth at a nano-scale.
The other direction of investigation is to utilize
protein and peptide chemistry. The amino acid
sequences of almost all high-molecular-weight matrix
proteins have been deduced by the nucleotide
sequences of their cDNAs. Protein and peptide
chemistry will be useful to clarify molecular
characteristics more precisely such as glycosylation,
phosphorylation, hydroxylation, amidation and
disulfide bond formation, which might be strongly
associated with the regulation of biomineralization. It
will also be important to determine secondary and
tertiary structures of macromolecules as mentioned
before, because direct interaction between crystals and
macromolecules at atomic levels should be analyzed in
order to understand the exact mechanism of
biomineralization. Recently, solution structure of a
model peptide representing 12-residue consensus
sequence found near the N-terminus of the major
domains of Lustrin A was analyzed by NMR (Zhang et
al., 2002). However, the results implied the molecular
characteristics of its elasticity but did not explain the
association with calcification. Protein and peptide
chemistry also involves chemical modification of
macromolecules such as fragmentation of
macromolecules to smaller peptides, removal of post-
translationally modified moieties, replacement of a
specific residue by other amino acid residues and
designing of peptides with unnatural sequences.
Peptide synthesis and recombinant DNA techniques
will be useful for production of natural and artificial
peptides, and proteins. Investigation of matrix
macromolecules is really an interdisciplinary research
field among mineralogy, chemistry and biology.
Therefore, it will be necessary to study this subject
from a variety of directions.
ACKNOWLEDGEMENT
This work was supported by a Grant-in-Aid for
Creative Basic Research (#12NP0201) from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.

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