Tài liệu Báo cáo Y học: Soluble silk-like organic matrix in the nacreous layer of the bivalve Pinctada maxima A new insight in the biomineralization field pptx

Soluble silk-like organic matrix in the nacreous layer of the bivalve
Pinctada maxima
A new insight in the biomineralization field
Lucilia Pereira-Mourie
`
s
1
, Maria-Jose
´
Almeida
1,3
, Cristina Ribeiro
3
, Jean Peduzzi
2
, Michel Barthe
´
lemy
2
,
Christian Milet
1
and Evelyne Lopez
1
1
Laboratoire de Physiologie Ge
´
ne
´
rale et Compare
´

e, UMR CNRS 8572, Muse
´
um National d’Histoire Naturelle, Paris, France;
2
Laboratoire de Chimie des Substances Naturelles, ESA CNRS 8041, Muse
´
um National d’Histoire Naturelle, Paris, France;
3
INEB – Instituto de Engenharia Biome
´
dica, Rua do Campo Alegre, Porto, Portugal
Nacre organic matrix has been conventionally classified as
both Ôwater-solubleÕ and Ôwater-insolubleÕ,basedonits
solubility in aqueous solutions after decalcification with acid
or EDTA. Some characteristics (aspartic acid-rich, silk-
fibroin-like content) were specifically attributed to either one
or the other. The comparative study on the technique of
extraction (extraction with water alone vs. demineralization
with EDTA) presented here, seems to reveal that this gen-
erally accepted classification may need to be reconsidered.
Actually, the nondecalcified soluble organic matrix, extrac-
ted in ultra-pure water, displays many of the characteristics
of what until now has been called Ôinsoluble matrixÕ.We
present the results obtained on this extract and on a
conventional EDTA-soluble matrix, with various charac-
terization methods: fractionation by size-exclusion and
anion-exchange HPLC, amino acid analysis, glycosami-
noglycan and calcium quantification, SDS/PAGE and
FTIR spectroscopy. We propose that the model for the
interlamellar matrix sheets of nacre given by Nakahara [In:

Biomineralization and Biological Metal Accumulation,
Westbroek, P. & deJong, E.W., eds, (1983) pp. 225–230.
Reidel, Dordrecht, Holland] and Weiner and Traub
[Phil.Trans.R.Soc.Lond.B(1984) 304, 425–434] may no
longer be valid. The most recent model, proposed by
Levi-Kalisman et al.[J. Struct. Biol. (2001) 135, 8–17],
seemed to be more in accordance with our findings.
Keywords: nacre; undecalcified soluble matrix; EDTA-
soluble matrix; hydrophobicity; silk-fibroin-like-proteins.
In the biomineralization field, the mollusk shell is one of
the best studied of all calcium carbonate biominerals.
Particular attention has been given to the organic matrix
[1–5]. The latter is thought to promote the nucleation of
the mineral component, to direct the crystal growth and to
act as glue, preventing fracture of the shell [6–9]. The main
biopolymers present in the organic matrix are essentially
proteins, either glycosylated or not, acidic polysaccharides
and chitin. In nacre, they represent 1–5% (w/w) of the
structure.
From the earliest experiments, it was believed that the
biochemical properties of matrix constituents depend of
the use of a decalcification procedure for removing the
mineral component, which is strongly associated with the
organic matrix [1,3]. Therefore, all investigations up until
now used either EDTA, acetic acid or hydrochloric acid
for this demineralization step and, subsequently, two
fractions of the organic matrix were separated, based on
their solubility in aqueous solutions. Accordingly, a
designation of matrix into two classes, the soluble matrix
and the insoluble matrix, has evolved from this extraction

[10–14].
This paper presents for the first time the results of a
comparative study on the organic matrix extracted from
the nacreous layer of the shell from the pearl oyster
Pinctada maxima by two very different methods. The first
is a nondecalcifying technique obtained by an extraction in
ultra-pure water. This unconventional approach arises
from previous in vivo and in vitro experiments where we
showed that biochemical signals from nacre chips were able
to diffuse in the surrounding media and to induce new
bone formation [15–22]. In an attempt to identify these
signal molecules, we have previously perfected this original
method of extraction of the organic matrix, without any
acid treatment or demineralization, in order to minimize
any possible alteration of the activity of the macromole-
cules [20,23]. The second method is one of the widely used
extraction techniques which involves a demineralization
with EDTA followed by intensive dialysis against distilled
water. The content of the respective soluble matrix extracts
were very different and seemed to raise important
questions about the actual conventional classification of
the soluble (known as acidic and aspartic acid-rich) and
insoluble (said to be hydrophobic and glycine, alanine-rich)
matrices and on the current model of nacre organic matrix
organization.
Correspondence to E. Lopez, Laboratoire de Physiologie Ge
´
ne
´
rale

et Compare
´
e, UMR CNRS 8572, Muse
´
um National d’Histoire
Naturelle, 7 rue Cuvier, 75231, Paris Cedex 05, France.
Fax: +33 1 40795620, Tel: +33 1 40793622,
E-mail:
Abbreviations:EDTA-IM,EDTA-insolublematrix;EDTA-SM,
EDTA-soluble matrix; GAG, glycosaminoglycan; PG, proteoglycan;
WIM, water-insoluble matrix; WSM, water-soluble matrix.
(Received 22 April 2002, revised 16 August 2002,
accepted 23 August 2002)
Eur. J. Biochem. 269, 4994–5003 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03203.x
MATERIALS AND METHODS
Organic matrix extraction
The powdered nacre (particle size 50–150 lm), obtained
from the inner shell layer of the pearl oyster P. maxima,was
extracted by either ultra-pure water or EDTA and then
fractionated into soluble and insoluble matrix by centrifu-
gation. Demineralization of powdered nacre with EDTA
was performed as described by Wheeler et al.[24].Fifty
grams of powdered nacre was dissolved in 100 mL 10%
EDTA disodium salt dihydrate, pH 8, with continuous
stirring for 24 h at room temperature. Then, the suspension
was transferred in a dialysis bag (Spectrapor 2, 12–14 kDa
molecular weight cut-off) and placed in 2 L of the same
EDTA solution (replaced by fresh solution every 12 h), with
stirring and at 4 °C, until the powdered shell was completely
demineralized (about 3 days). The extract was centrifuged at

27 000 g for 30 min to separate the EDTA-soluble matrix
(EDTA-SM) from the EDTA-insoluble matrix (EDTA-
IM).ToremoveEDTA,thetwosampleswereextensively
dialyzed against ultra-pure water (Milli-Q
TM
), at 4 °C, and
the EDTA-SM was freeze-dried. The water-soluble matrix
(WSM) was obtained as described in Almeida et al.[20].
Fractionation of soluble extracts by HPLC
The WSM and the EDTA-SM were separated by size-
exclusion high performance liquid chromatography
(SE-HPLC), and by anion-exchange HPLC (AE-HPLC),
as described elsewhere [20].
For the SE-HPLC, a solution of 30 mg (dry
weight)ÆmL
)1
was prepared for the two soluble extracts
and filtered through a 0.22 lm centrifuge tube filter (Spin-
XÒ, Costar) before injection. Aliquots of WSM (500 lL) or
EDTA-SM (250 lL) were injected onto the preparative
column. Proteins were eluted with ultra-pure water rather
than with a salt buffer in order to preserve the integrity of
the macromolecules [20,21].
Amino acid analysis
Samples of proteins were hydrolyzed at 110 °C under
vacuum with 6
M
HCl constant boiling (Sigma) for 24 h.
Phosphoserine was determined from hydrolysis in 4
M

HCl
for 6 h [25]. The resulting amino acids were separated on a
cation exchange PC6A resin (Pierce) and the o-phthaldial-
dehyde derivatives of amino acids were detected with a
Waters 420 fluorimeter. Proline, hydroxyproline and
hydroxylysine were examined at 254 nm by reverse-phase
HPLC of their phenylisothiocarbamate derivatives [26], as
reported previously [20]. The serine, threonine and tyrosine
contents of the hydrolysates were corrected for destruction
during the hydrolysis by extrapolation to zero time hydro-
lysis. The amino acid compositions, expressed as a mole
percent, represent the average of at least three independent
determinations. The amount of protein in each extract was
calculated from the amino acids’ molar yields.
Glycosaminoglycan analysis
Organic matrix samples were dissolved in 5 mL of 0.1
M
NaOH [27] at room temperature for 24 h with periodic
mixing and maceration, followed by centrifugation at
1000 g for 10 min. Sulfated and nonsulfated glycosamino-
glycans (GAGs) from the supernatant were estimated by the
Whiteman Alcian blue binding technique [28,29], using
chondroitin sulfate as standard. The assay was adapted to
the estimation of GAG in more dilute samples by increasing
the aliquot size, as proposed by Gold [30].
Calcium analysis
Calcium analyses were performed after nitric acid hydrolysis
of samples, by atomic absorption spectrophotometry, using
a GBC 904AA spectrophotometer.
Fourier transform infrared (FTIR) spectroscopy

Organic compounds binding groups from soluble matrix
samples were detected by FTIR spectrometry. The FTIR
spectra were obtained using a Perkin Elmer 200FTIR
spectrometer. All the samples were prepared as KBr discs
and were run at a spectral resolution of 4 cm
)1
.One
hundred scans were acquired for each sample.
Polyacrylamide gel electrophoresis
The proteins from the soluble samples were separated under
denaturating conditions (30 lg total protein per well) by
SDS/PAGE [31] using 12% polyacrylamide mini-gels (Mini
Protean III apparatus, Bio-Rad) of 0.75 mm thickness.
After electrophoresis, proteins were visualized by silver-
staining, as described by Morrissey [32], without the
glutaraldehyde step. The molecular masses were estimated
using the Amersham Pharmacia Biotech LMW-SDS
marker kit for electrophoresis.
RESULTS
Extraction of soluble matrix
After extraction and lyophilization, the two soluble extracts
(WSM and EDTA-SM) were re-suspended in ultra-pure
water for protein analysis. The protein recovery was similar
in the two cases, with about 0.05% by weight of powdered
nacre.
Fractionation of soluble extracts by HPLC
The size-exclusion chromatographic profiles for WSM and
EDTA-SM were very different (Fig. 1). As described in
Almeida et al. [20], the soluble matrix recovered by an
aqueous extraction (WSM) was separated in four distinct

fractions (Fig. 1A). On the contrary, under the same
conditions, the soluble matrix obtained after nacre demin-
eralization by EDTA (EDTA-SM) consisted of only two
different fractions (Fig. 1B). Absorbance values were very
high for EDTA-SM, in comparison with those obtained for
WSM, whereas the volume of EDTA-SM injected was half
the WSM volume. The low molecular weight peak due to
the use of EDTA, often mentioned in the literature [24,33],
was not observed in the size-exclusion profiles of the
EDTA-SM extract (Fig. 1B).
The fractionation by anion-exchange HPLC was also
different for WSM and EDTA-SM. Separation was better
Ó FEBS 2002 Soluble silk-like organic matrix in nacre (Eur. J. Biochem. 269) 4995
resolved for EDTA-SM than for WSM (Fig. 2). The main
peak from each separation, indicated by an asterisk in
Fig. 2, was collected and submitted to amino acid analysis.
Amino acid compositions
In accordance with previous studies on nacre EDTA-SM on
other mollusk shells [34,35], this extract was aspartate-rich
(nearly 40 mole percent) (Table 1) and exhibited a charge to
hydrophobic ratio (C/HP; Asx, Glx, His, Arg, Lys/Ala, Pro,
Val, Met, Ile, Leu, Phe) of 2.86. The main amino acids
found in EDTA-SM were aspartate and glycine (69.2% of
total amino acids). Previous studies with regard to soluble
organic matrix of mollusk shells indicated that more than
80% of the aspartate and glutamate is in the form of
aspartic and glutamic acid, respectively [34]. In order to
compare our results with published data [4,33,36], we also
determined the global amino acid composition of the
EDTA-IM. Here again, the composition was as expected,

i.e. very hydrophobic (C/HP ¼ 0.42) and glycine, alanine-
rich. Unexpectedly, the soluble matrix obtained by aqueous
extraction, WSM, likewise exhibited more than 60% of
glycine and alanine residues and a large proportion of
hydrophobic amino acids, resulting in a very low C/HP
value (0.29). Its content in Asx was moderate. These
features are exactly the opposite of that found in EDTA-SM
which exhibits a high Asx content (39.6%) and a low
content for alanine (6.5%). Thus, the alanine and glycine
content of WSM was rather similar to that of EDTA-IM. In
spite of the presence of mineral, it was possible to analyze
the amino acid composition of the water insoluble matrix
(WIM). Again we found a high content in Gly-Ala, and the
global composition was very similar to the WSM, and
consequently the EDTA-IM. Hydroxyproline, hydroxy-
lysine and phosphoserine were not detected in WSM, WIM,
EDTA-SM and EDTA-IM.
The comparison of the amino acid composition of the
main peak obtained from AE-HPLC of WSM and EDTA-
SM is given in Table 2. These two peaks were characterized
by a high content in glycine-alanine (31–30.6%) and serine.
As expected from an anion-exchange separation, the two
peaks contained acidic proteins. Nevertheless, they were
aspartic-rich in EDTA-SM whilst being quite glutamic acid-
rich in WSM.
Glycosaminoglycan analysis
Glycosaminoglycans are highly negatively charged because
of the presence of sulfate ester and/or carboxyl groups.
Therefore, they interact with cations and are precipitated by
cationic dyes like Alcian blue [37].

GAGs were found in WSM and EDTA-SM (Table 3).
Nevertheless, their amount in EDTA-SM was about 15
times as much as that of WSM, suggesting that they are
firmly tightened to the mineral or the aspartic acid-rich
Fig. 2. Anion exchange-HPLC elution profiles of the water-soluble
matrix (A) and the EDTA-soluble matrix (B) of Pinctada maxima nacre.
Samples (55 lL) containing 400 lg (WSM) or 200 lg(EDTA-SM)
protein mixture in 20 m
M
Tris/HCl pH 7.8 were loaded on a Mono Q
HR 5/5 column equilibrated with the same buffer. Proteins were eluted
at a flow rate of 1 mLÆmin
)1
with a 25-min linear gradient from 0 to
100% solvent (500 m
M
NaCl, 20 m
M
Tris/HClbuffer,pH7.8).
Absorbance was monitored at 226 nm. The main peak from each
separation is indicated by an asterisk.
Fig. 1. Size exclusion-HPLC elution profiles of the water-soluble matrix
(A) and the EDTA-soluble matrix (B) of Pinctada maxima nacre.
Samples of protein in ultra-pure water (500 lL and 250 lL, respect-
ively), were injected onto the preparative column (TSKGel G 3000
SW, 600 · 21.5 mm) and proteins were eluted with ultra-pure water at
2.5 mLÆmin
)1
flow rate. Absorbance was monitored at 280 nm. The
column was calibrated with alcohol dehydrogenase (150 kDa), bovine

serum albumin (66 kDa) and lysozyme (17 kDa).
4996 L. Pereira-Mourie
`
s et al. (Eur. J. Biochem. 269) Ó FEBS 2002
matrix. Very few GAGs were found in the pellet (EDTA-
IM) obtained after demineralization.
Calcium analysis
In the EDTA-SM, the amount of nondialyzable calcium
after decalcification was 51.2 lgÆmg
)1
dry weight (Table 3),
and one may suppose that some EDTA remained in the
soluble extract, associated with calcium, as suggested by
Wheeler et al. [24,35]. The presence of residual EDTA after
intensive dialysis of EDTA-SM was also observed in amino
acid composition determination as EDTA eluted near to the
histidine, precluding from detecting this amino acid. The
calcium content of the EDTA-IM was, as expected, much
higher; about 310 lgÆmg
)1
dry weight. This high value may
in part be related to the incomplete demineralization of
powdered nacre, during extraction. In contrast, WSM
contained only 1.1 lg calcium per mg dry weight, confirm-
ing that very small amounts of calcium were dissolved in
water and/or linked to the matrix components.
Fourier transform infrared (FTIR) spectroscopy
Infrared spectroscopy provides means for a characterization
at the molecular level of the structure and bonding of
surface functional groups and adsorbed species. In this

study, we used infrared spectroscopy to identify possible
differences in composition between the decalcified nacre
soluble matrix (EDTA-SM) and the aqueous, nondecalci-
fied, nacre organic matrix (WSM).
The FTIR spectrum of the EDTA-SM (Fig. 3A) was
characterized by two intense bands, one at 3432 cm
)1
(OH
and/or NH stretching modes of the organic matrix compo-
nents) and another, the most intense, at 1593 cm
)1
, possibly
corresponding to the COO coordinated asymmetric stretch-
ing band. The presence of this band resulted from the
EDTA, a potent metal chelator that was used to extract the
soluble matrix from the crystalline structure. The EDTA
molecule has six potential sites for bonding with a metal ion:
four carboxyl groups and the two amino groups. When
EDTA is dissolved in water, it behaves like an amino acid
such as glycine. From the infrared spectrum of a metal
chelated compound of EDTA, it is possible to distinguish
the coordinate and free COO stretching band. The union-
ized and uncoordinated COO stretching band occurs at
Table 2. Amino acid compositions (mole percent) of the main peak from
anion-exchange (AE) HPLC of water-soluble matrix (WSM) and
EDTA-soluble matrix (EDTA-SM). Cysteine, hydroxylysine, hydroxy-
proline, phosphoserine, proline and tryptophan were not determined.
Amino acid AE-WSM AE-EDTA-SM
Asx 9.7 21.0
Thr 3.3 3.7

Ser 27.6 18.3
Glx 13.4 9.5
Gly 22.4 22.9
Ala 8.6 7.7
Val 2.6 2.7
Met 0 0.5
Ile 1.7 2.1
Leu 3.9 2.9
Tyr 1.5 1.5
Phe 1.9 1.4
His 1.7 2.3
Lys 1.3 2.6
Arg 0.8 0.9
C/HP
a
1.44 2.10
a
Ratio charged to hydrophobic residues (see Results, section
Amino acid composition, for details).
Table 1. Amino acid compositions (mole percent) of the water-soluble matrix, water-insoluble matrix, the EDTA-soluble matrix and the EDTA-
insoluble matrix of Pinctada maxima nacre. Results are expressed as a mole percent and represent the mean of at least three independent
determinations. Cysteine and tryptophan were not determined. Hydroxyproline, hydroxylysine and phosphoserine were not detected in all samples.
Amino acid
Water-soluble
matrix
Water-insoluble
matrix
EDTA-soluble
matrix
EDTA-insoluble

matrix
Asx 7.3 10.8 39.6 10.8
Thr 0.9 1.1 1.4 1.1
Ser 3.9 4.0 3.6 4.4
Glx 2.2 2.2 4.3 2.2
Gly 37.6 28.7 29.6 31.5
Ala 30.0 31.8 6.5 30.5
Pro 1.5 1.1 1.2 1.3
Val 2.2 2.1 1.4 1.9
Met 1.2 1.1 1.3 1.0
Ile 1.2 1.3 1.5 1.2
Leu 6.7 6.3 3.4 5.9
Tyr 0.8 1.9 1 1.7
Phe 1.3 1.7 1.4 1.4
His 0.1 0.2 ND
a
0
Lys 1.2 1.7 1.7 1.2
Arg 1.9 4.1 2.1 3.9
C/HP
b
0.29 0.42 2.86 0.42
a
ND, not determined. After intensive dialysis the sample still contained residual EDTA.
b
Ratio charged to hydrophobic residues (see
Results, section Amino acid composition, for details).
Ó FEBS 2002 Soluble silk-like organic matrix in nacre (Eur. J. Biochem. 269) 4997
1750–1700 cm
)1

whereas the ionized and coordinated COO
stretching band appears at 1650–1590 cm
)1
[38]. The latter
frequency depends on the nature of the metal. Although the
EDTA-SM was subjected to intensive dialysis against ultra-
pure water to remove EDTA, the fractions isolated from the
oyster matrix seem to be, for the most part, EDTA protein
complexes. This finding recommends that caution must be
taken in interpreting binding data of EDTA-extracted
matrix. Another strong band appeared at 1409 cm
)1
and
corresponds to the COO symmetric stretching band. We
could also identify the presence of sulfate groups absorbing
at 1284, 1260, 927 and 855 cm
)1
. Absorption bands that can
be attributed to the amide vibrations, namely 1328 cm
)1
(C–N stretching vibration, amide III), 808 cm
)1
(amide
VII), 708 cm
)1
(amide V or VII), 639 and 621 cm
)1
(amide
IV), were also observed. Several bands were present in the
1000–1150 cm

)1
zone, which is the major polysaccharide
absorption region. A band at 1180 cm
)1
was probably due
to in-plane NH
2
rocking. It is also possible that the small
bands located at 963, 985 and 1032 cm
)1
in the EDTA-SM
and the bands 997, 1032 and 1047 cm
)1
in the WSM
correspond to PO
4
3–
vibrations. Phosphate as well as sulfate
groups are potential calcium-binding moieties and are
reported to be present in mollusk shell soluble fractions
[1,39].
The FTIR spectrum of the WSM (Fig. 3B) was very
different from that of the EDTA-SM, although some of
the bands are common to both samples. These corres-
pond to the band at 3431 cm
)1
(OH and/or NH
stretching modes of the organic matrix components)
and those in the 2800–3000 cm
)1

region (C–H stretching
modes). In the WSM spectrum, a strong band at
1656 cm
)1
, also assigned as a shoulder in the EDTA-
IM and powdered nacre spectra (data not shown), was
present and corresponds to the amide I groups (C¼O
stretching vibration in the associated state). The absorp-
tion occurred in the high-frequency wing of the amide II
band and was sometimes partly merged with it [40]. A
band at 1542 cm
)1
was also visible and is characteristic of
the amide II groups. In the WSM spectrum, other bands
were clearly assigned, namely at 1455 cm
)1
(CH
2
scissor-
ing) and 1384 cm
)1
(C–N stretching vibration, amide III).
Most of the absorption of the later band came within the
region 1390 ± 40 cm
)1
, in which the methyl or methy-
lene deformations are also active. The WSM sample
absorbed less in the region between 1000 and 1150 cm
)1
compared to the EDTA-SM sample and therefore

appeared to contain a smaller proportion of polysaccha-
rides in its composition. That confirms the GAGs analysis
results.
Polyacrylamide gel electrophoresis
Proteins from shell soluble matrices are generally not easy to
visualize after SDS/PAGE separation [41]. In the present
study, most of the proteins from both EDTA-SM and
WSMmigratedinthegelwithnodistinctpattern,leavinga
dark continuous smear after silver staining (Fig. 4). No
discrete individual bands were observed in the WSM
sample. However, the EDTA-SM revealed two distinct
proteins around 14 and 20 kDa, still presenting with the
dark smear background.
Table 3. Glycosaminoglycan analysis and calcium measurements of the water-soluble matrix, the EDTA-soluble matrix and the EDTA-insoluble
matrix of Pinctada maxima nacre. Sulfated and nonsulfated glycosaminoglycans from the supernatant were estimated by the Whiteman Alcian
blue binding technique [28,29], using chondroitin sulfate as standard. Calcium analyses were performed on samples digested with nitric acid,
by atomic absorption spectrophotometry. Results are expressed as lgÆmg
)1
organic matrix dry weight (mean value ± standard deviation of three
determinations).
Water-soluble
matrix
EDTA-soluble
matrix
EDTA-insoluble
matrix
Glycosaminoglycans 1.59 ± 0.41 24.38 ± 1.10 0.18 ± 0.02
Calcium 1.1 ± 0.5 51.2 ± 1.7 312.1 ± 155.7
Fig. 3. FTIR spectra of the EDTA-soluble
matrix (A) and the water-soluble matrix (B) of

Pinctada maxima nacre. Samples were pre-
pared as KBr discs and were run at a spectral
resolution of 4 cm
)1
.
4998 L. Pereira-Mourie
`
s et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
For several years, mollusk shell biomineralization has been
studied with demineralized structures, in order to obtain the
organic material. In this way, the matrix molecules have
been classified conventionally into two types based on their
solubility in aqueous solutions after demineralization, the
insoluble matrix being characterized by the presence of
highly hydrophobic molecules and by a rich content in
glycine and alanine residues. It is thought to be largely
intercrystalline [42] and to provide a framework where
mineralization occurs. The soluble matrix is characterized
by the predominance of acidic glycoproteins. It is known as
intracrystalline and is considered to play an important role
in induction of oriented nucleation, inhibition of crystal
growth and control of aragonite-calcite polymorphism [43].
At present, only a few constituents of these organic
matrices have been identified. One of the reasons for this is
that shell proteins are very difficult to isolate by means of
traditional biochemical methods (chromatography, electro-
phoresis, enzymatic cleavage) due to self-aggregation of the
molecules or an unusual resistance to temperature, chem-
icals and enzymes. Essentially, the recent advances in

isolation and characterization of matrix molecules have
been possible due to the genetic approach and cDNA
cloning. In a recent paper, Marin et al.[44]describeda
combined technique of preparative electrophoresis and
Western blot on individual proteins which enables the
purification of different proteins in relative large amounts.
In this work, we compare the nacre organic matrix
obtained by the traditional demineralizing extraction
method, to an original method of studying matrix mole-
cules, without previous decalcification. We showed that it is
possible to extract and study organic compounds of the
biomineral nacre, by bypassing the demineralization step.
We think that it may present a new perception of how the
different fractions of the organic matrix are organized in the
biomineral structure.
First, the aqueous method affected neither the yield of
organic material extraction in general nor the extraction of
proteins. Also, the WSM has a low calcium content,
confirming that the molecules extracted are not associated
with minute particles of CaCO
3
that had not been removed
by centrifugation. What changed significantly was the
content of the organic material and, presumably, its original
location in the biomineral itself. In fact, the FTIR spectra,
the amino acid compositions, the chromatographic and
electrophoretic fractionations of EDTA-SM and WSM
showed a first sign of this dissimilarity. For the FTIR
spectra, the main differences were as follows: first, the
presence of sulfate groups and several bands corresponding

to polysaccharides in the EDTA-SM, which may be
assigned to the high content of GAGs, observed in the
quantification assay. Second, the organic matrix extracted
solely in water, i.e. WSM, exhibited fewer polysaccharide
bands, but showed strong protein peaks (amide I and amide
II bands) that were previously observed in the insoluble
matrix from decalcified nacre [45] or shell material [46]. The
position of the amide I band, the major protein absorption
band, depends on the conformation of the polypeptide
chain [47]. The presence of this amide I absorption band at
1656 cm
)1
suggests that the proteins in WSM are in the
a-helix or randomly coiled form, two conformations not
distinguishable by IR spectrometry [48]. Until very recently,
it was thought that the aspartic acid-rich proteins from the
decalcified soluble matrix were in part in the b-sheet
conformation [6,43], as well as the decalcified, silk-fibroin-
like insoluble matrices [49]. Recently Levi-Kalisman et al.
[5] modified these assumptions in suggesting that the ÔsilkÕ
would coat the chitin core in a homogeneous and
completely disorganized phase. Does the random coiled
form of WSM correspond to that disorganized phase? If so,
the WSM would be related to the ÔsilkÕ matrix and not to the
soluble aspartic acid-rich matrix.
The electrophoretic pattern of WSM did not give
significant information on its protein composition. The
continuous smear suggests the presence of GAGs or other
sugars bound to the proteins, despite a low GAG content in
this extract. Western blot analysis with a WSM fraction-

specific antibody revealed the complexity of this kind of
matrix, with several closely separated bands [50]. The
EDTA-SM showed a 14-kDa band, probably the N14
protein found by Kono et al. [14], and another one around
20 kDa. Attempts to purify and characterize these proteins
are presently in progress.
Above all these distinctions, the global amino acid
composition showed clearly that the proteins extracted by
the two methods are not the same. On the one hand, the
soluble (aspartic acid-rich proteins) and insoluble (glycine,
alanine-rich, hydrophobic proteins) matrices extracted after
demineralization with EDTA are in accordance with similar
results in corresponding literature [7,51,52]. On the other
hand, the amino acid composition of WSM, obtained by an
aqueous extraction, was completely different to that of the
EDTA-SM and the so-called soluble matrices in general.
This extract was highly hydrophobic with a C/HP value of
0.29 and exhibited more than 65% glycine and alanine
residues. To begin with, such a characteristic is strange for a
Fig. 4. SDS/PAGE of Pinctada maxima nacre soluble matrices. (1)
LMW calibration kit; (2) EDTA-SM (30 lg protein); (3) WSM (50 lg
protein). Samples in Laemmli buffer were loaded on a 12% poly-
acrylamide mini-gel 0.75-mm thick, and silver stained.
Ó FEBS 2002 Soluble silk-like organic matrix in nacre (Eur. J. Biochem. 269) 4999
soluble extract. Again, these are predominantly features of
what has been called up until now Ôinsoluble matrixÕ,andof
the known silk-fibroin-like molecules. When we looked to
the WIM, we found that it was similar in amino acid
composition to the WSM and the EDTA-IM extractions.
This result means that the silk-like matrix is not completely

extracted with WSM, being present in the two phases. The
acidic matrix would be still enclosed in the mineral phase of
the WIM and would not be accessible for quantification.
However, when we placed the extracted particles (the pellet)
in pure water once again, no more matrix was released (data
not shown). This confirms the hypothesis that the silk is
indeed present in two states, soluble and insoluble, the latter
being in some way protected from dissolution in water. The
peculiarity of the WSM led to the question: how can such a
hydrophobic pool of molecules be dissolved in pure water?
Recently we hypothesized [50] that sugars might be
associated with the apolar proteins of WSM and would
be responsible for their solubilization in water. Here we
showed that GAGs are present in WSM, but in minor
amounts compared with the EDTA-SM.
GAGs and, more specifically, proteoglycans (PGs) are
supposed to be present in mollusk shell organic matrix.
Their presence was indicated by IR spectrometry and Alcian
blue staining [48,53], though without direct quantification.
Our results confirmed their presence in P. maxima nacre
organic matrix and showed that GAGs are mainly released
with EDTA and less with water. This is in accordance with
the observations of Golberg and Takagi [54] who have also
detected a loss of PGs in dentine during EDTA deminer-
alization. They concluded that it is necessary to study PGs
distribution on material fixed either with cryotechniques,
where PGs appear as an amorphous substance in dental
organic matrix, or with cationic dyes. The role of GAGs is
not really understood, but their presence in the organic
matrices of bones, teeth, avian eggshell, otoconia and

kidney stones shows their importance in biomineralizing
systems. Acidic mucopolysaccharides have been considered
as possible candidates for the initiation of the crystal
formation [55]. Sulfate, as well as carboxylate groups, may
cooperate in the induction of oriented crystal nucleation
[56]. These molecules may be responsible for fixation of
calcium in the shell [57]. Also, PGs which are GAGs
associated to a core protein, may act in cell signaling and
metabolic activity [58,59].
To proceed with our argument on the nacre organic
matrix organization, we may understand why the WSM
obtained by an aqueous extraction does not contain acidic
proteins, like the other soluble matrices obtained after
demineralization. The acidic proteins are thought to be
firmly linked to the mineral, and without decalcification it
seems difficult to dissociate them from the whole structure.
Thus, in WSM, we theoretically extracted the molecules that
were directly accessible to water around the sides of the
small mineral particles. In the five-layered model of nacre
organic matrix organization [7,60] the surface layers, also
called the ÔenvelopeÕ by the authors, are the aspartic acid-
rich proteins. However, the surface molecules extracted in
WSM correspond to the core of the organic matrix layer of
the previous model, the silk-fibroin-like molecules. Thus, we
may think of a different structure for nacre organic matrix,
where the silk (maybe WSM or at least part of it) would not
be so deeply located. From the cryo-transmission electron
microscopy studies of the matrix of the bivalve Atrina,anew
model for the nacreous layer organic matrix was recently
proposed by Levi-Kalisman et al. [5]. In this model, which

would be in accordance with our findings, a hydrated silk-
gel phase would be located between the mainly composed
b-chitin interlamellar sheets (Fig. 5). The aspartic acid-rich
glycoproteins would be present both within the silk gel prior
to mineralization and also as electron-dense patches at the
surface of b-chitin. The presence of chitin combined with the
silk-fibroin-like molecules was unfortunately not investi-
gated in P. maxima WSM. Still, its presence was demon-
strated in P. maxima as a chitin–protein complex, which
precipitated after demineralization of the nacreous layer
with hydrochloric acid [61]. The characterization of the
proteins associated to this complex by amino acid analysis
of the precipitate after two deacetylation steps revealed the
predominance of two amino acids: alanine (39.2%) and
glycine (30%). Interestingly, the proportions are very similar
to those found in WSM for alanine and glycine.
Curiously, the presence of a gel phase in shells was rarely
mentioned in the literature until now, although the forma-
tion of a jelly like substance within the shells of cultured
Crassostrea gigas is frequently reported by farmers. This
phenomenon is usually accompanied by a thickening of the
shell, with spaces filled by a nonmineralizing organic matrix
(a jelly-like substance) and is due to exogenous factors such
as exposure to the tributyltin contained in anti-fouling
paints. The few data available on the composition of this
kind of gel showed that it can not be compared to a silk-
fibroin-like substance [62–66]. Though this jelly-like sub-
stance is produced in abnormal situations, it shows that
bivalve organisms do possess the biochemical machinery
needed to produce gel in the calcification process.

In our opinion, the importance of the silk-fibroin-like
matrix has been neglected until now, in part because of its
inaccessibility. The first insoluble molecules, after decalcifi-
cation, to be purified from mollusk shell were MSI 60 and
MSI 31, both from Pinctada fucata nacreous and prismatic
Fig. 5. Schematic representation of the new model for the nacreous layer
organic matrix structure, as proposed by Levi-Kalisman et al.[5].The
putative silk gel phase is located between the interlamellar sheets
of b-chitin. See details in Discussion (courtesy of Professor Steve
Weiner and coworkers). Reprinted from Journal of Structural Biology
135, Levi-Kalisman et al., Structure of the nacreous organic matrix,
pp 8–17, 2001, with permission from Elsevier Science.
5000 L. Pereira-Mourie
`
s et al. (Eur. J. Biochem. 269) Ó FEBS 2002
layers, respectively [67]. At about the same time, Shen et al.
[68] isolated lustrin A, a modular and multifunctional
protein from Haliotis rufescens nacre. Lastly, N16 [69] and
its homologous soluble protein N14 [14] have been charac-
terized in the nacreous layer of P. fucata and P. maxima,
respectively, and would constitute a new protein family.
Many more of the studies on the organic matrix of
biominerals were focused on the attempt to characterize
and purify the aspartic acid-rich molecules [70–72]. One of
the obvious reasons is the solubility of the aspartic acid-rich
matrix under the conditions imposed by the decalcification
step. Another reason, related to the first one, may be the
accessibility of the acidic molecules. Being easily solubilized,
it was possible to use these molecules to perform tests in vitro
for their control in the biomineralization process. Some

important roles were ascribed to them as the control of
nucleation, the growth and the inhibition of crystal forma-
tion [8,73]. The other compounds of the nacre organic
matrix, present in WSM, also possess important biological
activities on cellular mechanisms involved in biominerali-
zation. Indeed, we have shown that the WSM is able to
induce in vitro the differentiation pathway of osteoblasts
from precursor cells like fibroblasts, bone marrow cells and
preosteoblasts [20,21,74,75]. As silk-fibroin is insoluble after
demineralization, it is difficult to isolate these molecules.
The significance of the findings presented here is that, in
practice, the silk fraction can now be analyzed for primary
and secondary conformations, as well as for other biological
and physical properties.
With our comparative study on P. maxima nacre, it
seems that a classification of the organic matrix into soluble
and insoluble, to distinguish the acidic proteins from the
hydrophobic glycine-alanine-rich molecules, is no longer
valid and may even lead to misunderstandings. Some results
support the idea that the amino acid sequence of proteins
extracted from soluble and insoluble matrices share com-
mon features [4]. Such a characteristic indicates that some of
these proteins may in fact belong to the same family.
ACKNOWLEDGMENTS
We would like to express our thank to Professor Steve Weiner and
Dr Lia Addadi for providing the illustration in Fig. 5. We are grateful
to Dr Sophie Berland and Sandrine Borzeix (Laboratoire de
Physiologie, MNHN, Paris, France) for all their useful comments
and iconography, respectively. This study was partly funded by a Siera
SA/CNRS Research and Development contract and by grants

(PRAXIS XXI/BPD/11811 and PRAXIS XXI/BD/20023) from the
Science and Technology Foundation of Portugal.
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