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
´

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

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: [email protected]
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

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

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,

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

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

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

)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

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

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

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

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

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

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