Soluble silk-like organic matrix in the nacreous layer of the bivalvePinctada maxima A new insight in the biomineralization field Lucilia Pereira-Mourie`s1, Maria-Jose´ Almeida1,3, Crist
Trang 1Soluble silk-like organic matrix in the nacreous layer of the bivalve
Pinctada maxima
A new insight in the biomineralization field
Lucilia Pereira-Mourie`s1, Maria-Jose´ Almeida1,3, Cristina Ribeiro3, Jean Peduzzi2, Michel Barthe´lemy2, Christian Milet1and Evelyne Lopez1
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 water-soluble and water-insoluble, based on its
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 insoluble matrix 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 maximaby 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: lopez@mnhn.fr
Abbreviations: EDT A-IM, EDT A-insoluble matrix; EDT A-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)
Trang 2M A T E R I A L S A N D M E T H O D S
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 4C, 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
SM) from the EDTA-insoluble matrix
(EDTA-IM) T o remove EDT A, the two samples were extensively
dialyzed against ultra-pure water (Milli-QTM), at 4C, 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-X, 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 110C under
vacuum with 6M HCl constant boiling (Sigma) for 24 h
Phosphoserine was determined from hydrolysis in 4MHCl
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 0.1M
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
R E S U L T S
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
Trang 3resolved 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/HP¼ 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 profilesof the water-s oluble matrix (A) and the EDTA-soluble matrix (B) of Pinctada maxima nacre Samples (55 lL) containing 400 lg (WSM) or 200 lg (EDT A-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)1with a 25-min linear gradient from 0 to 100% solvent (500 m M NaCl, 20 m M T ris/HCl buffer, pH 7.8) 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)1flow 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).
Trang 4matrix 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)1dry 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)1dry 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
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 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
a
ND, not determined After intensive dialysis the sample still contained residual EDTA.bRatio charged to hydrophobic residues (see Results, section Amino acid composition, for details).
Trang 51750–1700 cm)1whereas 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)1and
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)1was probably due
to in-plane NH2rocking It is also possible that the small
bands located at 963, 985 and 1032 cm)1in the EDTA-SM
and the bands 997, 1032 and 1047 cm)1 in the WSM
correspond to PO43–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)1region (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)1was also visible and is characteristic of the amide II groups In the WSM spectrum, other bands were clearly assigned, namely at 1455 cm)1(CH2 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 WSM migrated in the gel with no distinct pattern, leaving a 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)1organic 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
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.
Trang 6D I S C U S S I O N
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] described a
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 CaCO3that 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 silk 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 silk 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.
Trang 7soluble extract Again, these are predominantly features of
what has been called up until now insoluble matrix, and of
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 envelope 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, a new 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.
Trang 8layers, 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
A C K N O W L E D G M E N T S
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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