Molecular Evolution of Mollusc Shell Proteins: Insights from Proteomic Analysis of the Edible Mussel Mytilus

Page 1

Molecular Evolution of Mollusc Shell Proteins: Insightsfrom Proteomic Analysis of the Edible Mussel Mytilus

Benjamin Marie • Nathalie Le Roy •

Isabelle Zanella-Cleon • Michel Becchi •

Frederic Marin

Received: 15 February 2011 / Accepted: 23 May 2011

� Springer Science+Business Media, LLC 2011

Abstract Shell matrix proteins (SMPs) that are embed-

ded within calcified layers of mollusc shells are believed to

play an essential role in controlling the biomineral syn-

thesis and in increasing its mechanical properties. Among

the wide diversity of mollusc shell textures, nacro-pris-

matic shells represent a tremendous opportunity for the

investigation of the SMP evolution. Indeed, nacro-pris-

matic texture appears early in Cambrian molluscs and is

still present in the shell of some bivalves, gastropods,

cephalopods and very likely also, of some monoplacoph-

orans. One key question is to know whether these shells are

constructed from similar matrix protein assemblages, i.e.

whether they share a common origin. Most of the molec-

ular data published so far are restricted to two genera, the

bivalve Pinctada and the gastropod Haliotis. The shell

protein content of these two genera are clearly different,

suggesting independent origins or considerable genetic

drift from a common ancestor. In order to describe puta-

tively conserved mollusc shell proteins, here we have

investigated the SMP set of a new bivalve model belonging

to another genera, the edible mussel Mytilus, using an

up-to-date proteomic approach based on the interrogation

of more than 70,000 EST sequences, recently available

from NCBI public databases. We describe nine novel

SMPs, among which three are completely novel, four are

homologues of Pinctada SMPs and two are very likely

homologues of Haliotis SMPs. This latter result constitutes

the first report of conserved SMPs between bivalves and

gastropods. More generally, our data suggest that mollusc

SMP set may follow a mosaic pattern within the different

mollusc models (Mytilus, Pinctada, Haliotis). We discuss

the function of such proteins in calcifying matrices, the

molecular evolution of SMP genes and the origin of mol-

lusc nacro-prismatic SMPs.

Keywords Biomineralization � Proteomics � Mollusc

shell nacre � Organic matrix � Evolution

Background

The explosive radiation of metazoan taxa during the Pro-

terozoic–Cambrian transition was shaped on the appear-

ance of several innovations, including the ability to form

biomineralized skeletons. Among them, the calcium car-

bonate shell, that protects the mollusc soft tissues, consti-

tutes an excellent model for studying the process of

biomineral formation and its evolution. The wide mor-

phological diversity of shell-bearing molluscs (bivalves,

gastropods, cephalopods, monoplacophorans and scapho-

pods, about 100,000? species (Ponder and Lindberg

2008)) also extends to a tremendous diversity of shell

micro-textures, including ‘‘prismatic’’, ‘‘nacreous’’, ‘‘foli-

ated’’, ‘‘cross-lamellar’’, ‘‘granular’’ ‘‘composite-pris-

matic’’ and ‘‘homogeneous’’ structures (Bøggild 1930;

Carter 1990; Chateigner et al. 2000). Despite this diversity,

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-011-9451-6) contains supplementarymaterial, which is available to authorized users.

B. Marie (&) � N. Le Roy � F. Marin (&)

UMR 5561 CNRS Biogeosciences, Universite de Bourgogne,

6 bd. Gabriel, Dijon 21000, France

e-mail: [email protected]; [email protected]

F. Marin

e-mail: [email protected]

I. Zanella-Cleon � M. Becchi

IFR 128 BioSciences Gerland-Lyon Sud, UMR 5086 CNRS,

Institut de Biologie et Chimie des Proteines, Universite

de Lyon 1, Lyon, France

123

J Mol Evol

DOI 10.1007/s00239-011-9451-6

Page 2

all molluscan shell layered structures are extracellular and

synthesised according to the same physiological pathway:

they result from the secretory activity of an evolutionarily

homologous organ known as the mantle. In short, the

mantle epithelium extrudes the ionic precursors of the shell

minerals (Bielefeld et al., 1992), together with an extra-

cellular ‘cell-free’ organic matrix that is incorporated into

and surrounds nascent CaCO3 crystals during the shell

growth. Although the organic shell matrix represents only a

small part of the CaCO3 shell weight (between 0.1 and 5%

w/w according to the different species and microstruc-

tures), it is well known to be essential for the control of the

biomineral formation (Mann 1988). It is, in particular,

involved in the arrangement of the organic framework

(Sudo et al. 1997), in the regulation of the CaCO3 pre-

cipitation (Wheeler et al. 1981) and in the control of the

crystal polymorph—aragonite and/or calcite (Falini et al.

1996). The biochemical characteristics of the organic

matrix, usually purified and studied following decalcifica-

tion of the shell, indicate that it comprises a heterogeneous

set of macromolecules including chitin, hydrophobic

‘framework’ proteins, soluble proteins and glycoproteins

(Crenshaw 1972; Weiner and Traub, 1984; Lowenstam and

Weiner 1989; Keith et al. 1993; Levi-Kalisman et al. 2001;

Bedouet et al. 2001; Marie et al. 2007, 2009a).

Because of its exceptional toughness (Jackson et al.

1988, Berthelat 2010), of its commercial value and

remarkable biocompatibility properties when implanted in

vivo (Atlan et al. 1997; Westbroek and Marin 1998), nacre

is among the most studied mineralized biomaterial (Mann

2001). Many authors consider it as the reference model for

understanding at the micro- and nano-scales how molluscs

control the regular deposition of calcium carbonate crystals

(Rousseau et al. 2005; Lin and Meyers 2005; Checa et al.

2009a; Gilbert et al. 2008). Nacre, also called mother-of-

pearl, is the calcified internal layer of several mollusc

shells. The mature nacreous layer consists of the super-

imposition of around 0.5-lm-thick aragonitic tablets,

embedded in a peripheral thin organic matrix (Nakahara

1991; Addadi et al. 2006; Nudelman et al. 2008; Weiss

2010). The prisms that are almost always associated to

nacre, are composed of calcitic or aragonitic needle-like

structures of various lengths and diameters that always

constitute the external calcified layer of shells, and that

grow inward by accretion of crystal units on the inner

surface of the periostracum (Marin et al. 2007; Checa et al.

2005). The individual prisms are stacked together in an

insoluble and hydrophobic organic sheath, which forms a

honeycomb-like structure. They also comprise a slight

intracrystalline organic fraction (Marin et al. 2005).

Nacro-prismatic shell microstructure assemblage appeared

in the Early Cambrian (Carter 1990; Feng and Sun 2003;

Vendrasco et al. 2010). Since then, it remained apparently

almost unchanged. Nowadays, nacro-prismatic microstruc-

tures are represented in at least three mollusc classes, bivalves,

gastropods and cephalopods. In monoplacophorans, true

nacreous layers are potentially observed only in one extant

genus (Checa et al. 2009b). One key question is to know

whether they are constructed from similar matrix protein

assemblages, i.e. whether they share a common origin. If so,

one can wonder whether the shell matrix proteins (SMPs) are

conserved within the different taxa.

Answering these questions is laborious, but will shed a

light on the process of recruitment of SMPs in the Cam-

brian, and on the evolutionary constraints exerted on these

proteins during the Phanerozoic. In fact, since the eluci-

dation of the primary structure of Nacrein (Miyamoto et al.

1996), the first-described nacre protein, the number of SMP

sequences has increased, but remains limited (Marin et al.

2008). Currently, pursuing discovery of mollusc SMPs is

particularly promising because of the genomic and tran-

scriptomic resources that are expending rapidly for many

phylogenetically diverse species that are also experimen-

tally tractable (Weiss and Schonitzer 2006; Jackson et al.

2007a; Suzuki et al. 2009, Auzoux-Bordenave et al. 2010;

Mamangkey and Southgate 2009; Inoue et al. 2010). Given

the high proportion of novel genes being reported from

non-model EST datasets and the flood of sequence data

from next generation technologies, these results emphasise

the importance of proteomic approaches for the validation

and the annotation of coding sequences. This is especially

relevant in the field of molluscan biomineralization where

all characterised biomineral-associated proteins have no

known homologues in any model species (Marin et al.

2008). There is already good precedent for such work in

global investigations of mollusc shell proteins based on

transcriptomics (Jackson et al. 2006, 2007a, 2010; Wang

et al. 2010), proteomics (Marie et al. 2009b, 2010a, b), or

both (Joubert et al. 2010). It is important to notice that

these works concern mostly two mollusc genera—the pearl

oyster Pinctada spp. and the abalone Haliotis spp.—which

comprise more than 90% of the molecular data for mollusc

SMPs (Marin et al. 2008). In a puzzling manner, despite

microstructural resemblance between the nacro-prismatic

shell layers of these two genera, no sequence homology has

been reported so far for mollusc SMPs. Moreover, recent

comparative EST approach performed on Haliotis and

Pinctada calcifying tissues have highlighted clear differ-

ences in the gene sets devoted to the control of shell for-

mation within the two genera (Jackson et al. 2010).

Interestingly, Joubert et al. (2010) have highlighted that

some biomineral-related proteins from gastropods share

sequence similarity with the pearl oyster mantle EST-

deduced proteins, but until now no direct evidence of their

implication in shell formation process has been produced.

On the other hand, although most of the protein domains of

J Mol Evol

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mollusc SMPs do not exhibit sequence similarity with

other known proteins, few of them present striking domain

homology with known extracellular matrix (ECM) proteins

from vertebrates, suggesting a deep Precambrian origin

(545 ? Ma). For example, N66/Nacrein presents two car-

bonic anhydrase domains (Miyamoto et al. 1996), Perlu-

strin shows similarities with insulin-like growth factor

binding proteins (Weiss et al. 2001), Pif-177 presents a

Von Willerbrand A domain (Suzuki et al. 2009), Perlucin

exhibits a C-type lectin domain (Mann et al. 2000), and

Perlwapin possesses whey acidic protein (WAP) domains

(Treccani et al. 2006).

In the present study, we have investigated the SMPs of

the edible mussel Mytilus, in order to compare them with

those of the above-mentioned taxa. Despite the recent

increasing interest in mytilid shells for ocean acidification

purposes (Miller et al. 2009; Gazeau et al. 2010), very few

works focus on the calcifying shell organic matrix of rep-

resentatives of this group (Weiner et al. 1977; Weiner

1983; Keith et al. 1993). To date, only one extrapallial fluid

protein has been described (Hattan et al. 2001; Yin et al.

2005) and almost no data exist on Mytilus SMPs (Weiner

1983; Keith et al. 1993). By combining a proteomic

approach based on the parallel investigation of the SMPs of

three closely related species of edible mussel (M. edulis,

M. galloprovincialis and M. californianus), and by inter-

rogating the EST dataset recently published for this genera

(Tanguy et al. 2008; Vernier et al. 2009; Craft et al. 2010),

we report here for the first time the primary structure of

nine SMPs, associated with the nacreous and the prismatic

shell microstructures of Mytilus. These include three novel

proteins (one of them probably corresponding to the

N-terminus of P21 which was partially characterised by

Keith et al. (1993)), four homologous proteins of Pinctada

SMPs, and two homologous proteins of Haliotis SMPs.

These results constitute the first report of conserved SMPs

between Bivalvia and Gastropoda. We discuss the function

of such proteins in calcifying matrix, the molecular evo-

lution of SMP genes and the origin of mollusc nacro-

prismatic SMPs.

Materials and Methods

Shell Matrix Extraction

Fresh adult M. edulis, M. galloprovincialis and M. califor-

nianus shells (6–12 cm in length) were collected from the

Brittany coast (France), the Adriatic coast (Croatia) and the

Californian coast (USA), respectively. Superficial organic

contaminants as well as the periostracum were removed by

incubating intact shells in NaOCl (1%, v/v) for 24 h. Shell

calcified layers (nacreous ? prismatic layers) were then

thoroughly rinsed with deionised water, dried and then

roughly crushed into fine powder ([200 lm). All subsequent

extractions were performed at 4�C as previously described

(Marin et al., 2005), with some modifications. Shell powder

samples were decalcified overnight in cold dilute acetic acid

(5%, v/v), which was slowly added by an automated titrator

(Titronic Universal, Schott, Mainz, Germany) at a flow rate

of 100 lL every 5 s. The solutions (final pH around 4.2) were

centrifuged at 3,9009g (30 min). The resulting pellets,

corresponding to the acid-insoluble matrices (AIMs), were

rinsed 6 times with MilliQ water, freeze-dried and weighed.

The supernatants comprising the acido-soluble matrices

(ASMs) were filtered (5 lm) before being concentrated with

an Amicon ultrafiltration system on a Millipore� membrane

(YM10; 10 kDa cut-off). The concentrated solutions (about

5–10 ml) were extensively dialysed against 1 l MilliQ water

(3 days, several water changes) before being freeze-dried

and weighed.

Protein Cleavage

The trypsin digestion of the AIMs from the calcified shell

layers (nacre ? prisms) of M. edulis, M. galloprovincialis

and M. californianus was performed in solution (Marie et al.

2008, 2009a). The samples (0.1 mg) were reduced with

25 lL of 10 mM dithiothreitol in 50 mM NH4HCO3 for

30 min at 50�C. Alkylation was performed with 50 lL of

50 mM iodoacetamide in 50 mM NH4HCO3 for 30 min at

room temperature in the dark. Then the solution was treated

with 1 lg of trypsin (Sequence grade, Promega, USA) in 10

lL 50 mM NH4HCO3 overnight at 37�C. The sample was

dried in a vacuum concentrator and re-suspended in 30 lL of

0.1% trifluoroacetic acid and 4% CH3CN.

Mass Spectrometry Analysis

Mass spectrometry was performed using a Q-Star XL

nanospray quadrupole/time-of-flight tandem mass spec-

trometer, nanospray-qQ-TOF–MS/MS (Applied Biosys-

tems, France), coupled to an online nano liquid

chromatography system (Ultimate Famos Switchos from

Dionex, The Netherlands). One microlitre of samples were

loaded onto a trap column (PepMap100 C18; 5 lm; 100 A;

300 lm 9 5 mm, Dionex), washed for 3 min at 25

lL min-1 with 0.05% trifluoroacetic acid/2% acetonitrile,

then eluted onto a C18 reverse phase column (PepMap100

C18; 3 lm; 100 A; 75 lm 9 150 mm, Dionex). Peptides

were separated at a flow rate of 0.300 lL min-1 with a

linear gradient of 5–80% acetonitrile in 0.1% formic acid

over 120 min. MS data were acquired automatically using

Analyst QS 1.1 software (Applied Biosystems). Following

a MS survey scan over m/z 400–1600, MS/MS spectra were

J Mol Evol

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sequentially and dynamically acquired for the three most

intense peptide molecular ions over m/z 65–2000. 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?.

MS Data Analysis

Protein identification was performed using the MASCOT

search engine (Matrix Science, London, UK; version 2.1)

against a protein database comprising the around 70,000

nucleotide sequences derived from the EST libraries of

Mytilus spp. (mainly represented by the around 5,000,

19,000 and 42,300 sequences from M. edulis, M. gallo-

provincialis and M. californianus, respectively), down-

loaded (March 2010) from the NCBI server (http://

www.ncbi.nlm.nih.gov). LC–MS/MS data were searched

using carbamidomethylation as fixed modification, and

methionine oxidation as variable modification. The peptide

mass and fragment ion tolerances were set to 0.5 Da. The

peptide hits were manually confirmed by the interpretation

of the raw LC–MS/MS spectra with analyst QS software

(Version 1.1). Quality criteria were the peptide MS value,

the assignment of major peaks to uninterrupted y- and

b-ion series of at least 3–4 consecutive amino acids and the

match with the de novo interpretations proposed by the

software.

Sequence Analysis

Protein sequence identification was attempted using

BLASTp and tBLASTn analysis performed against Swiss-

Prot, GenBank’s nrdb and dbEST using the online tool

provided by UniProt (www.uniprot.org) and NCBI (http://

blast.ncbi.nlm.nih.gov/blast.cgi) servers. Signal peptides

were predicted using SignalP 3.0 (http://www.cbs.dtu.dk/

services/SignalP/), and conserved domains were predicted

using SMART (http://smart.embl-heidelberg.de/) and In-

terProScan (http://www.ebi.ac.uk/Tools/InterProScan/).

The sequence alignments were performed with Clustal-W

or hierarchical-clustering algorithms using UniProt (www.

uniprot.org) or the MULTALIN (http://bioinfo.genotoul.

fr/multalin/multalin.html) online tools, using default para-

meters.

Phylogenetic Analysis

Representative complete sequences of the major non-ver-

tebrate metazoan CAs were selected from the results of

BLAST searched performed with Mcal-CA, using UniProt

and NCBI online tools, against Swiss-Prot, GenBank’s nrdb

and dbEST, or the specific blast tool available from Lottia

gigantea genome web site (http://genome.jgi-psf.org/pages/

blast.jsf?db=Lotgi1). These selected sequences were com-

pared with the molluscan mantle-secreted and non-secreted

CAs and the M. californianus sequence detected in the

current data set. The multiple alignment was created using

T-Coffee (Notredame et al. 2000) set to standard parame-

ters, and then a phylogenic reconstruction was using the

maximum like-hood method PhyML (Guindon and Gascuel

2003) from the www.phylogeny.fr server (Dereeper et al.

2008). Accession numbers of the sequences used are: Elysia

timida HP152215; Plakobranchus ocellatus HP204215;

Strongylocentrotus purpuratus XP001179236; Capitella

teleta EY522037; Lottia gigantea Lotgi1|238082|, Lot-

gi1|239188| from genome assembly (http://genome.

jgi-psf.org/Lotgi1/Lotgi1.download.ftp.html); Crassostrea

gigas CU996533; Pinctada maxima EZ420150, Q9NL38;

Pinctada fucata Q27908; Mytilus californianus P86856;

Haliotis gigantea BAH58349, BAH58350; Turbo marmo-

ratus Q8N0R6; Callinectes sapidus A3FFY1; Panaeus

monodon A9XTM5; Culex quinquefasciatus B0W447;

Drosophila simulans Q3YMV3; Riftia pachyptila Q8MPH8;

Nematostella vectensis A6QR76; Amphimedon queenslan-

dica A6QR75, A6QR76, A6QR77; Ectocarpus siliculosus

D8LB10; Chlamydomonas reinhardtii P20507.

Results

The Mytilus Shell

Like for other Mytilidae, the outer wall of the shell of Mytilus

exhibits a multi-layered organo-mineral structure (Fig. 1).

While the thin external layer, called the periostracum, is

mostly organic and gives to the shell its dark brown colour

(Fig. 1a), the rest of the shell is highly calcified and com-

posed of an outer prismatic and an inner nacreous layer

(Mutvei 1980; Feng et al. 2000; Dalbeck et al. 2008). Prisms

are calcitic micro-needles, oblique to the external shell sur-

face, that are enveloped by an organic sheath (Fig. 1b). The

nacre consists of the brick-wall-like superimposition of

around 0.5-lm thick aragonitic tablets, embedded by a

peripheral thin organic matrix, together forming a cohesive

framework (Fig. 1c). We carefully removed the periostra-

cum with sodium hypochlorite treatment. After decalcifica-

tion of the shell powder with cold acetic acid 5% (4�C), we

subsequently extracted the matrix exclusively associated

with the whole calcified layers of M. edulis, M. gallopro-

vincialis and M. californianus. The AIMs represents around

1% by weight of the shell powder, while the ASMs represents

only 0.05–0.1% by weight of the shell powder, and was not

further investigated here.

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Proteomic Analysis

In order to investigate the largest part, if not all, of the

protein set of Mytilus shell calcified layers, the non-frac-

tionated AIM materials derived from the nacre ? prism

samples of M. edulis, M. galloprovincialis and M. califor-

nianus (representing around 95% of the total AIM ? ASM

amount) were similarly analysed by LC–MS/MS, after

digestion with trypsin enzyme. For all samples, the peak list

generated from the MS/MS spectra was directly interro-

gated against the Mytilus EST database using MASCOT

software (Version 2.1). Using this approach, we were able

to identify nine proteins of the Mytilus shell matrices

(Table 1). The three novel proteins that did not present

homology with already known shell proteins, nor putatively

biomineral-related EST sequences, were called MUSP-1,

MUSP-2 and MUSP-3, for Mytilus Uncharacterised Shell

Proteins. Some of these proteins were detected in the shells

of all three Mytilus species (MUSP-3 and chitin-binding-

like), in two shell matrices (MUSP-1) or in only one of the

three species. The MS/MS spectra corresponding to

matching peptides were individually checked to confirm

their peptide sequences. No additional peptides were iden-

tified by including phosphorylation as a variable modifica-

tion during the MASCOT searches, indicating that specific

enrichment and LC–MS procedures are needed to analyse

these post-translational modifications. We also found that

all conceptually translated EST sequences that match our

MS/MS peptides possess a signal peptide. This indicates

that these bioinformatically predicted proteins are likely to

represent the entire amino N-terminus, and are genuinely

secreted by the mantle epithelium.

Novel Mollusc SMPs

Three of the nine proteins that we have identified here

(MUSP-1, MUSP-2 and MUSP-3) do not exhibit sequence

similarity with other already described SMPs. MUSP-1

[P86853] was detected in M. galloprovincialis shell matrix.

Its EST (FL490251) encodes for the C-terminus incomplete

sequence of a protein of at least 181-AA long, presenting a

22-AA long signal peptide (Fig. 2a). Interestingly, two

MUSP-1 trypsin peptides were also detected in M. edulis

shell matrix, testifying that a homologue protein is also

present in other Mytilus species, or at least also in M. ed-

ulis. The two M. californianus EST sequences, GE755963

and GE750813, encode for similar incomplete protein

sequences that can be aligned to form a unique contig

(Supplementary data S1) in one unique complete sequence

of MUSP-2 [P86858], a 341-AA long protein containing a

24-AA long signal peptide (Fig. 2b) that was detected in

M. californianus shell matrix. When the signal peptide is

removed from Mcal-MUSP-2 sequence, the resulting pro-

tein exhibits a theoretical molecular mass of 36 kDa and a

calculated pI around 11. No significant hit could be found

for MUSP-1 and MUSP2, when sequence similarities and

protein domains were searched by using BLASTp,

tBLASTn and SMART domain tools, respectively. These

data suggest that MUSP-1 and MUSP-2 are entirely novel

proteins associated with nacro-prismatic structures that

very likely do not present known homologous protein in

other metazoan taxa, including pteriomorphid bivalves.

On the other hand, MUSP-3 [P86859] was detected in

the shell matrix of the three Mytilus species. Its EST

(GE749275) encodes for a complete sequence of a 174-AA

long protein with a 16-AA long signal peptide (Fig. 2c).

When the signal peptide is removed from the Mcal-MUSP-3

sequence, the resulting protein exhibits a theoretical

molecular mass of 17 kDa and a calculated pI around 10.

Although no significant hit was obtained with BLASTp or

protein domain searches, we noticed that the N-terminal

sequence of MUSP-3 presents remarkable 82% sequence

similarity (but with an insignificant E-value score) with the

30-AA long N-terminus of P21 protein (Q9TWS3), previ-

ously described from soluble matrix of M. edulis shell

(Keith et al. 1993). Additionally, nBLASTt search with

MUSP-3 against all metazoan EST sequences indicates

remarkable sequence identity (Fig. 2d) with the EST

PmaxCL82Contig1 (EZ420213) that encodes for a putative

protein predicted to be secreted by the mantle of the pearl

oyster Pinctada maxima (Jackson et al. 2010), and which

has not been, to date, detected in calcified shell layers using

Fig. 1 The shell layers of the mussel Mytilus galloprovincialis. a General external view of the shell. b Scanning electron micrograph of texture

detail of the external prismatic layer. c Scanning electron micrograph of texture detail of the internal nacreous layer

J Mol Evol

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Ta

ble

1Id

enti

fica

tio

no

fth

esh

ell

mat

rix

pro

tein

so

fM

ytil

us

by

MS

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ysi

s

Sh

ell

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[Sw

issP

rot

nu

mb

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ES

TA

cc.

No

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pec

ies

Mas

cot

sco

re(n

um

ber

of

pep

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es)

Co

mp

lete

seq

uen

ce/

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Th

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tica

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mas

s/p

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rote

in(h

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Med

uM

ga

lM

cal

Nac

rein

-lik

ea[P

86

85

6]

GE

75

12

62

––

23

3(5

)N

o/?

–/–

No

vel

(Car

bo

nic

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yd

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/CA

do

mai

ns)

GE

74

90

08

M.

cali

forn

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us

MU

SP

-1[P

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3]

FL

49

02

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97

(2)

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0(5

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No

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–/–

No

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(no

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/no

reco

gn

ised

do

mai

n)

M.

ga

llo

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MS

I60

-lik

e[P

86

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7]

GE

74

96

43

––

20

6(4

)N

o/?

–/–

No

vel

(MS

I60

/A-

and

G-r

ich

do

mai

ns)

M.

cali

forn

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us

MU

SP

-2a

[P8

68

58

]G

E7

55

96

3–

–2

03

(4)

Yes

a/Y

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6k

Daa

/pI

=1

1a

No

vel

(no

ho

mo

log

/no

reco

gn

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do

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GE

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08

13

M.

cali

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Per

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ike

[P8

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55

]F

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4–

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)–

Yes

/Yes

14

kD

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/2W

AP

do

mai

ns)

M.

ga

llo

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MU

SP

-3[P

86

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J Mol Evol

123

Page 7

a similar proteomic approach (B. Marie, unpublished data).

These observations suggest that MUSP-3-related proteins

constitute a novel family of pteriomorphid conserved

proteins.

Bivalve-Conserved SMPs

We have identified four Mytilus SMPs that exhibit high

sequence homologies with SMPs extracted from the nacro-

prismatic shells of Pinctada bivalves (carbonic anhydrase,

MSI60, chitin-binding and fibronectin). Peptides matching

Mcal-carbonic anhydrase (Mcal-CA) [P86856] were

detected in the M. californianus shell samples (Fig. 3a).

The putative ORF for this protein was deduced from the

alignment of the ESTs GE751262 and GE749008 in a

unique contig (Supplementary data S2). The conceptually

derived protein sequence of Mcal-CA exhibits a N-termi-

nus incomplete sequence of 321-AA long. The Mcal-CA

sequence exhibits a characteristic CA domain with high

sequence identity for AA position conserved in metazoan

CA and involved in the catalytic activity of the enzymatic

domain, suggesting that Mcal-CA is an active CA

(Fig. 3b). Figure 3c shows the phylogenetic reconstruction

of the relationships between different mollusc and meta-

zoan CAs (Fig. 3c). This analysis clearly indicates that

Mcal-CA belongs to a group of molluscan mantle-secreted

CAs that are likely to be included in shell or involved in

shell deposition that is distinct from other molluscan non-

secreted CAs.

Interestingly, our results confirm the presence of CA in

other pteriomorph bivalve shells, but also indicate that the

Mytilus shell CA does not present the GN-repeat sequen-

ces, characteristic of Pinctada Nacrein proteins (Smith-

Keune and Jerry 2009). However, in spite of the absence of

GN repeats in Mytilus shell CA, the best sequence align-

ment was observed with enzymatic CA domain of Nacrein,

the shell-specific CA from Pinctada fucata (Fig. 4a). The

specific role of CA in a bivalve nacre extracellular calci-

fying matrix is still puzzling and may be related to the fine

regulation of ionic balance at the vicinity of the biomineral

structure formation.

Four different peptides corresponding to the partial

sequence of Mcal-MSI60 [P86857] were detected in the

shell matrix of M. californianus. The conceptually deduced

sequence of the EST GE749643 encodes for the 189-AA

long C-terminus sequence of a poly-Ala protein that pre-

sents high sequence similarities with MSI60 (Fig. 4b),

previously described from the shell of Pinctada fucata

(Sudo et al. 1997). MSI60 is a nacre specific insoluble

framework protein that exhibits 11 poly-Ala blocks and 39

poly-Gly blocks dispersed throughout the sequence. The

poly-Ala blocks confer to MSI60 structural similarity with

silk fibroins.

The ES393395 and ES393550 EST sequences can be

aligned in a unique contig sequence (Supplementary data

S3) and the resulting sequence encodes for the C-terminus

of a 294-AA long incomplete sequence of Mcal-Chitin-

binding [P86860] that can be detected in the calcified shell

layers of M. edulis, M. galloprovincialis and M. califor-

nianus. A SMART search for protein domains indicates

that it contains a Peritrophin-A chitin-binding domain

(Pfam:CBM_14). Interestingly, the result of the tBLASTn

search indicates a high sequence similarity (if not a true

homology) with a putative protein (Fig. 4c) encoded by the

EST PmaxCL21Contig1 EZ420121 (Jackson et al. 2010),

that was in parallel detected by similar proteomic analysis

of the nacreous layer of the pearl oyster Pinctada maxima

(Marie B., unpublished data).

Additionally, one peptide corresponding of the partial

putative sequence of Mcal-Fibronectin [P86861], concep-

tually deduced from the EST GE759315, was detected in

the shell layer of M. californianus. This EST encodes for a

Fig. 2 Sequence analysis of novel shell proteins: MUSP-1, MUSP-2

and MUSP-3. a The AA sequence of Mgal-MUSP-1 [P86853] is

deduced from the translation of the EST entry FL490251. b The AA

sequence of Mcal-MUSP-2 [P86858] is deduced from the alignment

of the translated sequences of the entry GE755963 and GE750813 in a

unique contig (Supplementary data S1). c The AA sequence of Mcal-MUSP-3 [P86859] is deduced from the translation of the EST entry

GE749275. The predicted signal peptides are underlined. The

peptides identified by MS/MS are indicated in red/grey. The asterisksmark the stop codons. Missing sequence information is indicated by

‘‘?’’. d Alignment of AA sequences of MUSP-3 [P86859] with the

deduced sequence from GE749275 of Pinctada maxima. The

predicted signal peptides are underlined. Conserved AA positions

are shaded in blue (Color figure online)

J Mol Evol

123

Page 8

224-AA long N-terminus sequence presenting a 17-AA

long signal peptide, for which SMART search indicates the

presence of a fibronectin-type 3 domain (FN3). The

tBLASTn search indicates a high sequence similarity with

a putative fibronectin-containing protein (Fig. 4d), encoded

by the EST PmaxCL366Contig1 EZ420486 (Jackson et al.

2010), that was also detected by proteomic analysis of the

prismatic layer of the pearl oyster Pinctada maxima (Marie

B., unpublished data).

Bivalve/Gastropod-Conserved SMPs

One of the most interesting results of our study was the

detection in Mytilus shell matrix of two proteins that

present high sequence homologies with SMPs extracted

from the nacro-prismatic shells of Haliotis gastropods,

constituting the first report of conserved SMPs between the

two taxa. Three and two different peptides corresponding

to the sequences of Mgal-Perlwapin [P86855] and Mgal-

Perlucin [P86854], respectively, were detected in the shell

matrix of M. galloprovincialis. Figure 5 illustrates the de

novo sequencing of the three peptides observed for Mgal-

Perlwapin. Following signal sequence removal, Mcal-Per-

lwapin and Mcal-Perlucin are characterised by theoretical

pIs of 10 and 6, and theoretical molecular weights of 14

and 16 kDa, respectively. The Mgal-Perlwapin EST

(FL494664) encodes a 141-AA long protein, presenting a

19-AA long signal peptide and two consecutive whey

acidic protein domains (WAP). Mgal-Perlwapin presents

high sequence similarity with Perlwapin proteins. Inter-

estingly, Perlwapin proteins have been previously descri-

bed from the shell of the gastropods H. laevigata (Treccani

et al. 2006) and H. asinina (Marie et al. 2010b). These

shell-extracted Perlwapin exhibit three successive WAP

domains and their sequence alignment with Mgal-Perlwa-

pin (Fig. 6a) indicates a good conservation of the Cys

residues of the WAP domains that are potentially involved

in protease inhibitor function. On the other hand, the Mgal-

Perlucin EST (AJ624413) encodes a 156-AA long protein,

presenting a 20-AA long signal peptide and a characteristic

C-type lectin domain (CTL). Mgal-Perlucin presents high

sequence similarity with the C-type lectin domain con-

taining proteins from various metazoans and especially

with the Perlucin protein (Fig. 6b) that has been previously

described from the shell of the gastropods H. laevigata

(Mann et al. 2000). The alignment of these two shell-

extracted Perlucin sequences (Fig. 6b) presents a good

conservation of the Cys- and Trp-rich regions that are

Fig. 3 Sequence analysis of Mytilus shell carbonic anhydrase (CA).

a The protein sequence of Mcal-CA [P86856] is deduced from the

alignment of the translated sequences of the entry GE751262 and

GE749008 in a unique contig (Supplementary data S3). The peptides

identified by MS/MS are indicated in red/grey. Missing sequence

information is indicated by ‘‘?’’ and asterisk marks the stop codon.

b Sequence alignment of a partial sequence of Mcal-CA with

representative CA from various metazoan taxa. We observed that the

enzymatic domains (shaded in purple/grey) are conserved in all

represented CA forms. c Phylogram of CAs from various metazoan

taxa. Included in the phylogenetic analysis are representative metazoan

CAs, the molluscan mantle-secreted and molluscan shell-extracted CAs

involved in biocalcification, other molluscan CAs, and two outgroup

sequences (brown and green alga). Numerals at each node show local

likelihood ratio values estimated by PhyML. The scale bar indicates an

evolutionary distance of 0.8 AA substitution per position in the

sequences. The Mytilus shell-extracted CA [P86856] is shown by a

black arrow. (SP) and (-) indicate that the protein presents a

characteristic signal peptide sequence or not, respectively. Starsindicate that proteins were extracted from calcified shell layers (Color

figure online)

J Mol Evol

123

Page 9

involved in the Ca2?-dependent carbohydrate recognition

ability of C-type lectins.

Discussion

Mollusc Nacro-Prismatic Shells

Nacre and prisms seem to be evolutionary-conserved

microstructures that could have been observed in the shell

of numerous molluscs from the early Cambrian to our days.

At first sight, these microstructures are described by simple

terminologies, ‘prism’ on one side and ‘nacre’ on the other

side. However, these terminologies are misleading. For

example, while gastropod and cephalopod nacres are

described as ‘‘columnar’’, the bivalve nacre is presented as

‘‘sheet nacre’’, with characteristic arrangement of nacre

tablets in a ‘brick-wall’ manner (Nakahara 1991). Fur-

thermore, the arrangements of the three axes that charac-

terise their crystal orientations differ between the different

mollusc classes (Chateigner et al. 2000, 2010). Indeed,

both gastropod and bivalve nacres orient the c axis per-

pendicular to the shell surface, but in the other hand, the

b axis is oriented in the direction of shell growth for

bivalves (Chateigner et al., 2000), whereas, in gastropod

nacre, b and c axis are gradually co-oriented from

the prismatic boundary (Gilbert et al. 2008). These

crystallographic differences suggest that the modes of

deposition of aragonite platelets could be different within

the different mollusc clades, at the molecular level.

Different Sets of Nacro-Prismatic SMPs

The organic matrices extracted from nacro-prismatic shell

of several gastropods, cephalopods and bivalves have been

the subject of many investigations since they were believed

to control the deposition of calcified shell layer (Crenshaw

1972; Mann 1988; Lowenstam and Weiner 1989). As the

amino acid analysis of nacro-prismatic shells of different

molluscs exhibited similar compositions, with characteris-

tically high contents of Gly, Ala and Asx residues (Keith

et al. 1993), it was postulated that the molecular mecha-

nism controlling the formation of these different shell

layers is identical from clade to clade. But the discovery of

an increasing number of SMPs has revealed an unexpected

diversity of nacro-prismatic associated proteins among the

different taxa (Marin et al. 2008), rendering this idea

oversimplified. Furthermore, a preliminary comparative

proteomic approach on four nacreous molluscs has sug-

gested that the nacre protein content of these four genera

are different (Marie et al. 2009b). More recently, Jackson

et al. (2010) have demonstrated, by using a specific EST

approach, the drastic differences in the respective shell

building gene sets of the bivalve Pinctada and of the

Fig. 4 The bivalve conserved shell proteins detected in Mytilus shell

matrices and their homologous proteins from Pinctada shells.

a Sequence alignment of Mcal-CA (alignment of GE751262 and

GE749008 in a unique contig, Supplementary data S1) with Nacrein

from Pinctada fucata [Q27908]. b Sequence alignment of Mcal-MSI60

[P86857] (GE749643) with MSI60 from Pinctada fucata [O02402].

c Sequence alignment of Mcal-Chitin-binding [P86860] (alignment of

ES393395 and ES393550 in a unique peptide, Supplementary data S1)

with chitin-binding from Pinctada maxima (PmaxCL21Contig1

EZ420121). C-bind = chitin-binding. d Sequence alignment of

Mcal-Fibronectin [P86861] (GE759315) with Fibronectin from Pinct-ada maxima (PmaxCL366Contig1 EZ420486). FN3 = Fibronectin of

type 3. The conserved AA positions are shaded in blue/grey. The

asterisks mark the stop codons (Color figure online)

J Mol Evol

123

Page 10

gastropod Haliotis. As underlined by these authors, the data

suggest that ‘‘the Bivalvia and the Gastropoda have either

independently evolved the ability to deposit nacre or that

subsequent to the genesis of the ability in a common

ancestor, bivalves or gastropods have significantly modi-

fied the molecular mechanism that guide this process’’

(Jackson et al. 2010). We generally agree with this

statement—based solely on two genera—but feel that it

should be balanced, with the introduction of the new data

on Mytilus, especially those that show unexpected simi-

larities between mussel and abalone Perlwapins and

Perlucins.

Figure 7a summarises the list of the around sixty SMPs

that are now known for the three main models of nacro-

Fig. 5 Example of de novo

sequencing for the three

peptides matching with Mgal-Perlwapin sequence. a MS/MS

spectrum of the CAAVTVNK

peptide (m/z 431.74). b MS/MS

spectrum of the FNCLFQK

peptide (m/z 478.74). c MS/MS

spectrum of the CAAVTVNKK

peptide (m/z 495.78). The de

novo sequencing was performed

by considering precise mass

differences between adjacent

b and y ion series

J Mol Evol

123

Page 11

prismatic molluscs, the bivalves Mytilus and Pinctada, and

the gastropod Haliotis. Beside the four common SMPs

between Pinctada and Mytilus (CA, MSI60, Chitin-binding

and Fibronectin), the two common SMPs between Pinctada

and Crassostea (Fibronectin and EGF-like, described in

Marie et al. 2011), we notice that the distribution of these

SMPs follows a ‘‘mosaic pattern’’, which means that some

SMPs are absent from, at least, one of the studied models

(Fig. 7b). The first example is that of N14/N16/Pearlin which

is present only in the pearl oyster shell matrix. N14/N16/

Pearlin represents one of the main components of the

Pinctada SMP set, and it is believed to be essential in the

deposition of the nacreous layer (Samata et al. 1999; Kono

et al. 2000). We have searched for N14/N16/Pearlin homo-

logue in Mytilus EST database (also in Crassostrea and

Haliotis EST db), using both BLASTn and tBLASTn, and no

hit was observed. Taken together, these data suggest that no

detectable homologous protein of N14/N16/Pearlin is pres-

ent in Mytilus, highlighting significant differences with

Pinctada in the molecular mechanisms of nacre deposition.

Similarly, the homologues of other Pinctada SMPs (e.g. Pif-

177, Shematrin, Tyrosinase and KRMP) were not detected

by our proteomic approach in Mytilus shell. However, we

cannot exclude here the possibility that the Mytilus EST

dataset does not represent the whole shell-forming tran-

scriptome. Indeed, the efficiency of such a proteomics

approach relies largely on the completeness of the EST data

set. In this study, we have exploited a pool of around 70,000

different ESTs from various tissues of M. edulis, M. gallo-

provincialis and M. californianus (Tanguy et al. 2008;

Vernier et al. 2009; Craft et al. 2010). We noticed that,

although homologous proteins were detected in different

Mytilus shells (e.g. MUSP-1, MUSP-3 and Chitin-binding),

the corresponding mRNAs only appeared in the EST of one

of these species (Table 1), testifying of important qualitative

differences in their respective EST dataset. Indeed, impor-

tant variations in biomineralising gene expression are likely

to occur between individuals according to their develop-

mental stage (Jackson et al. 2007a), to their respective

physiological condition or even depending on the moment of

the day (Miyazaki et al. 2008). This point should be carefully

considered, especially when sampling calcifying tissues for

transcriptomic analysis. Moreover, we are aware that the

EST data sets used in this study are not exhaustive, and future

efforts will likely reveal additional SMPs For example, a

recent proteomic analysis of the calcified skeleton of the sea

urchin Paracentrotus purpuratus evidenced an unexpected

diversity of matrix proteins, due to the availability of a

important dataset from Spur_v2.1 draft genome (Mann et al.

2008a, b).

CA is a ubiquitous metalloenzyme, essential in calcifi-

cation processes (Wilbur and Jodrey 1955; Medakovic

2000), that catalyses the production of bicarbonate ions,

that subsequently react with calcium ions to form calcium

carbonate. This enzyme has been observed in an increasing

number of calcifying epithelia and was also extracted from

calcified biominerals from different models belonging to a

wide range of metazoan species (Rahman et al. 2005;

Tambutte et al. 2007; Jackson et al. 2007b; Mann et al.

2008). In molluscs, a shell-specific form of CA, Nacrein,

containing both CA active domain and long GN-repeats,

has been isolated from the shell matrix of the pearl oysters

Pinctada (Miyamoto et al. 1996; Kono et al. 2000). Sim-

ilarly, a Nacrein-related protein sequence has been

Fig. 6 The bivalve/gastropod

conserved shell proteins

detected in Mytilus shell

matrices and their homologous

proteins from Haliotis shells.

a Sequence alignment of Mgal-Perlwapin [P86855]

(FL494664) with Perlwapin

from H. laevigata and

H. asinina, [P84811] and

[P86730], respectively.

b Sequence alignment of

Mgal-Perlucin [P86854]

(AJ624413) with Perlucin from

Haliotis laevigata [P82596].

The conserved AA positions are

shaded in green/grey. The

asterisks mark the stop codons

(Color figure online)

J Mol Evol

123

Page 12

described from the analysis of the mRNA of the mantle of

the gastropod Turbo marmoratus (Miyamoto et al. 2003),

but to date no CA has been directly detected from the shell

nacre of this gastropod nor from a cephalopod nacre (Marie

et al. 2009a). On the other hand, the proteomic investiga-

tion of the limpet Lottia gigantea SMPs reveals the pres-

ence of two different CAs in the calcifying matrix of this

gastropod. As shown in Fig. 3c, the phylogenetic recon-

struction of non-vertebrate metazoan CAs clearly distin-

guishes two groups of mollusc CAs—the mantle-secreted

CAs and the non-secreted CAs—suggesting a common

origin between bivalves and gastropods for the recruitment

of a specific CA for the process of shell deposition. Sur-

prisingly, and in spite of numerous studies, no shell CA has

ever been detected in the Haliotis shell matrices (for review

see Marin et al. 2008; Jackson et al. 2010; Marie et al.

2010b; Le Roy et al., unpublished data). However, we

cannot exclude the possibility that one or more mantle-

secreted CAs could be specifically involved in shell

deposition process, but remain absent of shell-integrated

matrix protein set, as suggested by molecular biology data

(Le Roy et al., unpublished data). This proves without

ambiguity that major calcifying matrix proteins, although

always in contact with the mineralization front, may be

ultimately not integrated within the calcifying shell matrix

during the deposition of the calcified shell layers.

Interestingly, our study emphasises the apparent absence

of EP (extrapallial protein, Q6UQ16) in the Mytilus SMP set,

in spite of the presence of EP mRNA in the Mytilus EST

dataset used for our proteomic investigation. EP is a His-rich

Ca2?-binding glycoprotein that was clearly detected from

extrapallial fluid of Mytilus edulis by both MALDI or ESI

MS/MS techniques, and that was previously supposed to be

part of the SMP (Hattan et al. 2001; Yin et al. 2005). Our data

suggests that this protein is not incorporated within the shell,

while it represents the main extrapallial fluid content and its

apparent strong interactions with calcium ions.

Evolution and Origin of Mollusc SMPs

The fact that Perlwapin and Perlucin homologues have

been observed from the nacro-prismatic shell matrix of

Fig. 7 Evolution of the composition of the calcifying matrix in

molluscs. a Comparison of shell matrix composition between the

nacro-prismatic shell models Pinctada, Haliotis and Mytilus. Blackand grey boxes indicate when the proteins were isolated from the

prismatic or the nacreous layer, respectively. b Presence/absence

mosaic pattern of mollusc SMPs in front of the phylogenetic

relationship of the main models. ‘‘?’’ indicates that although CA

was observed by the mantle epithelial cells, no CA was directly

observed from the shell of Haliotis spp. (Le Roy et al., unpublished).

Fibronect = Fibronectin; Chit-bind = chitin-binding; HUSP = Hal-iotis Uncharacterised Shell Protein

J Mol Evol

123

Page 13

both Mytilus and Haliotis may suggest that these proteins

were present in the calcifying matrix of the last common

ancestor of conchiferan molluscs. Alternately, we cannot

exclude the possibility that these proteins were recruited

twice independently in both classes. As the sequence

similarities of these proteins are restricted to the active site

residues—a fact that allows assigning them to their

respective family—it is still difficult to determine whether

they are orthologues or paralogues, and more work at the

gene level should be performed to give an unambiguous

answer.

Interestingly, the similarities detected between the

bivalve and gastropod Perlwapins and Perlucins find an

echo with previous findings on putatively conserved shell

proteins domains, such as Kunitz-like domains, detected in

both Pinctada and Haliotis (Liu et al. 2007; Marie et al.

2010b). Here again, such domains may be inherited from

the last common ancestor of bivalves and gastropods, or

may result from independent recruitments. So far, we

cannot yet definitively conclude on the evolutionary sce-

nario for calcifying matrix proteins of the different con-

chiferan molluscs.

SMP Description by Interrogating EST Dataset

with a Proteomic Approach

Our observations make obvious the value of EST libraries

when used in conjunction with a shotgun proteomic

approach for the investigation of calcifying matrix proteins.

By establishing that several of the predicted proteins from

the EST dataset are actually components of the shell, we

are able to make hypotheses about their direct contribution

to shell construction and the implications of their evolution

among calcifying shell matrices. Without this, the EST

dataset is simply a list of sequences that can be associated

to putatively secreted protein sequences, for which func-

tional assumption can be only attempted according to

sequence similarity with already described proteins, and is

not valuable for the description of novel proteins.

The main contribution of this article is that it is taking

the current push of sequencing huge numbers of ESTs to

the next step, which is to try and get some functional

information from these genes for biomineral formation

purposes. The challenge that now faces the field is to

characterise the function of novel biomineral associated

proteins, using in vivo or in vitro techniques.

Conclusion

Aside from the significant differences in the molecular

mechanisms used by the bivalve Pinctada and the gastro-

pod Haliotis for nacro-prismatic shell deposition (review

Marin et al. 2008, 2010b; Jackson et al. 2010), we observed

that the shell protein set of the nacro-prismatic bivalve

Mytilus is partly similar to that of other bivalves, but also

shares few similarities with that of the gastropod Haliotis.

The evolutionary picture that emerges is, for the moment,

patchy. We suggest that the mollusc SMP sets may follow a

mosaic phylogenetic pattern, suggesting that the process of

the integration in the shell of the mantle secreted proteins

may be a complex phenomenon, which does not take place

according to the taxonomic position of the considered

species. We believe, furthermore, that important molecular

functions for shell calcification may not be represented in

the shell, once formed.

The origin and evolution of molluscan SMPs appears to

be a complex phenomenon, which will require large-scale

comparisons across the whole Mollusca phylum, which

means, accurate systematic and wide sampling of mollusc

species, for deciphering the whole protein set (mantle

secreted proteins, extrapallial fluid proteins together with

shell-incorporated matrix proteins) involved in shell

calcification.

Acknowledgments The work of BM, NLR and FM is financially

supported by an ANR (ACCRO-EARTH, ref. BLAN06-2_159971,

coordinator Gilles Ramstein, LSCE) during the period 2007–2011.

The ‘‘Conseil Regional de Bourgogne’’ (Dijon, France) provided

additional supports for the acquisition of new equipment in the Bio-

geosciences research unit (UMR CNRS 5561). A complementary

financial support was provided by INSU (Action INTERRVIE 2010).

BM would like to thanks Davorin Medakovic for providing the fresh

shells of Mytilus galloprovincialis and Jerome Thomas for handling

shell pictures. The present protein sequences appear in the UniProtKB

under the accession numbers P86853–P86861.

References

Adamkewicz SL, Harasewych MG, Blake J, Saudek D, Bult CJ

(1997) A molecular phylogeny of the bivalve molluscs. Mol Biol

Evol 14:619–629

Addadi L, Joester D, Nudelman F, Weiner S (2006) Mollusk shell

formation: a source of new concepts for understanding biomin-

eralization processes. Chem Rev 12:980–987

Atlan G, Balmain N, Berland S, Vidal B, Lopez E (1997)

Reconstruction of human maxillary defects with nacre powder:

histological evidence for bone regeneration. C R Acad Sci Paris

320:253–258

Auzoux-Bordenave S, Badou A, Gaume B, Berland S, Helleouet MN,

Milet C, Huchette S (2010) Ultrastructure, chemistry and

mineralogy of the growing shell of the European abalone

Haliotis tuberculata. J Struct Biol 171:277–290

Bedouet L, Schuller J, Marin F, Milet C, Lopez E, Giraud M (2001)

Soluble proteins of the nacre of the giant oyster Pinctadamaxima and of the abalone Haliotis tuberculata: extraction and

partial analysis of nacre proteins. Comp Biochem Physiol B

128:389–400

Berthelat F (2010) Nacre from mollusk shell: a model for high-

performance structural materials. Bioinspir Biomin 20:035001

J Mol Evol

123

Page 14

Bielefeld U, Zierold K, Korje KH, Becker W (1992) Calcium

localization in the shell-forming tissue of the freshwater snail,

Biomphalaria glabrata: a comparative study of various methods

for localizing calcium. Histochem J 24:927–938

Bøggild O (1930) The shell structure of the mollusks. K Dan Vidensk

Selsk Skr Naturvidensk Math Afd 9:233–326

Carter JG (1990) Skeletal biomineralization: patterns, processes and

evolutionary trends. Van Nostrand Reinhold, New York

Chateigner D, Hedegaard C, Wenk HR (2000) Mollusc shell

microstructures and crystallographic textures. J Struct Geol

22:1723–1735

Chateigner D, Ouhenia S, Krauss C, Hedegaard C, Gil O, Morales M,

Lutterotti L, Rousseau M, Lopez E (2010) Voyaging around

nacre with the X-ray shuttle: from bio-mineralisation to

prosthetics via mollusc phylogeny. Mater Sci Eng A 528:37–51

Checa AG, Rodriguez-Navarro AB, Esteban-Delgado JF (2005) The

nature and formation of calcitic columnar prismatic shell layers

in pteriomorphian bivalves. Biomaterials 26:6404–6414

Checa AG, Cartwright JH, Willinger MG (2009a) The key role of the

surface membrane in why gastropod nacre grows in towers. Proc

Nat Acad Sci USA 106:38–43

Checa A, Ramirez-Rico J, Gonzalez-Segura A, Sanchez-Navas A

(2009b) Nacre and false-nacre (foliated aragonite) in extant

monoplacophorans (= Tryblidiida: Mollusca). Naturwissens-

chaften 96:111–122

Craft JA, Gilbert JA, Temperton B, Dempsey KE, Ashelford K,

Tiwari B, Hutchinson TH, Chipman JK (2010) Pyrosequencing

of Mytilus galloprovincialis cDNAs: tissue-specific expression

patterns. PLoS One 5:e8875

Crenshaw MA (1972) The soluble matrix from Mercenaria merce-naria shell. Biomineralization 6:6–11

Dalbeck P, England J, Cusack M, Lee MR, Fallick AE (2008)

Crystallographic and chemistry of the calcium carbonate poly-

morph switch in M. edulis shells. Eur J Mineral 18:601–609

Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F,

Dufayard J-F, Guindon S, Lefort V, Lescot M, Claverie J-M,

Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for

the non-specialist. Nucleic Acid Res 36:465–469

Falini G, Albeck S, Weiner S, Addadi L (1996) Control of aragonite

and calcite polymorphism by mollusk shell macromolecules.

Science 271:67–69

Feng W, Sun W (2003) Phosphate replicated and replaced micro-

structure of molluscan shells from the earliest Cambrian in

China. Acta Palaeontol Pol 48:21–30

Feng QL, Li HB, Pu G, Zhang M, Cui FZ, Li D (2000) Crystallo-

graphic alignment of calcite prisms in the oblique prismatic layer

of the Mytilus edulis shell. J Mater Sci 35:3337–3340

Gazeau F, Gattuso J-P, Pronker E, Peene J, Heip CH, Middelburg JJ

(2010) Effect of ocean acidification on the early stages of blue

mussel Mytilus edulis. Biogeosciences 7:2051–2060

Gilbert PU, Metzler RA, Zhou D, Scholl A, Doran A, Young A, Kunz

M, Tamura N, Coppersmith SN (2008) Gradual ordering in red

abalone nacre. J Am Chem Soc 130:17519–17527

Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm

to estimate large phylogenies by maximum likelihood. Syst Biol

52:696–704

Hattan SJ, Laue TM, Chasteen ND (2001) Purification and charac-

terization of a novel calcium-binding protein from the extrapal-

lial fluid of the mollusc, Mytilus edulis. J Biol Chem

276:4461–4468

Inoue N, Ishibashi R, Ishikawa T, Atsumi T, Aoki H, Komura A

(2010) Gene expression patterns and pearl formation in the

Japanese pearl oyster (Pinctada fucata): a comparison of gene

expression patterns between the pearl sac and the mantles

tissues. Aquaculture 308:S68–S74

Jackson AP, Vincent JV, Turner RM (1988) The mechanical design of

nacre. Proc R Soc Lond 234:415–440

Jackson DJ, McDougall C, Green KM, Simpson F, Worheide G,

Degnan BM (2006) A rapidly evolving secretome builds and

patterns a sea shell. BMC Biol 4:40

Jackson DJ, Worheide G, Degnan BM (2007a) Dynamic expression

of ancient and novel molluscan shell genes during ecological

transitions. BMC Evol Biol 7:160

Jackson DJ, Macis L, Reitner J, Degnan BM, Worheide G (2007b)

Sponge paleogenomics reveals an ancient role for carbonic

anhydrase in skeletogenesis. Science 316:1893–1895

Jackson DJ, McDougall C, Woodcroft B, Moase P, Rose RA, Kube

M, Reinhardt R, Rokshar DS, Montagnani C, Joubert C,

Piquemal D, Degnan BM (2010) Parallel evolution of nacre

building gene sets in molluscs. Mol Biol Evol 27:591–608

Joubert C, Piquemal D, Marie B, Manchon L, Pierrat F, Zanella-

Cleon I, Cochennec-Laureau N, Gueguen Y, Montagnani C

(2010) Transcriptome and proteome analysis of Pinctadamargaritifera calcifying mantle and shell: focus on biomineral-

ization. BMC Genomics 11:613

Keith J, Stockwell S, Ball D, Remillard K, Kaplan D, Thanhauser T,

Sherwood R (1993) Comparative analysis of macromolecules in

mollusc shells. Comp Biochem Physiol B 105:487–496

Kono M, Hayashi N, Samata T (2000) Molecular mechanism of the

nacreous layer formation in Pinctada maxima. Biochem Biophys

Res Comm 269:213–218

Levi-Kalisman Y, Falini G, Addadi L, Weiner S (2001) Structure of

the nacreous organic matrix of a bivalve mollusk shell examined

in the hydrated state using cryo-TEM. J Struct Biol 135:8–17

Lin A, Meyers MA (2005) Growth and structure in abalone shell.

Mater Sci Eng 390:27–41

Liu HL, Liu SF, Ge YJ, Liu J, Wang XY, Xie LP, Zhang RQ, Wang Z

(2007) Identification and characterization of a biomineralization

related gene PFMG1 highly expressed in the mantle of Pinctadafucata. Biochemistry 46:844–851

Lowenstam HA, Weiner S (1989) On biomineralization. Oxford

University Press, New York

Mamangkey NG, Southgate PC (2009) Regeneration of excised

mantle tissue by the silver-lip pearl oyster, Pinctada maxima(Jameson). Fish Shellfish Immun 27:164–174

Mann S (1988) Molecular recognition in biomineralization. Nature

332:119–124

Mann S (2001) Biomineralization: principles and concepts in

bioinorganic materials chemistry. Oxford University Press,

New York

Mann K, Weiss IM, Andre S, Gabius HJ, Fritz M (2000) The amino

acid sequence of the abalone (Haliotis laevigata) nacre protein

perlucin. Eur J Biochem 267:5257–5264

Mann K, Poustka AJ, Mann M (2008a) The sea urchin (Strongylo-centrotus purpuratus) test and spine proteomes. Proteome Sci

6:22

Mann K, Poustka AJ, Mann M (2008b) In-depth, high-accuracy

proteomics of sea urchin tooth organic matrix. Proteome Sci 6:33

Marie B, Luquet G, Pais de Barros J-P, Guichard N, Medakovic D,

Marin F (2007) The shell matrix of the freshwater mussel Uniopictorum (paleoheterodonta, unionoida): involvement of acidic

polysaccharides from glycoproteins in nacre mineralization.

FEBS J 274:2933–2945

Marie B, Luquet G, Bedouet L, Milet C, Guichard N, Medakovic D,

Marin F (2008) Nacre calcification in the freshwater mussel Uniopictorum: carbonic anhydrase activity and purification of a

95 kDa calcium-binding glycoprotein. ChemBioChem 9:2515–

2523

Marie B, Marin F, Marie A, Bedouet L, Dubost L, Alcaraz G, Milet C,

Luquet G (2009a) Evolution of nacre: biochemistry and

J Mol Evol

123

Page 15

proteomics of the shell organic matrix of the cephalopod

Nautilus macromphalus. ChemBioChem 10:1495–1506

Marie B, Le Roy N, Marie A, Dubost L, Milet C, Bedouet L, Becchi

M, Zanella-Cleon I, Jackson DJ, Degnan BM, Luquet G, Marin F

(2009b) Nacre evolution: a proteomic approach. Mater Res Soc

Symp Proc 1187:KK01–KK03

Marie B, Zanella-Cleon I, Le Roy N, Becchi M, Luquet G, Marin F

(2010a) Proteomic analysis of the acid-soluble nacre matrix of

the bivalve Unio pictorum: detection of novel carbonic anhy-

drase and putative protease inhibitor proteins. ChemBioChem

11:2138–2147

Marie B, Marie A, Jackson DJ, Dubost L, Degnan BM, Milet C,

Marin F (2010b) Proteomic analysis of the organic matrix of the

abalone Haliotis asinina calcified shell. Proteome Sci 8:54

Marie B, Zanella-Cleon I, Guichard N, Becchi M, Marin F (2011)

Novel proteins from the calcifying shel matrix of the pacific

oyster Crassostrea gigas. Mar Biotechnol. doi:10.1007/s10126-

011-9379-2

Marin F, Amons R, Guichard N, Stiger M, Hecker A, Luquet G,

Layrolle P, Alcaraz G, Riondet C, Woestbroek P (2005)

Caspartin and Calprismin, two proteins of the shell calcitic

prisms of the mediterranean fan mussel Pinna nobilis. J Biol

Chem 280:33895–33908

Marin F, Pokroy B, Luquet G, Layrolle P, De Groot K (2007) Protein

mapping of calcium carbonate biominerals by immunogold.

Biomaterials 28:2368–2377

Marin F, Luquet G, Marie B, Medakovic D (2008) Molluscan shell

proteins: primary structure, origin, and evolution. Curr Top Dev

Biol 80:209–276

Medakovic D (2000) Carbonic anhydrase activity and biomineraliza-

tion process in embryos, larvae and adult blue mussels Mytilusedulis. Hegol Mar Res 54:1–6

Miller AW, Reynolds AC, Sobrino C, Riedel GF (2009) Shellfish face

uncertain future in high CO2 world: influence of acidification on

oyster larvae calcification and growth in estuaries. PLoS One

4:e5661

Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T,

Matsushiro A (1996) A carbonic anhydrase from the nacreous

layer in oyster pearls. Proc Natl Acad Sci USA 93:9657–9660

Miyamoto H, Yano M, Miyashita T (2003) Similarities in the

structure of nacrein, the shell-matrix protein, in a bivalve and a

gastropod. J Moll Stud 69:87–89

Miyazaki Y, Usui T, Kajikawa A, Hishiyama H, Matsuzawa N,

Nishida T, Machii A, Samata T (2008) Daily oscillation of gene

expression associated with nacreous layer formation. Front

Mater Sci China 2:162–166

Mutvei H (1980) The nacreous layer in molluscan shells. In: Omori

M, Watabe N (eds) The mechanisms of mineralization in

biological systems. Tokay University press, Tokyo, pp 49–56

Nakahara H (1991) Nacre formation in bivalve and gastropod

molluscs. In: Suga S, Nakahara H (eds) Mechanisms and

phylogeny of mineralization in biological systems. Springer-

Verlag, New York, pp 343–350

Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel

method for fast and accurate multiple sequence alignment. J Mol

Biol 302:205–217

Nudelman F, Shimoni E, Klein E, Rousseau M, Bourrat X, Lopez E,

Addadi L, Weiner S (2008) Forming nacreous layer of the shells

of the bivalves Atrina rigida and Pinctada margaritifera: an

environmental- and cryo-scanning electron microscopy study.

J Struct Biol 162:290–300

Ponder W, Lindberg DR (2008) Phylogeny and evolution of the

mollusca. University of California Press, Berkeley

Rahman AM, Isa Y, Uehara T (2005) Proteins of calcified endoskel-

eton: II. Partial amino acid sequences of endoskeletal proteins

and the characterization of proteinaceous organic matrix of

spicules from the alcyonarian, Sinularia polydactyla. Proteomics

5:885–893

Rousseau M, Lopez E, Stempfle P, Brendle M, Franke L, Guette A,

Naslain R, Bourrat X (2005) Multiscale structure of sheet nacre.

Biomaterials 26:6254–6262

Samata T, Hayashi N, Kono M, Hasegawa K, Horita C, Akera S

(1999) A new matrix protein family related to the nacreous layer

formation of Pinctada fucata. FEBS Lett 462:225–229

Smith-Keune C, Jerry DR (2009) High level of intra-specific variation

in the NG repeat region of the Pinctada maxima N66 organic

matrix protein. Aquat Res 40:1054–1063

Sudo S, Fujikawa T, Nagakura T, Ohkubo T, Sakaguchi K, Tanaka M,

Nakashima K, Takahashi T (1997) Structure of mollusc shell

framework proteins. Nature 387:563–564

Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato

T, Nagasawa H (2009) An acidic matrix protein, Pif, is a key

macromolecules for nacre formation. Science 325:1388–1390

Tambutte S, Tambutte E, Zoccola D, Caminiti N, Lotto S, Moya A,

Allemand D, Adkins J (2007) Characterization and role of

carbonic anhydrase in the calcification process of the azooxan-

thellate coral Tubastrea aurea. Mar Biol 151:71–83

Tanguy A, Bierne N, Saavedra C, Pina B, Bachere E, Kube M, Bazin

E, Bonhomme F, Boudry P, Boulo V, Boutet I, Cancela L,

Dossat C, Favrel P, Huvet A, Jarque S, Jollivet D, Klages S,

Lapegue S, Leite R, Moal J, Moraga D, Reinhardt R, Samain J-F,

Zouros E, Canario A (2008) Increasing genomic information in

bivalves through new EST collections in four species: develop-

ment of new genetic markers for environmental studies and

genome evolution. Gene 408:27–36

Treccani L, Mann K, Heinemann F, Fritz M (2006) Perlwapin, an

abalone nacre protein with three four-disulfide core (whey acidic

protein) domains, inhibits the growth of calcium carbonate

crystals. Biophys J 91:2601–2608

Vendrasco MJ, Porter SM, Kouchinsky A, Li G, Fernandez CZ (2010)

New data on molluscs and their shell microstructures from the

Middle Cambrian Gowers Formation, Australia. Palaeontology

53:97–135

Vernier P, De Pitta C, Bernante F, Varotto L, De Nardi B, Bovo G,

Roch P, Novoa B, Figueras A, Pallavicini A, Lanfranchi G

(2009) Mytibase: a knowledgebase of mussel (M. galloprovin-cialis) transcribed sequences. BMC Genomics 10:72

Wang A, Wang Y, Gu Z, Li S, Shi Y, Guo X (2010) Development of

expressed sequence tags from the pearl oyster, Pinctada martensiDunker. Mar Biotechnol. doi:10.1007/s10126-010-9296-9

Weiner S (1983) Mollusk shell formation: isolation of two organic

matrix proteins associated with calcite deposition in the bivalve

Mytilus californianus. Biochemistry 22:4139–4145

Weiner S, Traub W (1984) Macromolecules in mollusc shells and

their functions in biomineralization. Philos Trans R Soc Lond

304:425–434

Weiner S, Lowenstam HA, Hood L (1977) Discrete molecular weight

components of the organic matrices of mollusc shells. J Exp Mar

Biol Ecol 30:45–51

Weiss I (2010) Jewels in Pearl. ChemBioChem 11:297–300

Weiss I, Schonitzer V (2006) The distribution of chitin in larval shells

of the bivalve mollusk Mytilus galloprovincialis. J Struct Biol

153:264–277

Weiss IM, Gohring W, Fritz M, Mann K (2001) Perlustrin, a Haliotislaevigata (abalone) nacre protein, is homologous to the insulin-

like growth factor binding protein N-terminal module of

vertebrates. Biochem Biophys Res Commun 285:244–249

Westbroek P, Marin F (1998) A marriage of bone and nacre. Nature

392:861–862

Wheeler AP, George JW, Evans CA (1981) Control of CaCO3

nucleation and crystal growth by soluble matrix of oyster shell.

Science 212:1397–1398

J Mol Evol

123

Page 16

Wilbur KM, Jodrey LH (1955) Studies on shell formation. V. The

inhibition of shell formation by carbonic anhydrase inhibitors.

Biol Bull 108:359–365

Yin Y, Hang J, Paine ML, Reinhold VN, Chasteen D (2005)

Structural characterisation of the major extrapallial fluid protein

of the mollusc Mytilus edulis: implication for function. Bio-

chemistry 44:10720–10731

J Mol Evol

123