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# Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization

Caroline Joubert, David Piquemal, Benjamin Marie, Laurent Manchon, Fabien Pierrat, Isabelle Zanella-Cléon, Nathalie Cochennec-Laureau, Yannick Gueguen, Caroline Montagnani

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## To cite this version:

Caroline Joubert, David Piquemal, Benjamin Marie, Laurent Manchon, Fabien Pierrat, et al. *Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization*. BMC Genomics, 2010, 11 (1), 10.1186/1471-2164-11-613. doi:10.1186/1471-2164-11-613

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Submitted on 26 May 2021

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# Transcriptome and proteome analysis of *Pinctada margaritifera* calcifying mantle and shell: focus on biomineralization

Caroline Joubert¹, David Piquemal², Benjamin Marie³, Laurent Manchon², Fabien Pierrat², Isabelle Zanella-Cléon⁴, Nathalie Cochennec-Laureau⁵, Yannick Gueguen¹*, Caroline Montagnani¹

## Abstract

**Background:** The shell of the pearl-producing bivalve *Pinctada margaritifera* is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in *P. margaritifera*, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.

**Results:** We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 *P. margaritifera* biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in *P. fucata*. Among these sequences, we could show the localization of two other biomineralization protein transcripts, *pmarg-aspein* and *pmarg-pearlin*, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the *P. margaritifera* shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the *Pinctada* genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.

**Conclusions:** This EST study made on the calcifying tissue of *P. margaritifera* is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of *P. margaritifera* shell, but also that the mantle transcripts encode proteins present in *P. margaritifera* shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

## Background

Mollusk shell is a natural biomaterial made up of a mineral phase - calcium carbonate (CaCO$_3$) - and an organic cell-free matrix (proteins, glycoproteins, lipids and polysaccharides) secreted by the external mantle

epithelium, the tissue layer underlying the shell. Although this matrix represents less than 2% of the total composition of the shell by dry weight [1], it interacts with the crystal surface to orientate its nucleation and control crystal polymorphism, in the form of aragonite or calcite, in the different structural layers of the shell [2]. The highly organized internal structure of the shell has led to a very interdisciplinary approach to the study of biomineralization. The secretion of shell by

<footnote>* Correspondence: ygueguen@ifremer.fr
¹Ifremer - Laboratoire de Biotechnologie et de Qualité de la Perle, Centre Océanologique du Pacifique - BP 7004, 98719 Taravao, French Polynesia
Full list of author information is available at the end of the article</footnote>
mollusks is one of the best examples of a matrix-
mediated mineralization process achieved outside living
tissues [3,4]. Models of mollusk shell biomineralization
have therefore been proposed based on histochemical
studies and ultrastructural observations of the shell,
combined with biochemical analysis of the extracellular
organic matrix. The current "chitin-silk fibroin gel pro-
teins-acidic macromolecules" model proposed by Levi-
Kalisman et al. [5], updated by Addadi et al. [6] and
recently reviewed by Furuhashi et al. [7], was established
from mollusk nacre analysis and involves the major
matrix components of the shell. According to this
model, the major components of biomineralization are
relatively hydrophobic silk proteins and a complex
assemblage of hydrophilic proteins (many of which are
unusually rich in aspartic acid), highly structured in a
polysaccharide β-chitinous framework. These compo-
nents of the organic matrix are thought to control var-
ious aspects of the biomineralization process: the
CaCO3 crystal polymorphisms (calcite and aragonite)
and the microstructures of shell layers [8]. Since the
publication of the first complete amino-acid sequence of
a nacre-shell protein in 1996 [9], major advances in the
field of molecular biology have led to the identification
of an increasing number of shell matrix proteins [8].
However, the molecular aspects of shell building are still
far from being fully understood.

As marine bivalves are organisms of major economic
interest, attention has been turned to the study of their
genomics during the last decade [10]. In particular, var-
ious sequence-based strategies have been developed
for transcriptome studies. Among them, Expressed
Sequence Tag (EST) sequencing programs have proven
to be an effective method for gene discovery and have
been widely used for initiating genomic research in non-
model organisms [11]. EST collections provide informa-
tion on the part of the genome that is expressed, and
can be valuable in a number of ways, e.g. gene fishing,
genome annotation and analysis, discovery of single
nucleotide polymorphisms (SNPs), and expression stu-
dies such as microarrays. An EST approach to biominer-
alization offers the opportunity to rapidly identify
transcripts encoding secreted shell proteins, proteins
specific to the pallial space and proteins implicated in
calcium regulation in mantle cells, as well as transcrip-
tion factors responsible for the regulation of the process.
EST programs have recently been developed for aqua-
culture bivalve species, in particular the Eastern oyster
(Crassostrea virginica) [12-15], the Pacific oyster (Cras-
sostrea gigas) [14,16], and the common blue mussel
(Mytilus galloprovincialis) [17], but these have mainly
been aimed at investigating the mollusk immune
response in the context of environmental or genome
evolution studies. To date, only five studies report the

analysis of EST programs performed on calcifying tis-
sues with the aim of providing more insight into the
biomineralization process. Suppression subtractive
hybridization (SSH) studies were performed on the
bivalve pearl oysters *Pinctada fucata* [18] and *P. mar-
garitifera* [19]. Two other studies, involving the vetigas-
tropod *Haliotis asinina* [20,21] and the bivalve pearl
oyster *Pinctada maxima* [21], revealed the high
complexity of the calcifying mantle transcriptome, sug-
gesting extensive differences between Bivalvia and Gas-
tropoda in the molecular composition of the organic
matrix guiding the deposit of calcium carbonate poly-
morphs within the shell. The most recent study [22]
described the transcriptome of the mantle tissue of
*Laturnela elliptica*, focusing on the datamining of genes
involved in calcium regulation and shell deposition.
Despite these genomic approaches, there is still a small
amount of genomic data available on bivalve species and
this limits our understanding of the dynamic process of
biomineralization.

With the aim of increasing the genomic resources for
the pearl-producing bivalve *P. margaritifera*, we con-
ducted a pyrosequencing program to analyze the first
EST library produced from the calcifying mantle of this
bivalve. Here we report the functional analysis of 276
738 EST sequences, leading to the constitution of a
*P. margaritifera* mantle transcript catalog of 82
sequences potentially implicated in the biomineralization
process. Further structural characterization of a set of
proteins was undertaken in addition to transcript locali-
zation and proteomic mass spectrometry analysis of pro-
teins isolated from the shell matrix. Our results show
that protein repertoire of the biomineralization process
is conserved within pearl oysters, but also provide direct
evidence that our EST data set covers most of the diver-
sity of the shell matrix protein in *P. margaritifera* shell.

**Methods**

1. Mantle RNA Extraction and ESTs library construction

P. margaritifera pearl oysters raised in the Vairao lagoon
were brought to the Ifremer laboratory in Tahiti, French
Polynesia. Total cellular RNA was extracted from 12
mantle samples taken from separate P. margaritifera
individuals, using TRIZOL® Reagent (Life Technologies)
according to manufacturer's recommendations. RNA
integrity and purity were assessed in a Bioanalyzer 2100
(Agilent - Bonsai Technologies) and using agarose gel
analysis. RNA was quantified using a NanoDrop® ND-
1000 spectrophotometer (NanoDrop® Technologies Inc).
A pool of 24 µg total RNA (2 µg per sample) was used
to construct a cDNA library. Five µg of full-length dou-
ble-stranded cDNA was processed by the standard Gen-
ome Sequencer library-preparation method using the GS
DNA Library Preparation Kit to generate single-stranded
DNA ready for emulsion PCR (emPCR™). The cDNA library was pyrosequenced using GS FLX technology (454/Roche, http://www.454.com/).

**2. Contig assembly and functional annotation**

EST sequence analysis and assembly were performed by the Skuldtech Company http://www.skuldtech.com. ESTs were assembled into clusters using TGICL (TIGR Gene Indices Clustering tools), freely available on the sourceforge website http://sourceforge.net/projects/tgicl/ [23]. Overlapping identity percentage and minimum overlapping length parameters was set to 98% and 60 bp, respectively, in order to obtain highly reliable consensus sequences. Data were archived at NCBI Sequence Read Archive (SRA) under accession SRP002635. ESTs that did not form contigs (singletons) and contigs resulting from the assembly of multiple sequences are referred to as unique sequences. These unique sequences were translated into six reading frames and used as a query to search the non-redundant protein databases available at the National Center for Biotechnology Information (NCBI) using the BlastX algorithm with an E-value ≤10⁻³ (version # 2.2.15, GenBank release number #166) http://www.ncbi.nlm.nih.gov. Sequences with BlastX hits were manually assigned to the following five sequence categories: known, uncharacterized, predicted, unknown or unnamed, and hypothetical proteins. This classification was based on the information definition lines in each homologous sequence provided by NCBI. All unique sequences with BlastX hits (E-value ≤10⁻³) were functionally annotated using Blast2GO http://www.blast2go.org/[24] by mapping against gene ontology (GO) resources.

**3. Identification of biomineralization-related proteins in P. margaritifera mantle EST library**

Candidate genes from the biomineralization process were locally identified in the *P. margaritifera* mantle ESTs library using BlastX, according to the following parameters: E-value ≤10⁻³, expect feature set to a default value of 10, and low-complexity filter determined by the SEG program [25]. For this purpose, we collected all available sequences regarding biomineralization in mollusks (bivalvia and gastropoda) from the literature or from public databases. The *Pmarg*-Pif nucleotide sequence was obtained by assembling ESTs with an overlapping identity percentage and minimum overlapping length parameters set to 100% and 60 bp, respectively. Motifs and conserved domains of *Pmarg*-Pif protein sequence were used as a query to search the non-redundant protein databases available at the National Center for Biotechnology Information (NCBI) using the BlastP algorithm, according to the following parameters: expect feature set to a default value of 10, and low-complexity

filter determined by the SEG program [25]. Sequence alignments were performed using the ClustalW program setting parameters to default for the gap criterions (gap open, no gap end, gap extension, gap distance, pairgap), followed by manual correction with BioEdit software http://www.ebi.ac.uk/Tools/clustalw2/index.html [26]. The presence of signal peptides was inferred using the SignalP 3.0 server http://www.cbs.dtu.dk/services/SignalP/[27]. Conserved domains were identified using Prosite http://www.expasy.ch/prosite/[28]. Percentage identity and biochemical similarity between sequences were calculated using ProtParam http://www.expasy.ch/tools/protparam.html [29]. Repeat detection in protein sequences was performed using RADAR http://www.ebi.ac.uk/Tools/Radar/index.html [30].

**4. In situ hybridization analyses**

**a) Tissue preparation**

*P. margaritifera* mantle tissues were fixed for 24 h in Davidson fixative (22% formalin, 33% ethyl alcohol, 11.5% glacial acetic, 33% sterile sea water), embedded in paraffin wax, and serially sectioned at 7 µm. Sections were collected onto polylysine coated slides (Silane-prep™, Sigma-Aldrich), dried overnight at 60°C and treated with proteinase K (10 µg.mL⁻¹) in TE buffer (Tris 50 mM, EDTA 10 mM) at 37°C for 25 min. Slides were then dehydrated by immersion in an ethanol series and air dried. The sections were prehybridized for 1 h at 42°C with 500 µL hybridization buffer (4 × SSC, 50% formamide, 1× Denhardt's solution, 250 µg.mL⁻¹ yeast tRNA, 10% dextran sulfate). The solution was replaced with 120 µL of the same buffer, containing 6 µL of the digoxigenin-labeled sense or antisense probes. The slides were incubated overnight at 42°C for hybridization. The sections were washed twice for 5 min in 2x SSC at room temperature and once for 10 min in 0.4x SSC at 42°C. The detection steps were performed according to manufacturer's instructions (Dig nucleic acid detection kit, Roche Molecular Biomedicals). Slides were finally counter-stained with a solution of Bismark Brown Yellow and mounted in Eukitt. The slides were examined using a DM4000B Leica microscope.

**b) Specific probe preparation**

In order to synthesize probes for *in situ* hybridisation, we used the PeS4 (GACATAGAGAGAGACAGA-TATGA)/PeAS4 (ATTCACCATTTCCGTTACCGT) primer set, specific to the *pmarg-pearlin* ORF (265bp), and AspF1 (CTCTTACACCAAAATGAAGGGG)/AspR1 (TCCGTCATCATTATCTGC), specific to the *pmarg-aspein* transcript (253 bp). These primers (4 µM final volume) were used in PCR reactions with the iQ™ Supermix (BIO-RAD) and *pmarg-pearlin* full-length cDNA as template. After DNA denaturation at 94°C for
5 min, 35 cycles were run with an MJ-Research thermocycler as follows: 94°C for 30 s; 55°C for 30 s; 72°C for 45 s ended by a final elongation step at 72°C for 10 min. Probes (sense or antisense) were synthesized by asymmetric PCR (using the same amplification program) in the presence of Dig-dUTP (0.7 mM), in a PCR reaction mixture containing a unique primer (sense or antisense, 2 µM final volume), 2 µL of the previously purified PCR fragment (Mini Quick Spin Columns, Roche Diagnostics), a mix of dGTPs-dCTPs-dATPs (200 µM each final), dTTPs (130 µM final), and Taq polymerase (Promega, 2.5 u). Labelling efficiency was assayed using the DIG high prime DNA labelling kit (Roche Diagnostics).

## 5. Purification and identification of proteins from *P. margaritifera* shell

Organic matrix was extracted from fresh shells of *P. margaritifera* specimens aged 3-5 years, after acid acetic decalcification [31]. The acido-insoluble matrix was digested with trypsin prior to reduction and alkylation [32]. Samples were injected into a nano LC-nanoESI-MS/MS system for analysis. Mass spectrometry (MS) was performed using a nanoESI-qQ-TOF, and data acquired automatically using Analyst QS 1.1 software (Applied Biosystems). A 1 s TOF-MS survey scan was acquired over 400-1600 amu, followed by three 3 s product ion scans over a mass range of 65-2000 amu. The three most intense peptides, with a charge state of two to four above a 30 count threshold, were selected for fragmentation and dynamically excluded for 60 s with ± 50 mmu mass tolerance. The collision energy was set by the software according to the charge and mass of the precursor ion. The MS and MS/MS data were recalibrated using internal reference ions from a trypsin autolysis peptide at *m/z* 842.51 [M + H]$^+$ and *m/z* 421.76 [M + 2H]$^{2+}$. Protein identification was done using the Mascot database-searching software (Matrix Science, London, UK; version 2.2.04) using our database of the pyrosequencing-based EST mantle library from *P. margaritifera*. Carbamidomethylation and oxidation were set as fixed and variable modifications, respectively. The mass tolerance was set to 0.5 Da and the MS/MS tolerance to 0.2 Da.

## Results and Discussion

During recent decades, high-throughput techniques have been used to examine a broad range of physiological processes and applications in diverse fields of biology [33,34]. To examine the biomineralization process in pearl oyster *P. margaritifera*, we performed transcriptome pyrosequencing of its calcifying tissue combined with a proteome analysis of the shell.

<table><caption>**Table 1 Summary statistics for pyrosequencing and annotation of *P. margaritifera* mantle ESTs**</caption><thead><tr><th>Feature</th><th>Number</th><th>Percentage</th></tr></thead><tbody><tr><td>Total number of ESTs sequenced</td><td>276 738</td><td>-</td></tr><tr><td>Average lenght of ESTs (bp)</td><td>234</td><td>-</td></tr><tr><td>Number of assembled EST</td><td>219 205</td><td>79.2%</td></tr><tr><td>Number of contigs</td><td>19 257</td><td>-</td></tr><tr><td>Number of singletons</td><td>57 533</td><td>20.8%</td></tr><tr><td>Number of unique sequences</td><td><strong>76 790</strong></td><td>-</td></tr><tr><td>Ratio of singletons per unique sequences</td><td>-</td><td>74.9%</td></tr><tr><td>Number of contigs containing 2 ESTs</td><td>8 717</td><td>45.3%</td></tr><tr><td>Number of contigs containing 3 ESTs</td><td>3 419</td><td>17.8%</td></tr><tr><td>Number of contigs containing 4 ESTs</td><td>1 779</td><td>9.2%</td></tr><tr><td>Number of contigs containing 5 ESTs</td><td>1 119</td><td>5.8%</td></tr><tr><td>Number of contigs containing > 6 ESTs</td><td>4 223</td><td>21.9%</td></tr><tr><td>Number of annotated unique sequences:</td><td><strong>29 479</strong></td><td><strong>38.4%</strong></td></tr><tr><td>- Known protein</td><td>13 064</td><td>44.3%</td></tr><tr><td>- Uncharacterized</td><td>6 010</td><td>20.4%</td></tr><tr><td>- Predicted</td><td>4 795</td><td>16.3%</td></tr><tr><td>- Unknown, Unnamed</td><td>2 880</td><td>9.8%</td></tr><tr><td>- Hypothetical protein</td><td>2 730</td><td>9.3%</td></tr><tr><td>Number of annotated contigs</td><td>10 007</td><td>52.2%</td></tr><tr><td>Number of annotated singletons</td><td>19 472</td><td>33.8%</td></tr></tbody></table>

## 1. Transcriptome analysis of *P. margaritifera* calcifying mantle

### a) Generation of ESTs and contig assembly

We constructed and pyrosequenced a *P. margaritifera* mantle cDNA library, resulting in the production of 276 738 sequences of an average size of 234 bp (Table 1). Sequences in the library ranged from 33 to 406 bp, with the most abundant group of sequences (70%) in the 225-290 bp range and only 3% of the sequences longer than 300 bp. The 276 738 ESTs were assembled into clusters using TGICL, which gave 19 257 contigs and 57 533 singletons. Our *P. margaritifera* mantle EST collection thus contains 76 790 unique sequences (Table 1). The number of EST sequences generated here using pyrosequencing is similar to numbers obtained in other transcriptome pyrosequencing based studies [35]. The May 2010 GenBank release only contained 116 sequences from *P. margaritifera* including both the “nucleotide” and “EST” sections, and a total of 15 742 sequences from the genus *Pinctada* as a whole. Once released on public databases, the present 76 790 mantle unique sequences will account for 99.8% of all sequences available for *P. margaritifera* and 83% of all sequences available for the genus *Pinctada*. Pyrosequencing is, therefore, both a rapid and powerful way to dramatically increase transcriptomic resources for non-model organisms lacking detailed genomic data.

Of the 19 257 contigs, 8717 (45.3%) contained 2 ESTs, 3419 (17.8%) contained 3 ESTs, 1779 (9.2%) contained 4
ESTs, 1119 (5.8%) contained 5 ESTs, and 4223 (21.9%) contained more than 6 ESTs (Table 1). In our study, 79.2% of the 276 738 ESTs were successfully assembled and remaining singletons only represented 20.8% of the reads, and a large part (74.9%) of the 76 790 unique sequences was singletons. In other recent 454 transcriptome studies, results showed that the remaining singletons represented 10 to 40% of the reads [36,37]. It has already been observed that many ESTs resulting from deep sequencing of transcriptomes with 454 sequencing technology fail to assemble [38]. These unassembled singletons could result from sequencing errors, contaminants from other sources, or can even from technical difficulties in assembling with overlaps that are too short in length or which contain highly repeated sequences. Interestingly, however, these singletons can also represent rare transcripts of genes expressed at low levels [39], and therefore constitute an interesting source of genomic data.

b) *Putative identities of ESTs*

BlastX searches of the 76 790 unique sequences in the non-redundant protein databases available at the National Center for Biotechnology Information (NCBI) revealed 29 479 (38.4%) significant matches (E-value ≤10⁻³). Among these 29 479 matches, 13 064 (44.3%) are known proteins, but 6010 are uncharacterized (20.4%), 4795 are predicted (16.3%), 2880 are either unknown or unnamed (9.8%), and 2730 are hypothetical proteins (9.3%) (Table 1). This apparently low rate of identification is common among mollusk EST databases, with which rates usually range from 15 to 40% [15,17,22,40], although this is lower than for vertebrates [41], or even EST collections from model plants [42].

Although the lack of annotation can result from the
difficulty of annotating some short length sequences, it
can largely be explained by the lack of sequences avail-
able for mollusk species, and by the fact that a vast
majority of genes on public databases come from taxa
(in particular vertebrates species) whose amino
acid sequences show great divergence with those of
protostomians.

c) **Functional Gene Ontology annotation**

Gene Ontology (GO) assignment was carried out on
unique sequences in order to categorize transcripts from
*P. margaritifera* mantle by putative function. The GO
project provides a structured and controlled vocabulary
of terms (ontologies) for describing gene product char-
acteristics and gene product annotation data [43]. In our
study, 10 004 unique sequences (13.0%) were success-
fully assigned to one or more GO terms. Among these,
following the functional classification with the three
unrelated GO ontologies, 5976 (59.7%) are involved in
biological processes, 6855 (68.5%) have molecular func-
tions and 5737 (57.3%) are cellular components. For

each of these three ontologies, annotated sequences are
mainly distributed among two or three of the general
term categories. Within the 5976 unique sequences
involved in biological processes, 5006 (83.8%) and 4191
(70.1%) are dedicated to cellular processes and meta-
bolic processes, respectively (Figure 1A). Similarly, in
the molecular functions sub-ontology, 5208 (76.0%) and
3704 (54.0%) of the 6855 unique sequences have binding
and catalytic activity, respectively (Figure 1B). Finally, of
the 5737 unique sequences predicted to be cellular com-
ponents, 5656 (98.6%) and 3868 (67.4%) are related to
cell and organelle components, respectively (Figure 1C).
These results constitute common features among EST
databases available from marine organisms, and in parti-
cular mollusks [40,44].

The distribution of the sequences between specialized
terms in the binding section of the molecular function
category showed that the greatest numbers fell under
protein-binding (35%) and nucleotide-binding (19%).
Interestingly, the third greatest number of the binding
section fell into ion-binding (17%) (Figure 1D). Biomin-
eral crystal matrix macromolecules play a key role in
biologically-controlled biomineralization processes.
*In vitro* crystallization experiments, microscopic and
analytical methods revealed stereochemical properties of
matrix proteins, which allow them to bind calcium ions
and calcium carbonate, and therefore perform frame-
work building and crystal growth during the construc-
tion of the molluskan shell[45-48]. A significant
proportion of sequences in our mantle EST collection
are implicated in binding, and particularly in ion bind-
ing. This result is consistent with observations from a
previous study performed on the calcifying mantle of
the bivalve *L. elliptica* [22]. We therefore hypothesize
that this classification could be a pattern typical of tis-
sues of a secretory nature implicated in biomineraliza-
tion processes.

2. Identification of transcripts encoding proteins involved
   in the biomineralization process of *P. margaritifera*

a) Identification of a catalogue of 82 proteins potentially
   involved in the biomineralization process

To obtain an integrated view of the transcriptional
events of the biomineralization process in *P. margariti-
fera* mantle, we made BlastX searches with our EST
mantle library focusing on proteins known to be
involved in these mechanisms. For this purpose, we first
collected all available sequences regarding biomineraliza-
tion in calcifying invertebrates from the literature or
from public databases. In mollusks, we found 140
bivalve and 103 gastropod proteins potentially impli-
cated in biomineralization processes. These 243 mollus-
kan sequences were isolated from shell or mantle tissue
in previous studies, using either biochemical or
<figure><img src="image_4.png" /></figure>

molecular biology approaches. BlastX searches of the 140 bivalves and 103 gastropods proteins in our EST database revealed 121 and 56 significant matches (E-value ≤10⁻³), respectively. Analyzing these 177 sequences together with sequences from our EST library, we identified 82 *P. margaritifera* non-redundant unique sequences potentially implicated in the biomineralization process. Among these, 69 and 13 sequences could be recovered by homology with sequences from bivalve and gastropod, respectively.

Among the 69 unique *P. margaritifera* transcripts that were recovered by homology with the bivalve sequences, 55 sequences were obtained by homology with sequences from the *Pinctada* genus (Additional file 1). The overall identity percentage between *P. margaritifera* protein sequences potentially implicated in the biomineralization process and protein sequences from the *Pinctada* genus is ranging from 24% (C-type lectin 2 from *P. fucata*) to 95% (Ferritin-like protein from *P. fucata*). This level of identity is similar to percentages already observed for homolog proteins from the N66/ Nacrein and N14/N16 families [49-51]. The N66 sequence from *P. maxima* and Nacrein sequence from

*P. fucata* (*P. maxima* N44 homolog sequence) displayed identity percentages of 82% and 69%, respectively, with *P. margaritifera* homolog sequence. Similarly, the N14 sequence from *P. maxima* and N16 sequence from *P. fucata* displayed an identity percentage of 93% and 71% respectively with *P. margaritifera* homolog sequence, Perline matrix protein. Considering all sequences from the *Pinctada* genus, the identity percentage seems to be higher between *P. margaritifera* and *P. maxima* sequences than between *P. margaritifera* and *P. fucata* sequences.

Extending our analysis to biomineralization proteins from other bivalves led us to the identification of the 14 remaining sequences out of the 69 unique *P. margaritifera* transcripts that were recovered by homology with the bivalve sequences (Additional file 1). The overall identity percentage between *P. margaritifera* protein sequences potentially implicated in the biomineralization process and protein sequences from the other bivalves ranges from 28% (EP protein precursor from *Mytilus edulis*) to 58% (bone morphogenic protein type 2 receptor from *Crassostrea gigas*). This level of identity is lower than that observed between proteins within the
*Pinctada* genus, except for proteins implicated in calci-
cium regulation or signal transduction. For example,
Calmodulin sequences from *Hyriopsis schlegelii* (gen-
bank accession number: ACI22622) displayed an identity
percentage of 99% with the *P. margaritifera* homolog
sequence.

Finally, we identified 13 *P. margaritifera* unique
sequences by homology with sequences of gastropod
(Additional file 1). The overall identity percentage
between *P. margaritifera* protein sequences potentially
implicated in the biomineralization process and protein
sequences from gastropods ranged from 27% (Veliger
mantle 1 from *H. asinina*) to 100% (Calmodulin from
*Conus cuneolus*). Interestingly, some sequences homolo-
gous to abalone (*H. laevigata*) proteins could be found
in our EST database, namely Perlucin [52,53], Perlustrin
[53,54] and Perlawpin [55] from *Haliotis laevigata*. Per-
perlucin, Perlustrin and Perlwapin sequences were obtained
by direct protein sequencing of proteins purified from
the nacreous layer of abalone shell. All of the *P. margar-
*itifera* homolog sequences for each of these 3 proteins
found in the *P. margaritifera* EST library display the
same motif and numerous conserved cystein positions
as in the sequences from *H. laevigata*. Perlucin is a 155-
amino acid protein which exhibits similarities with cal-
cium dependent lectins (C-type). The *P. margaritifera*
homolog sequence for Perlucin (*Pmarg*-perlucin) is not
a complete sequence. However, of the 6 cysteins present
in the abalone sequence, 3 are conserved between
*Pmarg*-perlucin and Perlucin sequences. Moreover,
*Pmarg*-perlucin displays an E-value of 9.00E-9 and an
identity percentage of 38% (27/71 a.a.) with Perlucin
and also has a C-type lectin domain. Perlustrin is a
small protein (84 a.a.) with similarities to vertebrate
insulin-like growth factor-binding protein (IGF-BP)
sequences. The *P. margaritifera* homolog sequence for
Perlustrin (*Pmarg*-perlustrin) is a complete 142-amino
acid sequence with an E-value of 7.00E-6, and 39% (25/
64 a.a.) identity with Perlustrin; it also exhibits a insu-
lin-like growth factor binding proteins (IGFBPs). On the
12 cysteins scattered across the Perlustrin sequence, 11
(of the 14 cysteins of *Pmarg*-perlustrin) are conserved
between *Pmarg*-perlustrin and the Perlustrin sequences.
Finally, the Perlawpin protein consists of 134 amino
acids that contain 3 repeats of 40 amino acids very simi-
lar to the well-known whey acidic protein (WAP)
domains. The *P. margaritifera* homolog sequence for
Perlwapin (*Pmarg*-perlwapin) is a complete 139-amino
acid (a.a.) sequence with an E-value of 2.00E-11, 37%
identity (40/107 a.a.) with Perlawpin, and two WAP
domains. Out of the 25 cysteins spread along the Perl-
wapin sequence, all 14 cysteins of *Pmarg*-perlwapin are
conserved between the *Pmarg*-perlwapin and Perlwapin
sequences. These results would suggest that Perlucin,

Perlustrin and Perlwapin are present in *P. margaritifera*.
Previous studies have shown that there are significant
differences in the molecular mechanisms in different
mineralizing species and, therefore, between the proteins
they use. Such differences may even exist among species
that are phylogenetically very close, like the Mollusca.
The cause of this "evolvability" remains a controversy,
and it is still uncertain whether the biomineralization
"molecular tool box" required for shell construction is
inherited from an ancestral function, or whether this
ability is the result of an adaptive convergence. Recent
studies have explicitly demonstrated that shell or skele-
tal proteins had evolved independently among metazo-
ans [8,21,56]. However, the identification of homolog
proteins between bivalvia and gastropoda could support
the idea that at least some of the shell component could
have appeared early in the evolution of the molluscan
phylum.

Taken together, this candidate approach allowed us to
isolate 82 unique sequences potentially implicated in the
biomineralization process in *P. margaritifera*. This study
considerably increases the amount of transcriptomic
data available in this field, making *P. margaritifera* the
best documented marine protostomian with regard to
biomineralization.

b) Identification of proteins from the "chitin-silk fibroin gel-
acidic macromolecule" model

Mollusk shell construction is the result of biologically-
controlled mineralization, a highly dynamic process
mediated by an extracellular organic matrix secreted by
the mantle epithelium [3]. Histochemical studies and
ultrastructural observations of the shell, together with
biochemical analysis of the extracellular organic matrix,
provided a better understanding of shell structure and
led to the identification of proteins composing it,
thereby allowing mollusk shell biomineralization models
to be developed. The currently accepted "chitin-silk
fibroin gel-acidic macromolecule" model involves the
major matrix components of the shell, i.e. relatively
hydrophobic silk proteins plus a complex assemblage of
hydrophilic proteins (many of which are unusually rich
in aspartic acid), highly structured in a polysaccharide
β-chitinous framework [6].

In our study, beyond the consideration of protein
homologies between species, it is interesting to note
that our *P. margaritifera* EST mantle library includes
sequences coding for proteinaceous components of the
matrix following this model. Firstly, a sequence showing
78% identity with MSI60 from the silk fibroin matrix
component could be retrieved. MSI60 is an insoluble
framework protein purified from the nacreous layer of
the shell [57] and expressed in the more dorsal region
of the mantle [58]. Poly-Ala and poly-Gly blocks confer-
ring MSI60 homologies with spider silk fibroins are
present in the *P. margaritifera* homologous sequence. MSI31 [57] and Shematrins [59], displaying silk/fibroin-like domains, could be also retrieved. Secondly, a sequence showing 87% identity with the unusually acidic protein Aspein from *P. fucata* could be recovered in the EST database from *P. margaritifera* [60]. This sequence homologous to Aspein is the first extremely acidic shell protein identified in *P. margaritifera*. In *P. fucata*, Aspein is specifically expressed in the mantle region, which secretes the calcite prism matrix [58]. The main body of this protein includes a high proportion of Asp (60.4%) punctuated with Ser-Gly dipeptides, which are conserved in the *P. margaritifera* homologous sequence. Finally, recent electron microscopy studies on nacre have detected the presence of chitin in the shell of *P. margaritifera* [61], and chitin synthase gene has been cloned from *P. fucata* [62], *Atrina rigida* and *Mytilus galloprovincialis* [63]. A *P. margaritifera* homolog sequence of chitin synthase from this species could be retrieved, revealing that chitin synthase sequences are well conserved among bivalves. More precisely, the chitin synthase sequences from *Atrina rigida* and *Mytilus galloprovincialis* displayed identity percentages of 91% and 84%, respectively, with the homologous *P. margaritifera* sequence.

Taken together, searches realized on the EST mantle library allowed us to identify proteinaceous components of the calcifying matrix from *P. margaritifera*. These results demonstrate how EST-based studies are a powerful way of dramatically increasing knowledge about proteins implicated in the biomineralization process, which constitutes an important prerequisite for establishing relevant biomineralization models.

### c) *Pmarg-Pif* encodes an homolog of *Pif-177* from *P. fucata*, a protein involved in nacre formation

Pif-177 is an acidic matrix protein that was identified in *P. fucata* nacre shell and is known to specifically bind to aragonite crystals. Results from immunolocalization, RNA interference and *in vitro* calcium carbonate crystallization strongly indicate that Pif-177 regulates nacre formation; making Pif-177 the first mineralization protein in this species whose function was identified *in vivo* [64]. A Pif-177 homolog, which we named *Pmarg-Pif*, was identified in the *P. margaritifera* mantle EST library using BlastX. A comparison between *Pmarg-Pif* and Pif-177 protein structures is shown in figure 2A. A consensus furin cleavage site at position 555-558 (RIKR) was identified in the *Pmarg-Pif* sequence. In *P. fucata*, a similar furin-cleavage domain, observed between amino acid positions 544-547 (RMKR) is required for Pif-177 cleavage into Pif-80 and Pif-97. This suggests that, as with Pif-177, *Pmarg-Pif* cDNA is very likely to encode a precursor protein that will be post-translationally cleaved to produce Pif-97 and Pif-80 homologs (which

we named *Pmarg-Pif-97* and *Pmarg-Pif-80*, respectively). Alignments of *P. fucata* and *P. margaritifera* Pif sequences revealed 83.0% of identity between Pif-97 and *Pmarg-Pif-97* domains, and 60.0% identity between Pif-80 and *Pmarg-Pif-80*. Like Pif-97 and Pif-80, *Pmarg-Pif-97* and *Pmarg-Pif-80* are acidic proteins. *Pmarg-Pif-97* consists of 536 amino acid residues, including a high proportion of charged amino acid residues, Asp (15.3%), Glu (7.1%), Lys (10.3%), and Arg (6.0%), with a calculated isoelectric point (pI) value of 4.7. This sequence contains two conserved domains: a von Willebrand type A (VWA) domain and a chitin-binding domain, similar to those in Pif-97 (figure 2B). There are also 22 Cys residues, of which 21 are conserved between the two homologs. *Pmarg-Pif-80* consists of 456 amino acid residues, and also contains a high proportion of charged amino acid residues, Asp (26.1%), Glu (5.0%), Lys (16.2%), and Arg (11.6%), with a calculated isoelectric point (pI) value of 5.13. *Pmarg-Pif-80*, like Pif-80, displays a high Asp ratio and may, therefore, be involved in aragonite-binding processes, since Pif-80 was shown to bind aragonite crystals and Pif-177 to be implicated in the regulation of nacre formation. A cluster of acidic amino acid residues is also present near the center of the molecule, but this shows a higher number of Asp residues (14) in *Pmarg-Pif-80* than in Pif-80 (10). Only 9 repeats of the four-amino-acid motif (DD-R/K-R/K) could be found before the cluster of acidic amino acid residues in *Pmarg-Pif-80*, whereas 17 are found scattered throughout Pif-80 sequence, and only three of these are conserved in the same position between the two sequences. Interestingly, an 18 amino acid residue sequence (LVKEIERRKSDDK-K/I-S-F/L-DD) is repeated three times (742-816) in the *Pmarg-Pif-80* sequence. This highly charged amino acid sequence could not be retrieved in the Pif-80 sequence, and BlastP results showed no homology with any other protein in the public database. Considering that this consensus sequence is localized in a protein sequence suspected to play a role in aragonite binding in *P. fucata*, it potentially constitutes an interesting new motif with regard to biomineralization processes.

Taken together, the numerous conserved sequence motifs, conserved cystein residue positions, charged amino acid residue composition and common isoelectric properties between *Pmarg-Pif-97* and Pif-97 support the hypothesis that *Pmarg-Pif* might have a similar activity to Pif-177, and regulate nacre formation in *P. margaritifera*. However, the presence of the repeated 18 amino acid residues sequence specific to *Pmarg-Pif-80* and the distinct number of repeats of the four-amino-acid motif (DD-R/K-R/K) between *Pmarg-Pif-80* and Pif-80 also suggest that *Pmarg-Pif-80* might have a function specific to *P. margaritifera*. Considering these features, further research needs to be
<figure><img src="image_5.png" /><figcaption>**Figure 2 Pif-177 and Pmarg-Pif protein sequence comparison.** (A). Schematic representation of Pif-177 and Pmarg-Pif protein structures. Pif-97 consists of 525 amino acids (from a.a. 23 to 547) and Pmarg-Pif-97 consists of 536 amino acids (from a.a. 23 to 558). Pif-80 consists of 460 amino acids (from a.a. 548 to 1007) and Pmarg-Pif-80 consists of 456 amino acids (from a.a. 559 to 1014). Purple box is the signal peptide. Blue VWA box is von-Willebrand type A domain. Green CHIT box is a chitin-binding domain. Yellow box is aragonite-binding protein. The four-amino-acid motifs (DD-R/K-R/K) are indicated by asterisks, and arrows indicate motifs at conserved positions between the two sequences. Red box, in the aragonite-binding protein, indicates the cluster of acidic amino acid residues. (LVKEIERRKSDDK-K/I-S-F/L-DD) consensus motif repeated three times in Pmarg-Pif is boxed under aragonite-binding protein. (B) Alignments of the Pif-177 and Pmarg-Pif amino acid sequences of the von Willebrand type A domain and chitin-binding domain. Residues shaded in black are identical residues, and grey positions indicate conserved residues. Sequences are preceded and followed by numbers indicating the domain position in the entire sequence. Percentage of identity are indicated.</figcaption></figure>
undertaken in order to investigate *Pmarg-Pif* function and
its role in the biomineralization process.

3. Expression pattern of biomineralization-related protein transcripts

In order to focus on biomineralization processes, tran-
script localization was performed using isolated tran-
scripts from the *P. margaritifera* EST library. We
selected two highly documented proteins implicated in
biomineralization in the *Pinctada* genus: the Aspein and
N14/N16/Pearlin families. In adult oysters, the tissue
responsible for shell mineralization is the mantle outer
epithelium. This mantle can be divided into several
regions from the more proximal (dorsal) zone to the
more distal (ventral to mantle edge) zone. Studies have
shown that this zonation can be associated with distinct
gene expression patterns, suggesting a functional parti-
tion following the dorso-ventral axis, which might be
involved in production of specific calcium carbonate
polymorphs [57,65]. Our *in situ* analysis revealed that
these transcripts were specifically localized in the outer
epithelium of the mantle known to be bearing mineraliz-
ing cells (figure 3). More interestingly, these transcripts
were localized in two distinct areas of the outer epithe-
lium, the dorsal zone for *pmarg-pearlin* and the ventral

zone for *pmarg-aspein*. *Aspein* and *pearlin* genes are
known to produce proteins specific to the nacre and pris-
matic layers of the shell, respectively [49,58,60,66]. Our
observations confirm the functional subdivision within
the pearl oyster mantle outer epithelium, *pmarg-pearlin*
transcripts being specific to aragonitic nacre-forming
cells and *pmarg-aspein* transcripts being specific to calci-
tic prism-forming cells.

4. Mantle transcripts encode proteins identified in *P. margaritifera* shell

Using the *P. margaritifera* EST mantle library, identifi-
cation of shell matrix proteins was attempted by a com-
plementary proteomic approach. The shell matrix
proteins, extracted from decalcified shell powder, were
digested with trypsin and the resulting peptides were
analysed by MS/MS mode mass spectrometry. The raw
MS/MS data were directly interrogated against the EST
data set using Mascot software. After careful observation
of the MS/MS data on the 50 first most intensive pep-
tides, we estimated that almost all the main peptides
analysed led to contig identification. We only considered
matching proteins that presented at least 2 unambigu-
ously identified peptides, i.e. those presenting individual
scores superior to the threshold (calculated value of 32).

<figure><img src="image_6.png" /><figcaption>**Figure 3 Localization of pmarg-pearlin and pmarg-aspein gene transcripts in P. margaritifera mantle tissue by in situ hybridization.**</figcaption></figure>

Paraffin-embedded sections of oyster tissues were hybridized with antisense or sense single stranded cDNA probes labeled with digoxigenin and revealed using alkaline phosphatase-conjugated antibodies. Positive cells are stained in dark blue, sense probes showed no hybridization (data not shown). Stained cells enlargements are shown in A and B insets where scale bars are indicated. The expression partition limit is symbolized by a *. if: inner fold; mf: middle fold; of: outer fold; pg: periostracal groove; oe: outer epithelium; ie: inner epithelium; oec: outer epithelial cell.
<table><caption>Table 2 Protein identification in the shell matrix of *P. margaritifera* by a proteomic approach.</caption><thead><tr><th>Protein</th><th>Matching peptides</th><th>Mascot score</th><th>Homolog protein</th><th>Accession no.</th><th>Identity</th><th>E-Value</th><th>Species</th></tr></thead><tbody><tr><td><i>Pmarg-Pif177</i></td><td>26</td><td>1402</td><td>Pif177*</td><td>C7G0B5</td><td>72%</td><td>0.0</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-N66</i></td><td>16</td><td>759</td><td>N66*<br>Nacrein*</td><td>Q9NL38<br>Q27908</td><td>81% 67%</td><td>0.0 1 e-123</td><td><i>P. maxima</i><br><i>P. fucata</i></td></tr><tr><td><i>Pmarg-PFMG10</i></td><td>9</td><td>519</td><td>PFMG10</td><td>Q45TK0</td><td>56%</td><td>4 e-52</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-Linkine</i></td><td>6</td><td>278</td><td>Linkine</td><td>B5KFE5</td><td>100%</td><td>4 e-62</td><td><i>P. margaritifera</i></td></tr><tr><td><i>Pmarg-Pearlin</i></td><td>6</td><td>183</td><td>Perline*<br>N14/Pearlin*</td><td>Q14WA6<br>Q9NL39</td><td>96%<br>92%</td><td>2 e-85<br>2 e-82</td><td><i>P. margaritifera</i><br><i>P. maxima</i></td></tr><tr><td><i>Pmarg-Shematrin-2</i></td><td>4</td><td>340</td><td>Shematrin-8<br>Shematrin-2*</td><td>B5KFD0<br>Q1MW95</td><td>96% 68%</td><td>1 e-142<br>2 e-85</td><td><i>P. margaritifera</i><br><i>P. fucata</i></td></tr><tr><td><i>Pmarg-Pfty-1</i></td><td>4</td><td>285</td><td>Pfty-1*</td><td>A1IHF0</td><td>59%</td><td>1 e-146</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-Shematrin-5</i></td><td>4</td><td>248</td><td>Shematrin-5</td><td>Q1MW92</td><td>54%</td><td>1 e-68</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-MSI60</i></td><td>3</td><td>181</td><td>MSI60*</td><td>O02402</td><td>78%</td><td>1 e-120</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-MPN88</i></td><td>3</td><td>126</td><td>MPN88</td><td>B7X650</td><td>47%</td><td>2 e-87</td><td><i>P. fucata</i></td></tr><tr><td><i>Pmarg-KRMP-2</i></td><td>3</td><td>121</td><td>KRMP-11<br>KRMP-2</td><td>A7X103<br>C4TPC8</td><td>88%<br>56%</td><td>1 e-30 7<br>e-14</td><td><i>P. margaritifera</i><br><i>P. fucata</i></td></tr><tr><td><i>Pmarg-Shematrin-1</i></td><td>2</td><td>189</td><td>Shematrin-9<br>Shematrin-1*</td><td>B5KFD1<br>Q1MW96</td><td>98%<br>65%</td><td>4 e-99<br>8 e-62</td><td><i>P. margaritifera</i><br><i>P. fucata</i></td></tr><tr><td><i>Pmarg-Prismalin-14</i></td><td>2</td><td>149</td><td>Prismalin-14*</td><td>Q6F4C6</td><td>68%</td><td>5 e-40</td><td><i>P. fucata</i></td></tr></tbody></table>

The trypsin-digest peptides were separated on nano-LC, prior to nanoESI-qQ-TOF analysis. The MS/MS spectra were used for searching against the pyrosequencing based EST mantle library with Mascot software. We only considered proteins that presented at least 2 matching peptides. Mascot protein scores are indicated together with the number of unique peptides that matched to the sequence. The homologies were determined by BlastP interrogations against UniProtKB/Swiss-Prot protein database (January 2010) using the UniProt on-line tool http://www.uniprot.org, setting parameters to default. We only show here the best matches for the contigs which protein sequences are the homologues of already known mollusk shell proteins. We notice that all matches are from *Pinctada* genus origin.

This shotgun proteomic approach allowed us to identify more than 30 contigs, but only 13 proteins presented homologies to previously characterized mollusk shell proteins (Table 2). This list of proteins contains almost all of the shell proteins previously described before from shell matrix protein analysis of the *Pinctada* genus. [8]. Among them, PFMG10, Linkine, MPN88 and KRMPs were only characterized at the transcriptional level until now, and direct evidence of their implication in shell biomineralization process was lacking. Our data shows that they are definitely part of the calcifying matrix, which is embedded within the biomineral structures in the shell of *P. margaritifera*. We noticed that the few missing matches from the list of the biomineralization-related protein of the *Pinctada* genus. [8] - Aspein, MSI7 and Prisilkin-39 - exhibit sequences that are remarkably deficient in trypsin cleavage sites, making them unsuitable for standard proteomic detection. However, it is worth noting that homologs of the latter proteins are observed in the EST mantle library (Additional file 1).

Our proteomic analysis enabled us to retrieve *in silico* all the sequences from *P. margaritifera* involved in the biomineralization process already published on databases in our peptide library, and we were also able to

find a match in our database for all proteins experimentally found from *P. margaritifera* shell in our EST library. These results demonstrate that our EST data set covers most of the diversity of the matrix protein of the *P. margaritifera* shell.

## Conclusion

This global approach combining transcriptome and proteome analysis of *P. margaritifera* calcifying mantle and shell is the first description of a pyrosequencing program performed on a pearl-producing bivalve species. It led to the functional analysis of 276 738 EST sequences, with the constitution of a *P. margaritifera* mantle transcripts catalog of 82 sequences potentially implicated in the biomineralization process. Our results showed that the biomineralization protein repertoire is conserved within pearl oysters, but also provided direct evidence that our EST data set covered most of the diversity of *P. margaritifera* shell matrix protein. These observations clearly demonstrate the high efficiency of this pyrosequencing-based EST library in accurately identifying shell proteins, in combination with shotgun proteomic analysis and automated database searches. These data represent the most comprehensive list of biomineralization-related

<footnote>* indicates proteins which the occurrence in shell matrix was previously demonstrated by direct biochemical characterization.</footnote>
sequences available among protostomian species, and
represent a major breakthrough in the field of mollus-
kan biomineralization.

Additional material

**Additional file 1: Table S1:** Summary of BlastX results of biomineralization-related protein in the EST *P. margaritifera* mantle database. A catalogue of 82 *P. margaritifera* mantle transcripts potentially implicated in the biomineralization process was constructed using BlastX (E-value < 10⁻³) with selected protein sequences identified from mollusks (bivalvia and gastropoda).

Acknowledgements

This study is part of a collaborative project (GDR ADEQUA) supported by the "Service de la liciperature" of French Polynesia. It is also supported by Ifremer, Skuldtech and University of French Polynesia. Authors are grateful to Frédéric Marin, Marcel Le Pennec, Alexandre Tayalé, Florentine Riquet, Cédrik Lo and Anne-Sandrine Talfer for helpful discussions and assistance.

Author details

¹Ifremer - Laboratoire de Biotechnologie et de Qualité de la Perle, Centre Océanologique du Pacifique - BP 7004, 98719 Taravao, French Polynesia.

²Skuldtech, 134, rue du Curat - Bat. Amarante, 34090 Montpellier, France.

³UMR CNRS 5561 Biogéosciences, Université de Bourgogne, 6, bd Gabriel, 21000 Dijon, France. ⁴IFR 128 BioSciences Gerland-Lyon Sud; UMR 5086 CNRS; IBCP, Institut de Biologie et Chimie des Protéines, Université de Lyon 1, 7 passage du Vercors, Lyon, F-69367, France. ⁵Ifremer, Centre de Nantes-rue de l'Ile d'Yeu - BP 21105, 44311 Nantes cedex 03, France.

Authors' contributions

NCL and CM wrote the grant proposal. NCL, CM and YG conceived the project. CJ, BM and DP contributed to conception and design of the experiments. DP coordinated the construction, sequencing and analyses of the EST library. CJ performed RNA extraction and EST library construction, assisted with the functional annotations, analysis and interpretation of data and drafted the manuscript. BM and IZC carried out proteomic analysis of the shell matrix proteins. LM and FP performed contig assembly and functional annotation. CM performed *in situ* hybridization analyses. YG, CM and DP contributed to supervision of the work and critical review of the manuscript. All authors read and approved the final manuscript.

Received: 8 July 2010 Accepted: 1 November 2010

Published: 1 November 2010

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doi:10.1186/1471-2164-11-613

Cite this article as: Joubert et al.: Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization. BMC Genomics 2010 11:613.

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