Charles Le Pabic
email: clepabic@mnhn.fr

Arul Marie

Benjamin Marie

Aline Percot

Laure Bonnaud-Ponticelli

Pascal Jean Lopez

Gilles Luquet
email: gluquet@mnhn.fr

First proteomic analyses of the dorsal and ventral parts of the Sepia officinalis cuttlebone

Keywords: FTIR Demineralization Cuttlefish, Biomineralization, Shell, Aragonite, Organic matrix, Sepia officinalis, Proteomics

Introduction

Mollusks are known for their ability to build shells having a huge diversity of sizes, forms and structures. The mollusk shells are mainly composed of calcium carbonate under calcite and/or aragonite polymorphs (rarely vaterite) associated to a small amount of organic compounds (mainly polysaccharides, proteoglycans, glycoproteins and proteins) [START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF].

Although constituting a very minor fraction of the biomineral, the shell organic matrix is thought to regulate the crystal nucleation, orientation, polymorph, growth and morphology in the calcification process [START_REF] Lowenstam | On biomineralization[END_REF]. In order to better understand how organic compounds control calcification and because of their promising applications in biotechnology, shell proteins have been widely studied these last years resulting in the description of a great diversity of protein compounds [e.g. 1,3-10]. However, most of studies describing shell proteins were realized on two groups of mollusksthe bivalves and the gastropodsthat share some shell particularities. Indeed, their shells are constituted by the superposition of few calcified layers used for protecting the animal against environmental pressures and predators as well as to support tissue. Moreover, the formation of their shell occurs externally between a thin organic layer (the periostracum) and a calcifying epithelium secreting compounds needed for the shell synthesis [START_REF] Marin | The formation and mineralization of mollusk shell[END_REF].

Among other shell-builder mollusks, some Cephalopoda form an original calcified shell constituted by superposed hollow chambers, used to stock gas and thus regulate their buoyancy. Nowadays only three extant cephalopod families conserved this particularity: the Nautilidae, which includes few species with an external coiled shell; the Spirulidae, which contains only 1 species with an inner coiled shell; and the Sepiidae (i.e. the cuttlefish), which counts more than 100 species that form an inner straight shell [e.g. [START_REF] Denton | On buoyancy and the lives of modern and fossil cephalopods[END_REF][START_REF] Kröger | Cephalopod origin and evolution: A congruent picture emerging from fossils, development and molecules[END_REF][START_REF] Allcock | The contribution of molecular data to our understanding of cephalopod evolution and systematics: a review[END_REF]. This latter structure (also called "cuttlebone"), essentially composed by aragonite, is involved in tissue support and buoyancy regulation as for Spirulidae and Nautilidae, but its morphology and structural organization is quite different. Indeed, Sepiidae shell consists in two distinct regions: the upper side called the dorsal shield and the ventral chambered part (Fig. 1). The dorsal shield is a dense and rigid layer playing an important mechanical role, whereas the chambered part is formed by the superposition of independent hollow chambers of few hundred micrometers height, separated by lamella called septa. Each chamber is open posteriorly allowing the animal to fill them with variable volumes of gas and liquid in order to adjust its buoyancy. Within the chambers, vertical pillars form the supporting elements of the septa. These pillars can be independent or linked together depending on the inner area observed [START_REF] Appellöf | Die schalen von Sepia, Spirula und Nautilus. Studien über den bau und das wachsthum[END_REF][START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Le Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF]. Although both cuttlebone parts associate lamellar and prismatic structures, their organization differs according to their mineralogy organization and microstructures (Fig. 1). The dorsal shield consists in three layers: the uppermost one is formed of prismatic calcareous tubercles, the central one is characterized by a lamellar organization and the inner one is made by prismatic crystals [START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Dauphin | Microstructures des coquilles de céphalopodes II -La seiche (Mollusca, Coleoidea)[END_REF]. In the chambered part, each septum consists of a prismatic layer on its lower side, similar as in the pillar, and a lamellar structure on its upper side (previously described as nacre [START_REF] Dauphin | Microstructures des coquilles de céphalopodes II -La seiche (Mollusca, Coleoidea)[END_REF][START_REF] Mutvei | Ultrastructure of the mineral and organic components of molluscan nacreous layers[END_REF]) resulting in a septa of around 20 µm-thickness (Fig. 1; for a more detailed description, see [START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Le Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF]). This intricate structure combines contradictory properties as high compressive strength, high porosity and high permeability [START_REF] Denton | The buoyancy of the cuttlefish, Sepia officinalis (L.)[END_REF][START_REF] Denton | Floatation mechanisms in modern and fossil cephalopods[END_REF][START_REF] Birchall | On the architecture and function of cuttlefish bone[END_REF][START_REF] Ward | Shell implosion depth and implosion morphologies in three species of Sepia (Cephalopoda) from the Mediterranean Sea[END_REF], but knowledge about organic compounds involved in its building remains limited. Although the total amount of organic matterdescribed as being composed of polysaccharides, glycoproteins and proteinsis known to be important in Sepiidae shells (4-10% of the shell dry weight) [START_REF] Birchall | On the architecture and function of cuttlefish bone[END_REF][START_REF] Hackman | Studies on chitin IV. The occurrence of complexes in which chitin and protein are covalently linked[END_REF][START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF][START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF] in comparison with other mollusks (0.01-5%) [START_REF] Keith | Comparative analysis of macromolecules in mollusc shells[END_REF][START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Marin | The formation and mineralization of mollusk shell[END_REF]. The main polysaccharide component, the ß-chitin, plays a major framework role that allows the set up of the shell [START_REF] Birchall | On the architecture and function of cuttlefish bone[END_REF][START_REF] Hackman | Studies on chitin IV. The occurrence of complexes in which chitin and protein are covalently linked[END_REF][START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF][START_REF] Okafor | Isolation of chitin from the shell of the cuttlefish, Sepia officinalis L[END_REF][START_REF] Dauphin | The compositional analysis of recent cephalopod shell carbohydrates by Fourier transform infrared spectrometry and high performance anion exchange-pulsed amperometric detection[END_REF] in association with protein compounds, proposed as organic precursors of the mineralization [START_REF] Checa | The cuttlefish Sepia officinalis (Sepiidae, Cephalopoda) constructs cuttlebone from a crystal precursor[END_REF]. Most of the organic compounds involved in this process are synthesized by a monolayered epithelium that surrounds the shell -named "shell sac", described as a complex tissue composed by 3 to 5 different cell-types [START_REF] Appellöf | Die schalen von Sepia, Spirula und Nautilus. Studien über den bau und das wachsthum[END_REF][START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Denton | The buoyancy of the cuttlefish, Sepia officinalis (L.)[END_REF][START_REF] Kawaguti | Electron microscopy on the cuttlebone-producing cells[END_REF][START_REF] Spiess | Organogenese des schalendrüsenkomplexes bei einigen coleoiden cephalopoden des mittelmeeres[END_REF] (Fig. 1). This particularity represents a major difference with other shell-builder mollusks studied thus far.

Despite the importance of proteins in the mollusk shells formation, no detailed protein description has been published yet for Sepiidae shells. Moreover, although obviously distinct and in direct contact with different shell sac tissues (Fig. 1) [START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Spiess | Organogenese des schalendrüsenkomplexes bei einigen coleoiden cephalopoden des mittelmeeres[END_REF], the dorsal shield and the chambered part have been rarely compared (most of studies focusing on the chambered part).

In this paper, we analyzed for the first time the shell protein composition of one of the betterknown cuttlefish model, the species Sepia officinalis. In addition, both shell parts described hereabove have been discriminated.

Material & Methods

Shell material and matrix extraction

The cuttlebones from eight adult S. officinalis that were used in this study were obtained from freshly fished specimens along the English Channel coastline. After cuttlebone removing, the superficial organic contaminants were eliminated by 24 h incubation in a 0.25% NaClO solution under constant agitation, and thoroughly rinsed with Milli-Q water.

Thereafter, cuttlebones were air-dried at room temperature for 24 h.

In order to investigate the total protein composition of the cuttlebone, an entire cuttlebone (i.e. without separation of the dorsal shield and the ventral chambered part) was weighted, grounded into fine powder and demineralized in cold 10% acetic acid for 24 h at 4°C. The solution was then centrifuged at 4°C, 30 min at 1700 g. The supernatant containing the acetic acid-soluble organic matrix (ASM) was filtered and concentrated with an Amicon ultrafiltration system on a Millipore ® membrane (Ultracell ® ; 5-kDa cut-off). After extent dialyses against Milli-Q water (at least 8 times), the ASM solution devoid of acetic acid, was freeze-dried and kept at 4°C until used. The pellet, corresponding to the acetic acid-insoluble matrix (AIM), was rinsed 10 times with Milli-Q water, freeze-dried, weighed and stored at 4°C. These two fractions were kept for direct MS analysis.

To perform a comparative analysis of the protein content of the two main parts of cuttlebone, we carefully separated the dorsal shield from the chambered part. Both parts were then respectively weighed, grounded into fine powder and treated as described above to obtain the AIM and ASM organic fractions from the two structurally different parts. Thus, each shell sample gave 4 fractions, i.e. the dorsal shield AIM and ASM (respectively AIM DS and ASM DS ) and the chambered part AIM and ASM (respectively AIM CH and ASM CH ).

Fourier transform infrared (FTIR) spectrometry analysis

ATR-FTIR spectra were recorded using a Bruker Equinox 55 spectrometer equipped with an ATR diamond crystal accessory (Golden Gate®, Specac) and purged with dried air.

The diamond is cut to obtain a single reflexion at the crystal/sample interface with an accessible area of 50 µm × 2 mm. A Peltier-cooled DTGS Mid-IR detector, a Mid-IR source and an extended KBr beamsplitter were used. An atmospheric compensation was applied with Opus 6.5 software in order to remove residual H 2 O/CO 2 vapor signal. A background was collected before each sample's spectra. For each lyophilized samples (i.e. AIM DS , ASM DS , AIM CH and ASM CH ), 32 scans were accumulated between 4,000 and 600 cm -1 with a 4 cm -1 resolution.

Protein matrix analysis on 1-D gels

The separation of matrix components of the AIM and ASM fractions were performed under denaturing conditions by 1-D SDS-PAGE in 12% polyacrylamide gels (Mini-PROTEAN TGX; Bio-Rad; Hercules, CA, USA). Samples were individually suspended in Laemmli sample buffer (Bio-Rad; Hercules, CA, USA) containing 5% ß-mercaptoethanol, heat denatured at 95 °C for 5 min [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF], centrifuged for 1 min and kept at 4°C until gel loading. After preliminary trials, the optimal amounts of organic matrix for gel electrophoresis separation were found to be 100 µg for ASM CH , 200 µg for ASM DS and 300 µg for both AIM fractions. Because the AIM fractions were only partly solubilized by the buffer, the supernatants collected were called Laemmli-soluble AIM (i.e. LS-AIM DS and LS-AIM CH ).

Gel separated proteins were visualized with CBB (Bio-Safe TM , Bio-Rad; Hercules, CA, USA) or silver nitrate according to Morrissey [START_REF] Morrissey | Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced unform sensitivity[END_REF]. In addition, glycosylations were studied qualitatively on gels, and saccharide moieties were detected by the Periodic Acid Schiff (PAS; Pierce TM Glycoprotein Staining kit; Fisher Scientific, Illkirch, France) and Alcian blue stainings. At pH 2.5, the Alcian blue staining highlights saccharide moieties of glycosaminoglycans carrying polyanionic groups such as carboxyl and sulfate groups, whereas at pH 1, only sulfated compounds were stained [START_REF] Lev | Specific staining of sulphate groups with Alcian blue at low pH[END_REF][START_REF] Wall | Alcian blue staining of proteoglycans in polyacrylamide gels using the "Critical electrolyte concentration" approach[END_REF].

Protein assay and matrix analysis on 2-D gels

In order to estimate the amount of proteins solubilized by the rehydration buffer (urea 8 M, CHAPS 2%, DTT 50 mM, Bio-Lyte ® 3/10 ampholytes 0.2% (w/v)) used in the ReadyPrep TM 2-D Starter kit (Bio-Rad; Hercules, CA, USA), the protocol for microtiter plates described in the Bio-Rad protein assay kit II was employed. To avoid possible interferences due to the high urea concentration of the rehydration buffer (that must be kept below 6 M, according to manufacturer protocol), all samples and protein standards were diluted to maintain the rehydration buffer/Milli-Q water ratio constant (1/1; v/v). The protein concentrations were measured at 595 nm on the supernatant of the different fractions after 1 min centrifugation using as standard a bovine serum albumin standard curve (50-350 µg mL -1 of Milli-Q water).

The four fractions were respectively separated on a 2-D gel PROTEAN ® IEF cell (Bio-Rad; Hercules, CA, USA), according to the manufacturer's instructions. AIM DS (500 µg organic matrix in 150 µL), ASM DS (700 µg in 150 µL), AIM CH (1.5 mg in 150 µL) and ASM CH (400 µg in 150 µL) were rehydrated in the rehydration buffer and briefly centrifuged to avoid pipetting non-dissolved organic matter. Because the AIM fractions were only partly solubilized by the rehydration buffer, the supernatants collected were called urea-soluble AIM (i.e. US-AIM DS and US-AIM CH ). Supernatants were then used to rehydrate overnight strips (7 cm linear, pH 3-10 IPG), and IEF was carried out (250 V for 20 min, 4,000 V for 2 h, followed by 4,000 V until 10,000 Vh). The strips were then transferred for 10 min to equilibration buffer I and II (ReadyPrep TM 2-D Starter kit), rinsed in 1X Tris/Glycine/SDS buffer (Bio-Rad; Hercules, CA, USA), and positioned on top of precast gradient gels (Mini-PROTEAN TGX, 12% polyacrylamide) covered with 0.5% low melting agarose (w/v) in 1X Tris/Glycine/SDS. Electrophoresis was then performed in standard conditions and the gels were subsequently stained with CBB.

Proteomic analysis of the organic matrix fractions

In order to identify protein compounds present in the dorsal shield and chambered part organic matrices, we separately analyzed the most prominent 1-D gel bands from LS-AIM DS , ASM DS , LS-AIM CH and ASM CH using MS. In addition, the total ASM and AIM (i.e. without dorsal shield and chambered part split) were directly analyzed by MS (i.e. without preliminary protein separation). Finally, the supernatant was collected and residual peptides contained in gels extracted by subsequent baths of 30 µL ACN 50%, formic acid 5% and ACN 100%, pooled with previously collected supernatant. Tryptic peptides were then dried with a SpeedVac TM concentrator and stored at -20 °C until MS analysis.

Band protein digestion and MALDI-TOF/TOF analysis

MS experiments were carried out on an AB Sciex 5800 proteomics analyzer equipped with TOF-TOF ion optics and an OptiBeam TM on-axis laser irradiation with 1,000 Hz repetition rate. The system was calibrated immediately before analysis with a mixture of Angiotensin I, Angiotensin II, Neurotensin, ACTH clip [START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF][START_REF] Lowenstam | On biomineralization[END_REF][START_REF] Addadi | Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization[END_REF][START_REF] Keith | Comparative analysis of macromolecules in mollusc shells[END_REF][START_REF] Marxen | The organic shell matrix of the freshwater snail Biomphalaria glabrata[END_REF][START_REF] Sudo | Structure of mollusc shell framework proteins[END_REF][START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Mann | In-depth proteomic analysis of a mollusc shell: acidsoluble and acid-insoluble matrix of the limpet Lottia gigantea[END_REF][START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Marin | Shellome": proteins involved in mollusc shell biomineralizationdiversity, functions[END_REF][START_REF] Marin | The formation and mineralization of mollusk shell[END_REF][START_REF] Denton | On buoyancy and the lives of modern and fossil cephalopods[END_REF][START_REF] Kröger | Cephalopod origin and evolution: A congruent picture emerging from fossils, development and molecules[END_REF][START_REF] Allcock | The contribution of molecular data to our understanding of cephalopod evolution and systematics: a review[END_REF][START_REF] Appellöf | Die schalen von Sepia, Spirula und Nautilus. Studien über den bau und das wachsthum[END_REF][START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Le Pabic | Overview of the shell development of the common cuttlefish Sepia officinalis during early-life stages[END_REF], ACTH clip (18-39) and mass precision was better than 50 ppm. Dry sample was re-suspended in 10 µL of 0.1% TFA. One µL of this peptide solution was mixed with 10 µL of CHCA matrix solution prepared in 50% ACN, 0.1% TFA. The mixture was spotted on a stainless steel Opti-TOF TM 384 targets; the droplet was allowed to evaporate before introducing the target into the mass spectrometer. All the spectra were acquired in automatic mode employing a typically laser intensity of 3,300 for ionization. MS spectra were acquired in the positive reflector mode by averaging 1,000 single spectra (5 × 200) in the masse range from 700 to 4,000 Da. MS/MS spectra were acquired in the positive MS/MS reflector mode by averaging a maximum of 2,500 single spectra (10 × 250) with a laser intensity of 4,200. For the tandem MS experiments, the acceleration voltage applied was 1 kV and air was used as the collision gas.

LC ESI-QTOF MS analysis of whole ASM and AIM

One mg of total ASM or AIM was reduced with 100 µL of 10 mM DTT (Sigma-Aldrich) in 100 mM NH 4 HCO 3 (Sigma-Aldrich) pH 8.1 for 30 min at 57°C, followed by alkylation with iodoacetamide (50 mM, final concentration; Sigma-Aldrich) for 30 min in the dark and at room temperature. Samples were then freeze-dried. The dry residues were dissolved in 200 µL of a 50 mM NH 4 HCO 3 buffer (pH 8.1) containing 5 µg of trypsin (Sigma-Aldrich) and 5% ACN (Sigma-Aldrich), and incubated for 18 h at 37°C. After centrifugation at 14,000 g for 30 min, the supernatants were lyophilized and stored at -20°C until MS analysis.

The peptide digests were re-suspended in 50 µL of a solution containing 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich) and 3% ACN. Two µL of the peptide digest from the total organic matrices were separated on a C 18 column (150 × 1 mm, Phenomenex, France) at a flow rate of 40 µL min -1 with 0.1% formic acid (solvent A) and ACN (solvent B), using a gradient that varied from 3 to 50% of B in 45 min. The eluted peptides were analyzed in an ESI-QqTOF mass spectrometer (pulsar i, Applied Biosystems, France), using information dependent acquisition mode. This mode allows switching between MS and MS/MS scans. A 1-s MS scan was followed by two 2-s MS/MS acquisitions using two most intense multiply charged precursor peptide ions (+2 to +4). The fragmented precursor ions were excluded for 60 s in order to avoid reanalysis. Minimum ion intensity for MS/MS experiments was set to 10 counts and collision energy for the peptide ions was determined automatically by the acquisition software. gigantea, C. gigas, E. complanata and P. fucata were chosen as reference shell builder noncephalopod mollusks because of the quality of their databases, the availability of their genomes (for L. gigantea [START_REF] Simakov | Insights into bilaterian evolution from three spiralian genomes[END_REF], C. gigas [START_REF] Zhang | The oyster genome reveals stress adaptation and complexity of shell formation[END_REF] and P. fucata [START_REF] Takeuchi | Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle[END_REF]) and their protein description considering the biomineralization process. In all cases, the peptide spectral matches were validated only if at least one peptide sequence matched to the translated nucleotide sequence using both Mascot and PEAKS programs, guaranteeing the robustness of the results. Identified nucleotide sequences were translated using the EXPASY translate tool (http://web.expasy.org/translate/) and the reading frame and coding sequence were manually validated. The signal peptides and conserved domains were predicted using SMART (http://smart.embl-heidelberg.de), and the recognized sequences attempted to be identified using BLASTp analysis performed against UniprotKB database provided by UniProt server (http://www.uniprot.org/blast/) using default parameters.

Protein identification and sequence analysis

Results

Organic matrix extraction and repartition

The results of the extraction reveals an important difference in the amount of organic matter between the two cuttlebone parts studied (i.e. dorsal shield and chambered part) with almost twice more organic matrix in the dorsal shield (6.2 ± 1.5%; w/w) compare to the chambered part (3.4 ± 0.7%; w/w). As described for other mollusk shells, most of this extracted matrix corresponds to an insoluble form independently of the part of the cuttlefish shell studied (Table 1).

FTIR profiles of the dorsal shield and chambered part AIMs and ASMs

The global FTIR spectra of organic insoluble and soluble fractions extracted from the dorsal shield and the chambered part of S. officinalis shell exhibit typical protein and polysaccharide absorption bands (Fig. 2; Table 2). Nevertheless, the AIM and ASM shell spectra appear to be different. Indeed, whereas the characteristic bands commonly associated with proteins (i.e. amide A, I, II and III) are clearly visible in the four spectra, the bands associated with carbohydrate compounds (between 950-1,200 cm -1 ), appear stronger in the AIM ones (Fig. 2, Table 2). Also, note that the absorption band at 1,375 cm -1 , which is more intense in the AIMs, could be attributed to chitin groups (CH bending, CH 3 symmetric deformation [START_REF] Amaral | Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study[END_REF][START_REF] Biniaś | Supermolecular structure of chitin and its derivatives in FTIR spectroscopy studies[END_REF]).

In the insoluble fractions, the AIM CH spectrum exhibits a stronger carbohydrate absorption band compared to the AIM DS , which suggests a more important saccharide fraction in this shell part (Fig. 2A). A slight difference can also be observed in the ASM extracts, where the relative intensity of the band at 1,030 cm -1 (and 1,375 cm -1 ) is higher in the chamber part than in the dorsal shield one.

However, these results have to be confirmed because similar patterns can be obtained for protein and polysaccharide mixtures (e.g. for chitin composed of amide groups and a carbohydrate skeleton or for proteins associated to chitin).

Characterization of matrices by 1-D SDS-PAGE

The four extracted fractions (LS-AIM DS , LS-AIM CH , ASM DS and ASM CH ) were analyzed by 1-D SDS-PAGE. Gels were subsequently stained with CBB, silver nitrate, PAS and Alcian blue at pH 2.5 and 1 (Fig. 3 and4), providing constitutive information on the protein composition of each fraction. The four profiles are found to be composed of various distinct macromolecular elements.

The LS-AIM DS shows 7 main bands migrating at apparent molecular weights around 120, 89, 72, 61, 40 and 38 and below 15 kDa with particular thickness of the bands 61, 40 and 38 kDa (Fig. 3A). Two other minor bands at around 240 and 27 kDa are also visible. It is noteworthy that silver nitrate negatively stained 72-and 61-kDa bands. The pH 1 Alcian blue staining of the compounds present in the 61-kDa band highlights that these compounds carried sulfated sugars. Unfortunately, our experiments did not allow to determine whether the 72-kDa compounds are also sulfated. Faint staining of 40-and 38-kDa bands with Alcian blue carried out at pH 2.5 suggests that they contain small amount of carboxylated sugars.

Faint PAS staining suggests that the polypeptides migrating at 120, 89, 61, 40 and 38 kDa are glycosylated (Fig. 3A).

The LS-AIM CH shows 3 main bands migrating at around 117, 61 and 38 kDa. Another faint band can also be distinguished around 45 kDa (Fig. 3B). Silver nitrate reveals a more intense staining of this latter band, allows to distinguish two bands at 38 kDa, one faint band just below the 117-kDa band, and strongly stain compounds migrating above 250 kDa.

Periodic Acid Schiff stains all bands observed using CBB, with more intense staining of the 117-and 38-kDa bands. Alcian blue staining performed at pH 2.5 reveals that the compounds with the highest molecular weight of this fraction carried carboxylated groups associated with sugar moieties, whereas no band was revealed with Alcian blue at pH 1 (data not shown).

The ASM DS shows 4 bands migrating at around 240, 105, 31, and 18 kDa. Notably, the migration front appears intensively stained by CBB. Also, 3 other faint bands can be distinguished around 42, 38 and 19 kDa (Fig. 4A). The 240-, 105-and 31-kDa bands appear clearly glycosylated. Alcian blue staining, at pH 2.5, suggests that compounds around 240 kDa and at the migration front probably carry carboxylated groups but not sulfated groups because no band was revealed with Alcian blue at pH 1 (data not shown). The protein band patterns of these four fractions suggest that the protein contents, from a qualitative point of view, are different from each other. Based on their apparent molecular masses and their staining reactivity, only two compounds seem shared by more than one fraction: the bands around 105 and 117-120 kDa with polysaccharide moieties observed respectively in the soluble and insoluble fractions. Of course, it remains possible that several proteoforms with similar apparent molecular mass constitute similar sized bands (e.g. sulfated and non-sulfated 61-kDa bands in LS-AIM DS and LS-AIM CH , respectively).

Characterization of matrices by 2-D SDS-PAGE

In order to further characterize the proteins and their putative post-translational modifications, each fraction was analyzed by 2-DE (Fig. 5 and6). As for 1-D gel, the four profiles were found to be composed of various distinct macromolecular elements.

In the case of AIMs, an extremely limited part of the matrix was dissolved in the rehydration buffer, especially considering AIM CH (Table 3). The US-AIM DS matrix is mainly characterized by 3 protein rich areas: a smear around 61 kDa with an pI around 5 (supporting the sulfated sugar moiety of this compound previously underlined), a series of spots around 40 kDa with pIs between 7 and 8, and a large spot at 38 kDa just below this latter (Fig. 5A).

These compounds are consistent with the most intense bands observed in 1-D electrophoresis (Fig. 3A). Other faint compounds are visualized at higher pI (120 kDa smear at pI around 8 and 3 spots at same levels than the 38, 40 and 61 kDa compounds visible on the right).

Although difficult to distinguish, compounds of different pIs (around 5 and 8) seem to be present in the protein fraction migrating below 15 kDa (Fig. 5A).

The US-AIM CH matrix is mainly characterized by 2 series of spots respectively around 61 and 38 kDa. The first spreads from pIs 5 to 8, whereas the second is a series of 5 spots spread between pIs 5 and 6 (Fig. 5B). According to 1-D gel PAS staining, these spot series could be due to different glycosylation states (Fig. 3B). Nevertheless, this could also be attributed to the presence of other post-translational modifications such as phosphorylations.

The 2-DE of both ASM matrices resulted in greater protein extraction and separation (Fig. 5). The main compounds observed in the ASM DS fraction are a series of 6 acidic polypeptides (pIs from 4.5 to 6.5) of around 31 kDa. Furthermore, small compounds intensively stained by CBB near the 1-D migration front (i.e. 18 and below 15 kDa), appear here at a high pI around 9 (Fig. 6A).

The ASM CH mainly exhibits 2 acidic smears around 27 kDa (pI = 4.5 to 6) and a series of spots around 17 kDa spread from pIs 4.5 to 8, but mainly located in the acidic area (Fig. 6B). These observations are consistent with the most CBB-stained compounds described using 1-D gel electrophoresis. Two thin smears are also observed at around 105 and 38 kDa and mean pIs at around 4 and 6.5, respectively.

Protein identification and sequence analysis

The biochemical characterization performed in this study was complemented by a proteomic analysis aiming to identify proteins involved in the cuttlebone formation. The whole ASM and AIM (i.e. without dorsal shield and chambered part split) were analyzed by HPLC-ESI-MS/MS, whereas most prominent gel bands were analyzed by MALDI-TOF/TOF (see Fig. 3 and4). The resulting peptide sequences were used for screening the NCBI nucleic acid databases described in section 2.5.3. by using Mascot search engine, excluding peptides assigned to trypsin and keratin. De novo sequencing of whole soluble and insoluble fractions allowed identifying 65 unique peptides of between 7 and 14 amino acids length (Table S1).

We observed neither some particular richness in Gly, Ser, Ala or acidic residues (Asp and Glu), nor peptides with repetitive residue blocs classically described in biomineralization proteins [e.g. [START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Marxen | Carbohydrates of the organic shell matrix and the shell-forming tissue of the snail Biomphalaria glabrata (Say)[END_REF]. However, almost 10% of these peptides present an over-representation of the Leu/Ile residues in their sequence.

Whereas no significant protein hit was obtained from the L. gigantea, C. gigas, E. complanata and P. fucata databases, the NCBI Cephalopod database matched for 5 acid nucleic translated sequences. It is noteworthy that all identified protein sequences provided from the embryonic S. officinalis ESTs library [START_REF] Bassaglia | ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca -Cephalopoda)[END_REF]. This underlined the low representativeness of shell-builder Cephalopods in the NCBI Cephalopod database (less than 15% of the sequence number), especially considering the most diversified group: the Sepiidae.

Peptides from ASM matched with 4 EST sequences (FO196371, FO182034, FO201581 and FO162285), whereas peptides from AIM matched only one EST sequence (FO198959; Table 4). Among the 29 analyzed gel bands, only peptides from one ASM DS band (migrating at around 31 kDa, and also found in the whole ASM) were found to match with the FO196371 EST sequence. Among these 5 recognized EST sequences, 4 encode for protein sequences exhibiting a signal peptide (FO182034, FO201581, FO198959, FO162285) and only one being complete (FO182034). This latter contains a type 2 chitin-binding domain (ChtBD2; SM000494) and presents 42.7% identity with a chitin-binding protein (DgCBP-1) of the squid Dosidicus gigas [START_REF] Tan | Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient[END_REF]. The protein sequence recognized with the highest score contains 1 transferrin domain (TR_FER; SM000094) and matches with more than 42% identity with fish serotransferrins [e.g. 40]. The three other sequences contain respectively 1 O-Glycosyl hydrolase (Glyco_18; SM000636), 1 von Willebrand factor type A (VWA; SM000327) and 3 Kunitz (SM00131) domains. The first one best matches with a Sepia esculenta chitinase with 63% identity, the second one, with a Lottia gigantea uncharacterized protein (42.2% identity) having a chitin binding GO function [START_REF] Simakov | Insights into bilaterian evolution from three spiralian genomes[END_REF], and the last one, with various serine protease inhibitors.

Discussion

Although the aragonitic shell of the cuttlefish presents intriguing features (i.e. inner position, straight form, hollow chambers structuration allowing buoyancy regulation, high strength, porosity and permeability combination), no detailed description of its protein compounds have been published thus far. Yet, a better understanding of the processes regulating the biomineralization of this shell-type could bring new perspectives for applications in biotechnology. In order to understand whether the building of the cuttlefish shell is regulated by the same mechanisms than other mollusks, we described the proteins present in the cuttlefish shell organic matrix distinguishing its two main parts: the dorsal shield and the chambered part.

Firstly, from a quantitative viewpoint, the total amount of organic matter extracted with our protocol (4.7 ± 1.1%; Table 1) appears consistent with data classically measured in other mollusk shells (0.01-5%) although in the high range [START_REF] Keith | Comparative analysis of macromolecules in mollusc shells[END_REF][START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Marin | The formation and mineralization of mollusk shell[END_REF]. However, this amount appears half as data reported by Florek et al. [START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF] in whole S. officinalis shell using thermogravimetric analyses (9.8%). This difference could be due to sample characteristics (i.e. shell part used and/or origin) or differences in the measurement technique used.

Considering the organic matter amounts obtained from the chambered part, our value (3.4 ± 0.7%) is in agreement with the 3-4.5% previously reported by Jeuniaux [START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF] and Birchall and Thomas [START_REF] Birchall | On the architecture and function of cuttlefish bone[END_REF] after HCl aragonite dissolution. Finally, we observed a relatively high amount of organic matter in the dorsal shield (6.2 ± 1.5%) compared to other mollusk shells [START_REF] Keith | Comparative analysis of macromolecules in mollusc shells[END_REF][START_REF] Marin | Molluscan shell proteins: primary structure, origin, and evolution[END_REF][START_REF] Marin | The formation and mineralization of mollusk shell[END_REF].

However, in view of this measure, the 30-40% estimated from the same shell part by Birchall and Thomas [START_REF] Birchall | On the architecture and function of cuttlefish bone[END_REF] seems largely overestimated. In the light of these discrepancies and according to the known cuttlebone intraspecific variations [START_REF] Boletzky | Effets de la sous-nutrition prolongée sur le développement de la coquille de Sepia officinalis L. (Mollusca, Cephalopoda)[END_REF][START_REF] Sherrard | Cuttlebone morphology limits habitat depth in eleven species of Sepia (Cephalopoda: Sepiidae)[END_REF], it appears important to consider a likely variation of the amount of organic matter found in S. officinalis shells in function of the animal living environment. In this study, all cuttlebones used came from a low-depth area (i.e. the English Channel). Thus, according to 1) the shell plasticity highlighted in some mollusks in function of their environment [START_REF] Charrier | Soil calcium availability influences shell ecophenotype formation in the sub-antarctic land snail, Notodiscus hookeri[END_REF], to 2) the absolute need for cuttlefish to have a shell resistant to ambient pressure [START_REF] Ward | Shell implosion depth and implosion morphologies in three species of Sepia (Cephalopoda) from the Mediterranean Sea[END_REF], as well as to 3) the role of organic matter in the mineral structure hardness/elasticity compromise [START_REF] Currey | The mechanical consequences of variation in the mineral content of bone[END_REF][START_REF] Jackson | The mechanical design of nacre[END_REF], it would be interesting to compare our data with S. officinalis populations living in deeper environments (e.g. from the Mediterranean Sea).

The FTIR spectra of the four fractions studied exhibit mainly protein and carbohydrate bands (Fig. 2; Table 2) as previously observed for organic compounds extracted from other aragonitic mollusk shells [e.g. 28,47-49] and cnidarian skeletons [START_REF] Goffredo | The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation[END_REF][START_REF] Ramos-Silva | The skeleton of the staghorn coral Acropora millepora: Molecular and structural characterization[END_REF]. Our analyses also suggest that the insoluble fractions contain more carbohydrates than the soluble ones (Fig. 2). This carbohydrate richness is likely due to ß-chitin that is known in cuttlefish shell for its scaffold role and was previously described as the main polysaccharide compounds of Coleoidea shells [START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF][START_REF] Dauphin | The compositional analysis of recent cephalopod shell carbohydrates by Fourier transform infrared spectrometry and high performance anion exchange-pulsed amperometric detection[END_REF][START_REF] Goffredo | The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation[END_REF]. Notably, the AIM CH seems to be the fraction with the highest polysaccharide proportion as previously suggested by Okafor [START_REF] Okafor | Isolation of chitin from the shell of the cuttlefish, Sepia officinalis L[END_REF]. However, such polysaccharide bands could also originate from sugars moieties linked with proteins as evidenced by PAS and Alcian blue gel staining (Fig. 3 and4).

Although non-exhaustive (i.e. because describing only urea-soluble fractions of both AIMs), our quantitative protein assays suggest that ASMs are richer in protein compounds than AIMs with ASM DS being the richest soluble fraction (Table 3). Such protein richness of the dorsal shield organic matrix has been previously suggested by several studies interested in the chitin-linked compounds in the S. officinalis shell [START_REF] Hackman | Studies on chitin IV. The occurrence of complexes in which chitin and protein are covalently linked[END_REF][START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF][START_REF] Okafor | Isolation of chitin from the shell of the cuttlefish, Sepia officinalis L[END_REF]. In view of our SDS-PAGE data (Fig. 4 and6), this protein amount is likely due (at least in part) to the high amount of 5-15 kDa polypeptides. Such richness in small protein compounds (< 8 kDa) has been reported in the nacre water-soluble matrix of the black-lip pearl oyster Pinctada margaritifera with self-organization and protease inhibition properties [START_REF] Bédouet | Identification of low molecular weight molecules as new components of the nacre organic matrix[END_REF][START_REF] Bédouet | Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre[END_REF]. Finally, the carboxylate moieties present in these small protein compounds suggest an ability to bind calcium ions (Fig. 4).

As for the diversity of proteins, our results of 1-and 2-D SDS-PAGE electrophoreses underlined the predominance of low pI proteins, especially considering ASMs, and a global richness in glycoproteins for the four fractions (Fig. 3 to 6). These properties are consistent with the current literature interested in mollusk biomineralization [e.g. [START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF][START_REF] Addadi | Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization[END_REF][START_REF] Marxen | The organic shell matrix of the freshwater snail Biomphalaria glabrata[END_REF][START_REF] Hackman | Studies on chitin IV. The occurrence of complexes in which chitin and protein are covalently linked[END_REF][START_REF] Jackson | The mechanical design of nacre[END_REF][START_REF] Marxen | Carbohydrates of the organic shell matrix and the shell-forming tissue of the snail Biomphalaria glabrata (Say)[END_REF][START_REF] Bédouet | Identification of low molecular weight molecules as new components of the nacre organic matrix[END_REF][START_REF] Bédouet | Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre[END_REF].

Notably, the only sulfated glycoprotein detected in our study has been found in the AIM DS whereas compounds with similar moieties are usually considered soluble (Fig. 3A) [START_REF] Marxen | Carbohydrates of the organic shell matrix and the shell-forming tissue of the snail Biomphalaria glabrata (Say)[END_REF][START_REF] Marie | Evolution of nacre: biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus[END_REF][START_REF] Marie | Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein[END_REF]. Moreover, it is noteworthy that the electrophoretic profiles from 20 kDa and below, observed on ASM DS 1-D gels (Fig. 4A), seems similar to those from Nautilus macromphalus (Cephalopoda) nacre presented by Marie et al. [START_REF] Marie | Evolution of nacre: biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus[END_REF]. Although the main acidic compounds from this previous study has not been observed in our 2-DE, the migration profile likenesses suggests the conservation of some organic compounds between Nautilidae nacre and Sepiidae dorsal shield (partly constituted by mineral organized similarly than nacre [START_REF] Dauphin | Microstructures des coquilles de céphalopodes II -La seiche (Mollusca, Coleoidea)[END_REF]; Fig. 1). Of course, only the complete identification of these putatively shared proteinaceous compounds could allow to assess the existence and the level of conservation.

Proteomics study carried out on the whole soluble and insoluble shell fractions and on the 29 prominent gel bands resulting from LS-AIM DS , LS-AIM CH , ASM DS and ASM CH SDS-nucleotide and protein databases (Table 4). Moreover, the detected peptides only matched with the embryonic S. officinalis ESTs library [START_REF] Bassaglia | ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca -Cephalopoda)[END_REF]. This low number of matches highlights the lack of reference databases for Sepiidae that are under-represented in the used Cephalopod data set. Nevertheless, among the 5 protein compounds identified here, 4 of them (i.e. aside from the transferrin protein) contain domains that were previously characterized in matrix proteins of aragonitic shell-builder bivalve and gastropod mollusks [e.g. 8,9,57] -namely ChtBD2, Glyco_18, VWA and Kunitz domainssuggesting ancient and conserved mechanisms of the aragonite biomineralization processes within mollusks.

The ChtBD2, Glyco_18 and VWA domains are known for their role in the interaction between organic shell components such as polysaccharides (mainly chitin) and proteins. The presence of ChtBD2 and Glyco_18 domains are consistent with the chitin scaffold role previously described in various mollusk shell matrices [START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF][START_REF] Hackman | Studies on chitin IV. The occurrence of complexes in which chitin and protein are covalently linked[END_REF][START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF][START_REF] Ehrlich | Chitin and collagen as universal and alternative templates in biomineralization[END_REF][START_REF] Peters | Occurrence of chitin in Mollusca[END_REF]. Their likely respective roles consist in the attachment of mineral precipitating compounds and in the chitin framework modification or in shell repair [START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Tanabe | New chamber formation in the cuttlefish Sepia esculenta Hoyle[END_REF]. Moreover, the chitin richness of the S. officinalis shell (around 25% of organic matrix is ß-chitin [START_REF] Jeuniaux | Chitine et chitinolyse : un chapitre de la biologie moléculaire[END_REF][START_REF] Okafor | Isolation of chitin from the shell of the cuttlefish, Sepia officinalis L[END_REF]) and its ubiquitous inner repartition (i.e. in the central layer of the dorsal shield, septa, inter-septa zones and pillars [START_REF] Bandel | A comparative study of the structure, development and morphological relationships of chambered cephalopod shells[END_REF][START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF][START_REF] Checa | The cuttlefish Sepia officinalis (Sepiidae, Cephalopoda) constructs cuttlebone from a crystal precursor[END_REF]) provide good correspondence with our identification of proteins bearing chitininteraction domain. Similarly, VWA domain have been previously retrieved in various mollusk shell matrices (especially in the Pif-177 nacre matrix protein [START_REF] Suzuki | An acidic matrix protein, Pif, is a key macromolecule for nacre formation[END_REF]) and is believed to contribute to biomineralization processes, as being involved in protein-protein interactions and subsequent formation of matrix protein complexes [e.g. [START_REF] Suzuki | Mollusk shell structures and their formation mechanism[END_REF][START_REF] Tanabe | New chamber formation in the cuttlefish Sepia esculenta Hoyle[END_REF]. In addition, various serine protease inhibitors containing Kunitz-like domains have been previously identified in shell organic matrix and tissues involved in mollusk shell synthesis with potential role(s) in inner proteases regulation and/or exogenous proteases protection [e.g. [START_REF] Mann | In-depth proteomic analysis of a mollusc shell: acidsoluble and acid-insoluble matrix of the limpet Lottia gigantea[END_REF][START_REF] Marie | Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell[END_REF][START_REF] Suzuki | An acidic matrix protein, Pif, is a key macromolecule for nacre formation[END_REF][START_REF] Chang | Pif97, a von Willebrand and peritrophin biomineralization protein, organizes mineral nanoparticles and creates intracrystalline nanochambers[END_REF].

To the best of our knowledge, a transferrin protein has not been described in mollusk shell organic matrix so far. The transferrin family is a group of monomeric glycoproteins defined by conserved residue motifs allowing the reversible binding of ferric ion (Fe 3+ ) synergistically with a carbonate anion [START_REF] Baker | Structure and reactivity of transferrins[END_REF][START_REF] Lambert | Evolution of duplications in the transferrin family of proteins[END_REF]. It has been firstly described as performing essential iron transportation and delivery functions. More recent studies underlined that transferrins are multifunctional proteins with diverse physiological roles only beginning to be understood [e.g. [START_REF] Marie | Unveiling the evolution of bivalve nacre proteins by shell proteomics of Unionoidae[END_REF][START_REF] Baker | Structure and reactivity of transferrins[END_REF]. Nevertheless, the function of some transferrin family members can be related to mineralization processes, as it is the case for the ovotransferrin found in the calcitic eggshell [START_REF] Gautron | Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer[END_REF] and for a mammalian transferrin with a carbonic anhydrase inhibitor activity [START_REF] Wuebbens | Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: A novel member of the transferrin family[END_REF]. In aragonite biomineralization, a transferrin protein has been identified as a major component of the fish otolith organic matrix necessary for otolith growth [START_REF] Murayama | Molecular cloning and expression of an otolith matrix protein cDNA from the rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Murayama | Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae[END_REF]. Thus, our finding suggests a transferrin role in S. officinalis shell biomineralization, especially in the dorsal shield part. Moreover, this observation is consistent with the relative high amount of iron previously reported in the S. officinalis shell (from 24 to 300 µg g -1 of shell, making the iron the second most concentrated trace metal in this shell [START_REF] Florek | Complementary microstructural and chemical analyses of Sepia officinalis endoskeleton[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis[END_REF]) and questions about a role of this trace element in such structure. Finally, according to the known synergistic ligation of carbonate anion in transferrin binding site [START_REF] Baker | Structure and reactivity of transferrins[END_REF][START_REF] Lambert | Evolution of duplications in the transferrin family of proteins[END_REF], it can also be hypothesized that transferrin achieves a carbonate supply function for calcium carbonate crystal formation.

Conclusion

This study is the first proteomic report on organic matrix compounds of a cuttlefish shell. Moreover, we have separately analyzed the proteins of the dorsal shield and chambered part, the two main parts of the shell, both being aragonitic but with clearly different architectures. The general description of the shell organic matrix highlights a similar composition compared to other previously analyzed mollusk shells, with some quantitative and qualitative differences between the dorsal shield and the chamber part. These differences suggest dissimilar processes of biomineralization between both shell parts, associated with different shell sac cells and secretory materials.

Our proteomic analysis identified protein domains already known to play some roles in biomineralization processes, suggesting an ancient and common origin of aragonitic shell biomineralization within mollusks. However, the number of matched proteins remained limited, highlighting the scarcity of databases considering the shell builder cephalopods (i.e. mainly the Sepiidae). In order to overcome this hurdle, the transcriptome of the S. officinalis shell sac has been recently sequenced and is under analysis. It will allow to complete the proteomic analysis presented here and to better understand how organic compounds are involved in the setting up of this intricate structure.

Tables:

Table 1: Quantification of organic matrix fractions extracted from the different part of the S. officinalis shell (i.e. whole organic matrix of shell; respective whole organic matrix of dorsal shield (DS) and chambered part (CH); and respective acid-insoluble and acid-soluble organic matrices of dorsal shield and chambered part; mean ± SD; n = 5).

Shell part

Fraction Mean organic matrix (%) * maximum score found (independently of the fraction studied)

Captions to figures:

  To remove CBB, excised 1-D gel bands were first unstained by at least 3 baths in a 200 µL solution of 25 mM NH 4 HCO 3 , 50% ACN (v/v) for 30 min under stirring. Thereafter, they were subsequently washed in 200 µL Milli-Q water and ACN 100%, each time for 15 min under stirring and at room temperature. After supernatant removal, this procedure was repeated a second time. Then, the samples were subsequently reduced with DTT (20 mM, 45 min, 56 °C in 50 mM NH 4 HCO 3 pH 8.1) and alkylated in the dark with iodoacetamide (50 mM, 30 min, at room temperature in 50 mM NH 4 HCO 3 pH 8.1). Excised gel fractions were then rinsed once in 300 µL of 25 mM NH 4 HCO 3 pH 8.1, and dehydrated using 300 µL ACN 100%. Proteins from dehydrated gel were digested by adding 25 µL of trypsin (6 µg mL -1 ; Sigma-Aldrich) in 25 mM NH 4 HCO 3 for 15 min at 4 °C. Gel was then completely immersed using 30 µL of 25 mM NH 4 HCO 3 solution, and incubated overnight at 37 °C under stirring.

  Data acquisition and analyses were carried out with Analyst QS software (version 1.1). The mass spectra data were searched against the NCBI non-redundant nucleic acid databases of the gastropod Lottia gigantea (188,590 sequences), the bivalves Crassostrea gigas (50,925 sequences), Elliptio complanata (138,349 sequences) and Pinctada fucata (31,477 sequences), and Cephalopods (360,946 sequences; February 2016), with MASCOT (2.1. version, Matrix Science, London, UK) and PEAKS studio (Canada, version 7.1). L.

  Cephalopod dataset was represented by 114,034 ESTs and 246,912 nucleotide sequences (ns) mainly originating from Octopus bimaculoides (197,284 ns), S. officinalis (43,625 ESTs + 512 ns), Euprymna scolopes (35,420 ESTs + 5240 ns), Octopus vulgaris (32,430 ns) and Doryteuthis pealeii (22,033 ESTs + 177 ns). The database search parameters used were: fixed modification = carbamidomethylation, variable modification = deamidation of asparagine and aspartic acid and oxidation of methionine, parent ion mass tolerance = 0.5 Da, fragment ion tolerance = 0.5 Da, missed cleavage = 1, with decoy calculation. Results from de novo sequencing (Peaks studio version 7.5) were filtered by setting average local confidence (ALC) to 80 and residue local confidence to 50%. Only peptides with at least 7 amino acids were considered as reliable sequences. PTM search function of the PEAKS Studio was used to look for unexpected modification in the peptides in order to increase the number of peptide spectral matches. Peptide threshold (denoted as -10lgP) for PEAKS DB was set to 30.

  The ASM CH shows 4 main bands migrating around 105, 38, 27 and below 15 kDa. Two gel areas also appear delimited by 53-45 and 20-17 kDa bands (Fig. 4B). The bands at 38, 27, 20 and 17 kDa, stained by CBB, appear negatively stained by silver nitrate. PAS stains the 105-kDa band and reveals a smear from 45 to 17 kDa with faint staining of bands 38, 27, 20 and 17 kDa and a new band around 35 kDa. No band was revealed using both Alcian blue stainings, whatever the pH, suggesting no carboxylation neither sulfation of sugars carried by ASM CH compounds.

Figure 1 :

 1 Figure 1: Schematic representation of the main constituents of the cuttlefish shell in sagittal plane, with associate mineral microstructures and repartition of the different shell sac cell types. The pillar distribution (i.e. vertical alignment and being closer near chamber openings) has been drawn respectively with our observations and previously made descriptions. Note that around 100 chambers traditionally constitute an adult S. officinalis shell. For convenience, only three chambers have been drawn here. Arrows indicate the shell sac area where gas and liquid exchanges occur. DS: dorsal shield, CH: chambered part.

Figure 2 :

 2 Figure 2: Infrared spectra of the acid-insoluble (AIM; A) and acid-soluble (ASM; B) matrices obtained by complete decalcification of the dorsal shield (solid line) and the chambered part (dashed line) from the Sepia officinalis shell.

Figure 3 :

 3 Figure 3: Electrophoretic analysis of the acid-insoluble matrices of the dorsal shield (A) and the chambered part (B) of S. officinalis shell. The gels were stained with CBB (lanes 1), silver nitrate (lanes 2), periodic acid Schiff (lanes 3), Alcian blue at pH 2.5 (lanes 4) and pH 1 (lane 5); MM: molecular mass markers. Because of no staining, lane 5 is not presented for B. All bands from these two extracts were excised from the gel for MALDI-TOF/TOF mass spectrometer analyses.

Figure 4 :

 4 Figure 4: Electrophoretic analysis of the acid-soluble matrices of the dorsal shield (A) and the chambered part (B) of S. officinalis shell. The gels were stained with CBB (lanes 1), silver nitrate (lanes 2), periodic acid Schiff (lanes 3) and Alcian blue at pH 2.5 (lanes 4); MM: molecular mass markers. Both matrices were stained with Alcian blue at pH 1 without bands appearance. All bands from these two extracts were excised from the gel for MALDI-TOF/TOF mass spectrometer analyses. Framed band size corresponds to the band allowing protein identification after tryptic digestion and MS analysis.

Figure 5 : 2 -

 52 Figure 5: 2-DE analysis of the acid-insoluble matrices of the dorsal shield (A) and the chambered part (B) of S. officinalis shell. The 1-D gels (left) with respective extracts show the correspondence between the protein bands and the spots observed on the 2-D gel (right) after CBB staining.

Figure

  Figure Click here to download Figure: Figures_CLP.docx

Figure 6 : 2 -

 62 Figure 6: 2-DE analysis of the acid-soluble matrices of the dorsal shield (A) and the chambered part (B) of S. officinalis shell. The 1-D gels (left) with respective extracts show the correspondence between the protein bands and the spots observed on the 2-D gel (right) after CBB staining. Framed band size corresponds to the band allowing protein identification after tryptic digestion and MS analysis.

  Figure 1

Figure 3 MM 1 A

 31 Figure 3

Table Click here to download Table: Tables.docx

 Click

Table 2 :

 2 Position and assignment of the FTIR major bands in the 600-4,000 cm -1 region for polysaccharides, chitin and proteins (vs: very strong, m: medium, w: weak).

	Band position	Classical	Chitin	Protein	Assignment
	(cm -1 )	polysaccharide	(+protein?)
	950-1,200	vs	vs	w	CC, CO, COC, COH stretching
	1,228-1,265			m	Amide III: NH bending and CN stretching
	1,310		m		Amide III: CH 2 wagging
	1,375		m		CH bending, CH 3 symmetric deformation
	1,445			m	CH 2 and CH 3 deformation
	1,510-1,550		m	s	Amide II: NH bending coupled to CN stretching
	1,600-1,700		m	s	Amide I: CO stretching
	2,800-2,950	w	w	w	CH stretching
	3,250-3,300		m	m	Amide A: NH stretching
	3,550-3,670	m	m	w	OH stretching

Table 3 :

 3 Protein concentrations (mg g -1 organic matter; mean ± SD; n = 5) of the dorsal shield and chambered part acid-insoluble (AIM) and acid-soluble (ASM) matrices of S. officinalis shell, extracted in the 2-D kit rehydration buffer (US: urea-soluble).

	Shell part	Fraction	Mean protein concentration (mg g -1 organic matter)
	Dorsal shield	US-AIM ASM	30 ± 5 462 ± 71
	Chambered part	US-AIM ASM	< 10 192 ± 34

Table 4 :

 4 Identification of acid-insoluble and acid-soluble matrix proteins of the S. officinalis shell by MS/MS analysis

	No.	Fraction	GenBank Acc. No.	Peptide sequence	Peptide score* (Mascott / Peaks)	Protein score* (Mascott / Peaks)	Signal peptide / Complete sequence	Theoritical mass / pI	Identified domain
		ASM /	FO196371	CLEETDADVAFVK	93 / 63	209 / 126	No / No	21.8 kDa / 6.4	1 Transferrin (193 aa)
		31 kDa band ASM DS		ADVTVLDGGDIYLAGK	98 / 63
				HLTFLDNPAK	80 / 48
				TSGWFVPMSVLFPNK	31 / 32
				HGNNLYYGYSGAAK	-/ 31
		ASM	FO182034	LFSEATGK	45	117 / 83	Yes / Yes	16.5 kDa / 8.2	1 Chitin-binding type 2
				LPGPGYLGDYIDECPYPK	39 / 39				(66 aa)
				CESFEPVSCGSR	-/ 47
		ASM	FO201581	QVFVTSTINFLR	63 / 48	123 / 81	Yes / No	20.8 kDa / 9.5	1 O-Glycosil hydrolase
				GSPIEDKENFAELLK	43 / 49				(178 aa)
		AIM	FO198959	DGTNTDIGINK	69 / 41	69 / 41	Yes / No	19.2 kDa / 9.3 1 von Willebrand factor type A
									(167 aa)
		ASM	FO162285	FDICSLDARPGK	34 / 31	62 / 42	Yes / No	16.2 kDa / 6.7	3 BPTI/Kunitz family
									(54, 54 and 28 aa)

Acknowledgments

This work was financially supported by the Muséum national d'Histoire naturelle ATM funding (ATM "Interactions Minéral-Vivant"). The authors thank all the members of the platform PROTEOGEN of the University of Caen for MALDI-TOF/TOF analysis as well as Dr Christelle Jozet-Alves for cuttlebone supply.

Supplementary material

Click here to download Supplementary material: Supplementary Table .docx