A novel phosphorylated glycoprotein in the shell matrix of
the oyster Crassostrea nippona
Tetsuro Samata, Daisuke Ikeda, Aya Kajikawa, Hideyoshi Sato, Chihiro Nogawa, Daishi Yamada,
Ryo Yamazaki and Takahiro Akiyama
Laboratory of Cell Biology, Faculty of Environmental Health, Azabu University, Sagamihara, Japan
Subsequent to the pioneering work of Miyamoto et al.
[1], Sudo et al. [2] and Shen et al. [3], more than 20
genes encoding the organic matrix (OM) components
of molluscan shells have been determined and their
deduced amino acid sequences clarified [4–12]. How-
ever, the information available to date has been
restricted to the nacreous and prismatic layers of pearl
oysters, leaving the other shell layers poorly investi-
gated at the molecular level. One exception is the find-
ing of acidic glycoprotein MSP-1 in the foliated layer
of Patinopecten yessoensis [4].
Through their control of nucleation, growth, mor-
phology and polymorphism of CaCO
3
crystals, these
OMs are commonly assumed to be intimately associ-
ated with every phase of molluscan biomineralization,
and thus with the overall regulation of the shell micro-
structure. More recent investigations have primarily
involved in vitro measurement of OM activities related
to crystal formation [13–18]. Although these studies
have clearly shown that OM modulates molluscan bio-
mineralization, the results nevertheless demonstrate
marked methodology-dependent variation. The func-
tion of OM thus remains unclear, even in vitro, and is
a topic of future research.

Disulfide-dependent MPP1 polymers occurring in the form of multimeric
insoluble gels are estimated to contain repetitive locations of the anionic
molecules of phosphates and acidic amino acids, particularly Asp. Thus,
MPP1 and its polymers possess characteristic features of a charged mole-
cule for oyster biomineralization, namely accumulation and trapping of
Ca
2+
. In addition, MPP1 is the first organic matrix component considered
to be expressed in both the foliated and prismatic layers of the molluscan
shell microstructure. In vitro crystallization assays demonstrate the induc-
tion of tabular crystals with a completely different morphology from those
formed spontaneously, indicating that MPP1 and its polymers are poten-
tially the agent that controls crystal growth and shell microstructure.
Abbreviations
CBB, Coomassie brilliant blue; GISM, translucent gelatinous insoluble organic matrix; ISM, insoluble organic matrix; MPP1, molluscan
phosphorylated protein 1; ntp, nucleotide position; OM, organic matrix; SM, soluble organic matrix.
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2977
prismatic, foliated, chalky and granular structures. In
particular, the shells of oyster species are composed of
a highly complex microstructure consisting of the
chalky layer in addition to the foliated and prismatic
layers. The foliated layer is formed by the aggregation
of units termed lath, each with a width of 2 lm and
length of 10 lm [19], whereas the chalky layer has a
homogeneous morphology composed of tiny calcite
granules [19]. A variety of studies, mostly based on
amino acid analysis of bulk soluble matrix (SM) and
insoluble matrix (ISM) [20–24], have shown the pres-
ence of OM in oyster species with particularly highly
acidic properties. This high acidity is due to Asp and

Biochemical characterization of the OM
components extracted from oyster shell
Fractionation of the bulk OM separated two fractions,
namely the SM at approximately 20 mg per 50 g of
shell and the ISM, which was further sub-divided into
two components: a predominant translucent gelatinous
insoluble organic matrix (GISM) pellet at approxi-
mately 120 mg per 50 g of shell and a small quantity
of fibrous precipitate at approximately 5 mg per 50 g
of shell.
After SDS ⁄ PAGE of GISM, which was largely-
solubilized in a sample buffer containing 2-mercaptoeth-
anol after boiling, and subsequent staining procedures
with negative staining, Stains-all and Methyl green visu-
alized an exclusive band of approximately 52 kDa,
which showed a negative reaction with Coomassie
brilliant blue (CBB) (Fig. 1).
SDS ⁄ PAGE of the 52 kDa component after enzy-
matic deglycosylation and dephosphorylation showed
apparent downward shifts in molecular masses of
2.5 and 3.5 kDa, respectively (Fig. 2).
Table 1 shows that the 52 kDa component in GISM
exhibits an amino acid composition, strikingly domi-
nated by Asx (aspartic acid plus asparagine), which,
together with Ser and Gly, accounted for more than
80% of the total residue. By contrast, the bulk SM
showed a different amino acid composition, which
comprised large amounts of Asx, Glx (glutamic acid
plus glutamine) and Gly, and a much smaller amount
of Ser than that of GISM.

protein and an additional sequence of DNNGDGNG
(score of 16) in the NG repeat sequence at the C-ter-
minal region were characteristic. The same result was
obtained using the Sequest search engine. The most
appropriate condition for Asp-N digestion was the
addition of 75 ng of enzyme to 15 lg of protein.
FTIR analysis of GISM showed the most intensive
absorption peaks at 1654 cm
)1
and 1561 cm
)1
, corre-
sponding to amides I and II, respectively, characteristic
in protein moiety (Fig. 3) [29]. The small peak at
1243 cm
)1
may represent amide III, sulfates or phos-
phates and that at 1408 cm
)1
may be associated with
carboxylate [29,30]. An additional large absorption
peak occurred at around 1097 cm
)1
, which was consid-
ered to be associated with carbohydrates [29,30].
Cloning and sequencing of cDNA encoding the
OM component in the foliated layer
A nucleotide fragment of approximately 320 bp was
amplified using the primer pairs of F1 and R1. Nucle-
otide sequences of the primer positions in this frag-

poly(vinylidene difluoride) membrane.
44 kDa
deduced
protein
52 kDa
component SM
Asx 26.77 28.49 32.87
Thr 0.40 0.63 1.31
Ser 33.80 30.29 4.72
Glx 3.42 2.83 11.51
Pro 0.60 1.16 0.69
Gly 28.97 25.93 32.40
Ala 0.40 0.98 1.38
Val 0.60 1.11 1.24
Met 0.00 0.73 1.12
Cys 2.62 1.92 0.86
Ile 0.00 0.41 0.33
Leu 0.00 0.67 0.98
Tyr 1.81 2.27 3.88
Phe 0.00 0.45 0.61
Trp 0.00 0.00 0.73
Lys 0.40 1.08 2.75
His 0.00 0.00 0.00
Arg 0.20 0.85 2.62
T. Samata et al. A novel acidic glycoprotein from the oyster shells
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2979
gene-specific primer F2 based on the nucleotide
sequence of fragment A amplified a fragment of
approximately 1040 bp (fragment B), in which the F1
primer annealed with the same sequence, located

Fig. 4. Nucleotide sequence of the 1.9 kb
cDNA and deduced amino acid sequence.
Numbers on the left indicate the nucleotide
positions in the 1.9 kb cDNA sequence
(upper) and positions of the amino acid resi-
dues in the deduced protein (lower). The
putative signal peptide is underlined. The
start codon (ATG), stop codon (TAG) and
putative polyadenylation signal (AATAAA)
are boxed.
A novel acidic glycoprotein from the oyster shells T. Samata et al.
2980 FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS
nucleotide and deduced amino acid sequences are
shown in Fig. 4.
Deduced protein structure encoded by the 1.9 kb
cDNA
The deduced protein encoded by the 1.9 kb cDNA
fragment encompassed 516 amino acid residues and
had a calculated molecular mass before post-transla-
tional modification of 46561.41 Da. Following the typ-
ical sequence for signal peptide, comprising 19 amino
acids, the N-terminal amino acid of the mature protein
was expected to be Ala based on the prediction using
neutral networks and hidden Markov models. Eventu-
ally, the molecular mass of the mature protein was
estimated to be 44490.85 Da, containing 497 amino
acid residues.
The amino acid composition of the deduced protein
was characterized by a high proportions of Ser
(33.80%), Gly (28.97%) and Asp (26.77%), which

over Glu, and a single Cys residue was located at its
center.
A search of the nonredundant GenBank CDS data-
base using blast (protein–protein blast and Search
for short, nearly exact matches) showed a similarity of
34.4% between the sequence throughout the molecules
of the deduced 44 kDa protein and MSP-1, with only
exceptional high similarity between the SG domain of
them (Fig. 6). Partially high correspondence with phos-
phophorin, a dentin Ca-binding phosphoprotein [31],
and Lustrin A [3], a molluscan OM protein from a
gastropod Haliotis rufescens, was observed over the 50
amino acids comprising the SG domain of this protein.
No clear homology with any other protein occurring
in the database.
Motif analyses by scanprosite (provided by Swiss
Institute of Bioinformatics, SIB, Geneva, Switzerland)
and netphosk (provided by Center for Biological
Sequence Analysis BioCentrum-DTU Technical Uni-
versity of Denmark, Lyngby, Denmark) suggested that
35 and 45 casein kinase II phosphorylation sites were
present, respectively. A motif of an N-glycosylation
site was detected at two positions of the molecule. An
additional motif of GAGs (glucose aminoglycans)-
binding indicated as DGSD was confirmed at two
positions of the C-terminal region.
With consideration of the phosphorylation sites
and excluding the putative signal peptide, use of the
scansite tools of the ExPASy server showed that the
deduced 44 kDa protein had a very low theoretical pI

crystal growth
assay’, characteristic inhibitory efficiency against crystal
formation was recognized after addition of the SM,
GISM and the 52 kDa component to the crystallizing
solution. Inhibition was observed as a change in crystal
morphology, from a characteristic rhombohedral shape
to a poor crystalline habit with rounded edges for calcite
crystals, and from a spherical shape with needle-like
structure to a spherulite shape with smooth surfaces for
aragonite crystals, and the complete loss of crystal shape
for both in an additive volume-dependent manner. One
interesting result obtained by contrast interference
microscopy was the induction of tabular crystals of oval
to quadrangular shape with rough edges and very fine
parallel stria along the bottom face when the three
above mentioned components were added to the arago-
nitic crystallizing solution with the underlying GISM-
derived membrane. These crystals were observed to be
tightly adhered to the membrane in a manner com-
pletely different from those inorganically formed or
those formed without fixative (Fig. 8A-1, 2). Scanning
electron microscopy of the edge of the crystals revealed
the presence of rod-like rectangular structures with
a striking morphological appearance and dimensions
closely comparable to those of the folia (Fig. 8B).
Consistent with the findings of Wheeler et al. [15], an
instantaneous decrease in pH was seen in the ‘CaCO
3
precipitation assay’ when CaCl
2

several purified components that comprise the SM, as
described above [20–24]. Because no other component
exhibits the same composition or is stained with both
negative staining and Methyl green, we assume that
the 52 kDa component, which accounts for a consider-
able part of the gelatinous material in the foliated
layer, is the main phosphorylated glycoprotein. A sec-
ond key component in oyster biomineralization might
be the polyanionic components contained in the SM,
although their primary structures are still unclear.
The predicted amino acid composition of the
deduced 44 kDa protein agrees well with that of the
52 kDa component in the foliated layer of C. nippona
(Table 1) and the 54 kDa phospholylated component
(RP-1) in the same layer of C. virginica [26], as well as
those of the bulk OMs reported from several oyster
species described to date [20–24]. In addition,
LC ⁄ MS ⁄ MS analysis of the endoproteinase Asp-N
digest of the 52 kDa component revealed the presence
of several peptides with amino acid sequences corre-
sponding to those in the sequence of the genetically
determined 44 kDa protein, although amino acid
sequence analyses using the peptide sequencer failed to
determine the N-terminal sequence of the 52 kDa
component, strongly suggestive of the presence of
N-terminal block. As noted in the present study,
FTIR, amino acid composition and motif analyses all
suggest that the size discrepancy between the deduced
A1
B

(pH)
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
Fig. 9. Recordings of CaCO
3
precipitation by ‘CaCO
3
precipitation
assay’. (A) Reference experiment performed by addition of distilled
water (DW) to the crystallizing solution. (B, D, E) Addition of the
52 kDa component to the crystallizing solution at 2.5 lg (B), 10 lg
(D) and 50 lg (E). (C) Addition of phosphovitin to the crystallizing
solution at 50 lg.
T. Samata et al. A novel acidic glycoprotein from the oyster shells
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2983
44 kDa protein and the 52 kDa component may be
attributed to post-translational phosphorylation and
glycosylation. This assumption was supported by the
results obtained for the enzymatic dephosphorylation
and deglycosylation experiments of the 52 kDa com-
ponent. These data indicate with high probability that
the 1.9 kb cDNA is the gene encoding the 52 kDa

proteins, only MSP-1 has been confirmed as being dis-
tributed in the shell, as demonstrated by the N-termi-
nal amino acid sequence of the OM component
matching that deduced from the nucleotide sequence
of the MSP-1 gene, although a band with a compara-
ble molecular size as that of MSP-1 could not be vali-
dated by SDS ⁄ PAGE.
Regarding the modular structure of MPP1, the
remarkable DE-rich sequence appears to be anoma-
lous, in that the continuous repeats of Asp are inter-
rupted by a single Cys residue, which is conserved in
all DE-rich sequences except one. This sequence con-
servation of Cys hints at its functional significance,
namely that it is incorporated in the formation of
intra- or inter-molecular disulfide bonds. In the latter
case, MPP1 monomer may be self-assembled to a poly-
mer, converting them to an insoluble form, although
the mechanism of this insolubility is unknown.
The secondary structure of MPP1 estimated by the
method of Chau and Fasman [33] consists predomi-
nantly of a loop structure, which mainly corresponds
to the repeated arrangement of the SG domain with
densely distributed phosphorylation sites inserted by
the DE-rich sequence. In turn, this gives rise to the
regular arrangement of the anionic molecules of
phosphates and acidic amino acids. Given this
assumption, disulfide-dependent MPP1 polymers
occurring in the form of multimeric insoluble gels
can be estimated to contain a massively repeating
acidic region. MPP1 polymers may thus participate in

in CaCO
3
crystal lattices, and thus controls crystal polymor-
phism. However, it should be noted that highly acidic
proteins have been associated with calcitic shell lay-
ers, indicating the potential involvement of the Asp-
and ⁄ or p-Ser rich components in calcite formation
not only in the prismatic layers, but also in the foli-
ated layers. This notion is supported by the results of
the present study.
By contrast to this notion, however, our in vitro
crystallization assay showed that the OM compo-
nents had an inhibitory effect against CaCO
3
crystal
formation. This does not necessari ly imply a negative
role for the OM components in oyster shell biomin-
eralization because, although the soluble and the
additive components inhibited crystal formation when
present in the isolated state, the same molecules
induced tabular crystals with a completely different
morphology from spontaneously formed crystals
when pre-mixed with underlying GISM-derived mem-
brane. Unfortunately, X-ray diffractional analysis of
the tabular crystals failed to determine their mineral-
ogy due to their small quantities, which were far less
than the minimum detectable quantity. The basement
membrane is an artificial material, which is prepared
from the gelatinous pellet by clumping together on
drying. The surface area of the membrane may

and highlight the control of CaCO
3
polymorphism
and shell microstructure in molluscs. In further trials
to obtain a whole figure of molluscan shell biominer-
alization, several additional factors must be taken
into consideration; namely, the behaviour of cells, the
composition of extrapallial fluids, functions of the
signal molecules regulating expression of the OM
component, as well as environmental factors, as
described by Kuboki et al. [40]. Genetical research
combined with an analyses of these factors may com-
prise a potential tool for the elucidation of molluscan
biomineralization in the future.
Experimental procedures
Molluscan materials
We used live individuals of C. nippona cultured at the hatch-
ery of Shimane Technology Center for Fisheries, Japan.
Extraction and purification of the organic matrix
proteins
Shell surfaces were cleaned with an electric rotary grinder
(JOY-ROBO, Cannock, UK) to roughly remove perios-
tracum and adherent hard tissues. Pieces of folia were
carefully separated from the powder of chalky material
and then immersed in 5% NaClO for 30 min to remove
organic contaminants. After rinsing with distilled water
(DW) and air-drying, folia were ground into powder with
a ball mill (ITO Manufacturing, Nagano, Japan). The
powdered folia was decalcified with 5% acetic acid for
3 days at 4 °C under constant stirring and with pH regu-

Enzymatic digestion and LC ⁄ MS ⁄ MS analysis
V8 protease (Pierce, Rockford, IL, USA) and endoprotein-
ase Asp-N protease (Roche, Basel, Switzerland) were added
to the gel pieces, which contained the 52 kDa component
dissolved in 50 mm sodium phosphate buffer (pH 7.8). The
amounts of the enzymes and proteins were changed at a ratio
between 1 : 50 and 1 : 200. After incubation at 37 °C for
18 h, the protease digests were dried and dissolved in 10 lL
of trifluoroacetic acid, and then cleaned up by Zip-tip (Milli-
pore). Purified digests were subjected to LC ⁄ MS ⁄ MS anal-
ysis on a Paradigm MS4 LC System coupled to a model
LCQ ion trap mass spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA) equipped with an electrospray inter-
T. Samata et al. A novel acidic glycoprotein from the oyster shells
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2985
face utilizing a C18 column (Michrom Bioresources, Auburn,
CA, USA).
Deglycosylation and dephosphorylation
experiments
PNgase F (Roche) digestion of GISM was carried out as
described below. After addition of 100 lL of incubation
buffer [50 mm sodium phosphate buffer (pH 7.8), 10 mm
EDTA (pH 8.0), 0.5% (v ⁄ v) Nonidet P40, 0.2% (w ⁄ v)
SDS, 1% (v ⁄ v) 2-mercaptoethanol] to an equivalent volume
of GISM, the mixture was incubated for 18 h at 37 °C with
2 units of PNgase F.
Alkaline phosphatase (Roche) digestion of GISM was
carried out according to the manufacturer’s instructions.
After addition of 5 l Lof10· phosphatase buffer to 45 lL
of GISM, the reaction mixture was incubated for 1.5 h at

of the SGSSSSS and GGNGGDG of the MSP-1 gene,
respectively. Primers were supplied by Texas Genomics
Japan (Tokyo, Japan). PCR amplification was performed
using KOD-Plus as an enzyme for extensive reaction with a
thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA).
3¢-RACE was carried out using a set of primers of an
adaptor primer (TCG AAT TCG GAT CCG AGC TCT)
and the gene-specific primer of F2 (forward 918) (5¢-TGC
GAT GAT GAT GAC AGC GGA-3¢), based on the nucle-
otide sequence of the cDNA fragment obtained from the
first PCR.
5¢-RACE was primed using a Smart Race Kit (Clontech,
Mountain View, CA, USA) using a set of an adaptor UPM
and the gene-specific primer of R2 (reverse 1056) (5¢-TGC
GAG GAT GGT GGT GAT GGA-3¢), designed from the
nucleotide sequence of the cDNA fragment amplified by 3¢-
RACE.
The full length of the cDNA encoding the oyster OM
protein was amplified using a set of the gene-specific prim-
ers of F3 (forward 136) (5¢-CCT AGA AGA ATA CAT
CGG GGT-3¢), and R3 (reverse 1827) (5¢-TCT GGC ATG
AAA CAC GAC AAC-3¢), based on the nucleotide
sequences of the 5¢ and 3¢ terminal regions, respectively.
TA cloning
After purification and A-tailing, the PCR products were
used for ligation with pGEM-T Easy Vectors (Promega),
and catalyzed with T4 DNA ligase at 4 °C for 16 h. The
ligation products were supplied for transformation of
JM109 high-efficiency competent cells (Promega). Positive
clones were selected by blue ⁄ white colour screening and

crystal growth
assay’, crystals were induced by incubation of an additive
SM, GISM or the 52 kDa component with or without
basement GISM-derived membrane as a fixative in a satu-
rated solution of CaCO
3
. Several kinds of experiments have
been attempted to use different solutions modulated for
crystal induction. We used the system described by Sekigu-
chi and Samata [44], which was an improvement of a sys-
tem originally developed by Kitano [45,46], in which super-
saturation for CaCO
3
could be maintained only in the ori-
ginal solution by bubbling CO
2
gas, followed by a gradual
decline in saturation by CO
2
removal. This experiment used
two types of crystallizing solutions, namely calcitic crystal-
lizing solution (10 mm CaCl
2
) to ensure 100% calcite
formation and aragonitic crystallizing solution (10 mm
CaCl
2
containing 12 mm MgCl
2)
to ensure 100% aragonite

Acknowledgements
We thank Dr T. Yamane for assistance and advice
on sample collection, Dr N. Wada for discussion of
in vitro crystallization assays, Dr R. Mineki for advice
on calculation of amino acid composition and Dr
D. Higo for analysis of LC ⁄ MS data. This work was
supported in part by the Promotion and Mutual Aid
Corporation for Private Schools of Japan, Grant-in-Aid
for Matching Fund Subsidy for Private Universities.
References
1 Miyamoto H, Miyashita T, Okushima M, Nakano S,
Morita T & Matsushiro A (1996) A carbonic anhydrase
from the nacreous layer in oyster pearls. Proc Natl Acad
Sci USA 93, 9657–9660.
2 Sudo S, Fujikawa T, Nagakura T, Ohkubo T, Sakagu-
chi K, Tanaka M, Nakashima K & Takahashi T (1997)
Structures of mollusc shell framework proteins. Nature
387, 563–564.
3 Shen X, Belcher AM, Hansma PK, Stucky GD & Morse
DE (1997) Molecular cloning and characterization of
lustrin A, a matrix protein from shell and pearl nacre of
Haliotis rufescens. J Biol Chem 272, 32472–32481.
4 Sarashina I & Endo K (2001) The complete primary
structure of molluscan shell protein 1 (MSP-1), an
acidic glycoprotein in the shell matrix of the scallop
Patinopecten yessoensis. Mar Biotechnol 3, 362–369.
5 Samata T, Hayashi N, Kono M, Hasegawa K, Horita
C & Akera S (1999) A new matrix protein family
related to the nacreous layer formation of Pinctada
fucata. FEBS Lett 462, 225–229.

proteins in the shell of the pearl oyster Pinctada
fucata. Comp Biochem Physiol B 144, 254–262.
T. Samata et al. A novel acidic glycoprotein from the oyster shells
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2987
13 Watabe N & Wilbur KM (1960) Influence of the
organic matrix on crystal type in mollusks. Nature 188,
334.
14 Wilbur KM & Watabe N (1963) Experimental studies
on calcification in mollusc and the alga Coccoliths
huxleyi. Ann NY Acad Sci 109, 82–112.
15 Wheeler AP, George JW & Evans CA (1981) Control
of calcium carbonate nucleation and crystal growth by
soluble matrix of oyster shell. Science 212, 1397–1398.
16 Belcher AM, Wu XH, Christensen RJ, Hansma PK &
Morse DE (1996) Control of crystal phase switching
and orientation by soluble mollusc-shell proteins.
Nature 381, 56–58.
17 Falini G, Albeck S, Weiner S & Addadi L (1996) Con-
trol of aragonite or calcite polymorphism by mollusk
shell macromolecules. Science 271, 67–69.
18 Heinemann F, Treccani L & Fritz M (2006) Abalone
nacre insoluble matrix induces growth of flat and ori-
ented aragonite crystals. Biochem Biophys Res Commun
344, 45–49.
19 Sikes CS, Wheeler AP, Wierzbicki A, Mount AS &
Dillaman RM (2000) Nucleation and growth of calcite
on native versus pyrolyzed oyster shell folia. Biol Bull
198, 50–66.
20 Wheeler AP, Rusenko KW & Sikes CS (1988) Regula-
tion of in vivo CaCO

27 Wheeler AP (1992) Phosphoprotein of oyster (Crassos-
trea verginica) shell organic matrix. In Hard Tissue Min-
eralization and Demineralization (Suga S & Watabe N,
eds), pp. 171–187. Springer-Verlag, Berlin Hederberg,
Tokyo.
28 Kawaguchi T & Watabe N (1993) The organic matrices
of the shell of the American oyster Crassostrea virginica
Gmelin. J Exp Mar Biol Ecol 170, 11–28.
29 Dauphin Y (2003) Soluble organic matrices of the cal-
citic prismatic shell layers of two Pteriomorphid bival-
ves. Pinna nobilis and Pinctada margaritifera. J Biol
Chem 278, 15168–15177.
30 Pouchert CJ (1970) The Aldrich Library of Infrared
Spectra. Aldrich Chemical Co. Inc., Milwaukee, WI.
31 Gu K, Chang SR, Slaven MS, Clarkson BH, Ruther-
ford RB & Ritchie HH (1999) Human dentin phospho-
phoryn nucleotide and amino acid sequence. Eur J Oral
Sci 106, 1043–1047.
32 Marin F, Amons R, Guichard N, Stigter M, Hecker A,
Luquet G, Layrolle P, Alcaraz G, Riondet C & Westb-
roek P (2005) Caspartin and calprismin, two proteins of
the shell calcitic prisms of the Mediterranean fan mussel
Pinna nobilis. J Biol Chem 280, 33895–33908.
33 Chou PY & Fasman GD (1978) Prediction of the sec-
ondary structure of proteins from their amino acid
sequence. Adv Enzymol 47, 145–148.
34 Greenfield GEM, Wilson DC & Crenshaw MA (1984)
Ionotropic nucleation of calcium carbonate by mollus-
can matrix. Amer Zool 24, 925–932.
35 Norizuki M & Samata T (2008) Distribution and func-

A novel acidic glycoprotein from the oyster shells T. Samata et al.
2988 FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS
42 Cutting JA & Roth TF (1973) Staining of phosphor-
proteins on acrylamide gel electrophoregrams. Anal
Biochem 54, 386–394.
43 Fernandez-Patron C, Calero M, Collazo PR, Garcia
JR, Madrazo J, Musacchio A, Soriano F, Estrada R,
Frank R & Castellanos-Serra LR (1995) Protein reverse
staining: high-efficiency microanalysis of unmodified
proteins detected on electrophoresis gels. Anal Biochem
224, 203–211.
44 Sekiguchi N & Samata T (2003) Functionrelated to
crystallization of the organic matrix isolated from
molluscan and larger foraminifera shells. J Fossil Res
36, 12–15.
45 Kitano Y (1968) Factors controlling the crystal type
and minor element content of carbonates formed in
marine biological systems – the geochemical significance
of carbonate fossil. Chem Ind 21, 1017–1028.
46 Kitano Y (1986) Geochemical study on the formation
of carbonate sediment. J Oceanogr Soc Japan 42, 402–
420.
T. Samata et al. A novel acidic glycoprotein from the oyster shells
FEBS Journal 275 (2008) 2977–2989 ª 2008 The Authors Journal compilation ª 2008 FEBS 2989