Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus Benjamin Marie 1,2 , Isabelle Zanella-Cle ´ on 3 , Marion Corneillat 4 , Michel Becchi 3 ,Ge ´ rard Alcaraz 2,4 , Laurent Plasseraud 2,5 , Gilles Luquet 1,2 and Fre ´ de ´ ric Marin 1,2 1 UMR 5561 CNRS, Bioge ´ osciences, Dijon, France 2 Universite ´ de Bourgogne, Dijon, France 3 IFR 128 BioSciences, UMR 5086 CNRS, IBCP, Universite ´ de Lyon 1, Lyon, France 4 UPSP PROXISS, De ´ partement Agronomie Environnement, AgroSup, Dijon, France 5 ICMUB, UMR CNRS 5260, Faculte ´ des Sciences Mirande, Dijon, France Keywords biomineralization; de novo sequencing; immunolocalization; mollusc shell nacre; organic matrix Correspondence B. Marie or F. Marin, UMR 5561 CNRS Bioge ´ osciences, Universite ´ de Bourgogne, 6 Boulevard Gabriel Dijon 21000, France Fax: +33 3 80 39 63 87 Tel: +33 3 80 39 63 72 E-mail: benjamin.marie@u-bourgogne.fr; frederic.marin@u-bourgogne.fr (Received 29 September 2010, revised 18 March 2011, accepted 11 April 2011) doi:10.1111/j.1742-4658.2011.08129.x In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohe- sive and functional macromolecular assemblage, containing mainly pro- teins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several rea- sons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demon- strate that this soluble protein is glycine-aspartate-rich, that it is highly gly- cosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the mor- phology of CaCO 3 crystals precipitated in vitro but, unexpectedly, it exhib- its an extremely weak ability to inhibit in vitro the precipitation of CaCO 3 . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits ‘hybrid’ biochemical properties that are found both in the soluble and insoluble proteins, rendering it diffi- cult to classify according to the standard view on nacre proteins. Database The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702. Abbreviations AIM, acid-insoluble matrix; ASM, acid-soluble matrix; CBB, Coomassie brillant blue; EST, expressed sequence tag; HPAE-PAD, high performance anion exchange-pulsed amperometric detection; LSB, Laemmli sample buffer; N63, Nautilin-63; SEM, scanning electron microscopy; TFMS, trifluoromethanesulfonic acid. FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2117 Introduction The calcified shells, which protect the mollusc soft tis- sues, comprise layered structures that are produced extracellularly by the calcifying epithelium of the man- tle. The shell layers are composites of calcium carbon- ate crystals, which are densely packed together with an array of biomacromolecules that form a 3D frame- work. Although the organic shell matrix (comprising mainly proteins, glycoproteins and polysaccharides) represents only a very small part of the CaCO 3 shell weight (between 1% and 5% for the nacreous layer), it is now well known to be essential for the control of the biomineral formation [1]. In particular, it is assumed to interact in different ways with the mineral phase at the nano- to microscale. Indeed, the organic shell matrix is considered to create a suitable environ- ment for mineralization to occur [1–3], to promote or inhibit crystal nucleation [4], to select calcium carbon- ate polymorph (aragonite and ⁄ or calcite) [5], to allow crystals to grow in privileged directions [3] and to con- tribute to the spatial arrangement of crystals to form well-defined microstructures [2,3]. At the atomic scale, this matrix slightly modifies the crystal lattice parame- ters, although this effect is poorly understood [6]. Because of its admirable biomechanical properties [7], its use in pearl industry and, finally, its potential use in dentistry and bone surgery [8,9], nacre is by far the most studied nonhuman organo-mineral biocomposite. It has a remarkable regular lamellar structure consisting of uniformly thick layers (approximately 0.5 lm) of tablet- like aragonite crystals separated by interlamellar layers of organic matrix. This apparent simple geometry facili- tates various structural investigations from micro- to nanoscales [10–12]. Nacre, or its precursor, ‘foliated ara- gonite’, appeared early in mollusc history, somewhere in the Cambrian [13]. It constitutes the inner layer of sev- eral extant mollusc shells, including that of bivalves, gastropods, cephalopods and monoplacophorans. There are, however, structural differences between cephalo- pod, gastropod and bivalve nacres. Although the bivalve exhibits a characteristic ‘brick-wall’ nacre microstruc- ture, those of cephalopods and gastropods are a contin- uous superimposition of tablets forming characteristic columnar microstructures. Observations of growing nacre show that each tablet nucleates at a specific loca- tion on the matrix surface [14]. Today, the general con- sensus is that nacre tablets grow from their center and expand laterally until reaching the confluence with neighboring tablets [10]. Histochemical observations of Nautilus nacre [15,16] indicate a concentric distribution of reactive groups, similar to carboxylates or sulfates, from the center to the periphery of each single tablet. A recent ultrastructural study has shown that nacre tab- lets are individually coated by a 5 nm thick layer of amorphous calcium carbonate [17]. Atomic force microscopy studies by Rousseau et al. [18] have shown that each tablet is constituted of nanograins encapsu- lated in a continuous network of an organic intracrystal- line phase. Summarizing the different recent advances on molluscan nacre, Addadi et al. [19] have proposed a coherent and dynamic model for nacre formation, as described below. The organic matrix constitutes the framework in which nacre tablets form. The major constituents of the matrix are the polysaccharide b-chitin, together with a relatively complex assemblage of hydrophobic and hydrophilic proteins. These macromolecules, which control the crystal deposition and microstructure self- assembly, are finally occluded either between the super- imposed parallel lamellae (‘interlamellar matrix’), at the boundary of adjacent mature nacre tablets (‘intertabu- lar matrix’), or within the crystallites (‘intracrystalline matrix’). First, the interlamellar matrix is assumed to be predominantly constituted of b-chitin fibrils that are aligned with the a-axis of the growing aragonitic tablets [20], suggesting that they can be, directly or indirectly, implicated in the control of the crystal orientation [21]. On the other hand, the b- and c-axes of the nacre tablets are oriented in parallel to the growing front in bivalves, whereas this is not the case in gastropods [22]. These data suggest that chitin is therefore either not fulfilling this role in nacre formation or that not all nacres are constructed in the same way, as recently sug- gested by Jackson et al. [23]. However, structuring the interface between the formed mineral front and the secreting mantle tissues, these different matrices are considered to precede immediately the new minerali- zation [19]. Second, the hydrophobic matrix, which contains silk-like proteins [2], is rich in Gly and Ala, or Gly alone, constituting one of the major protein frac- tions of the matrix, which can be extracted by decalcifi- cation of the nacre. These hydrophobic silk-like proteins are considered to form a hydrogel phase, supersaturated in calcium ions, between the chitin sheets. In this gel, the nacre tablets nucleate and grow [11]. During their growth, they push aside and com- press the silk-like protein gel. When adjacent tablets come to confluence, the gel polymerizes and remains ‘sandwiched’, forming the intertabular matrix [24]. Third, acidic hydrophilic proteins containing carboxyl- ate or sulfate reactive groups [25] are dispersed in the gel; they are considered to act as nucleating centers for each tablet; at the same time, they constitute a tenuous Nautilin-63, a novel shell nacre protein B. Marie et al. 2118 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS organic lace inside which mineral nanograins (initially amorphous) self-organize, orient and crystallize in a coordinated manner. Once formed, each tablet contains this intracrystalline matrix. b-chitin, the silk-like proteins and the acidic proteins are considered to be the three major components of the nacre organic matrix. However, several other nacre proteins have been identified and characterized over the past 16 years; a review on mollusc shell proteins is provided by Marin et al. [26]. These proteins, which exhibit diverse putative functionalities according to their sequences, are not taken in account in the model. Furthermore, from the dozen primary structures of nacre proteins that have been described so far, most of them correspond to proteins of the pearl oyster or the abalone models. None of them were described from the cephalopod nacre. Although the Nautilus nacre has already been the focus of several ultrastructural [15,16,27–29] or bio- chemical investigations of the bulk shell matrix [30–32], only a few studies have dealt with the detailed charac- terization of its shell proteins or their amino acid sequence characterization [33,34]. Because one of the keys to elucidating the molecular mechanisms of bio- mineralization depends on a detailed characterization of matrix proteins, as well as on the understanding of their functions, we chose to focus on Nautilin-63 (N63), a major protein of the nacre of the cephalopod Nautilus macromphalus. N63 comprises an acid-soluble acidic shell matrix glycoprotein, which is specific to the nacreous layer. Results N63 purification by preparative electrophoresis Because N63 was found to be one of the main proteins of the nacre acid-soluble matrix (ASM) [34], it was investigated further. In our previous study, using a 2D gel, we determined that N63 corresponded to a single acidic protein, and not a mixture of proteins of the same molecular weight but with different isoelectric points: indeed, this protein migrated as a single acidic spot. The fractionation of the nacre ASM preparative electrophoresis resulted in the effective one-step purifi- cation of N63. The purity of the N63 extract was checked by monodimensional gel electrophoresis with silver nitrate staining (Fig. 1A). FTIR Figure 1B shows the FTIR profile of N63 and of the nacre ASM. Both samples exhibit characteristic bands of proteinaceous and ⁄ or glycoproteinaceous compo- nents [35]: the thick bands around 3270 cm )1 are attributed to the -OH and the amide A groups (N-H bonds), the two small bands at 2915 and 2850 cm )1 were assigned to the C-H bonds, and the two notewor- thy bands near 1640 and 1530 cm )1 were ascribed to the amide I (C=O bond) and the amide II (C-N bond) groups, respectively, which are commonly associated with proteins. Carboxylate (COO ) ) and sulfate (SO 4 2) ) absorption bands are also present in both samples, around 1420 and 1235 cm )1 . We note that N63 exhib- its a remarkably strong carbohydrate absorption band (C-O bond) around 1060 cm )1 . These observations suggest that N63 is an acidic glycoprotein. Amino acid composition of N63 The purified N63 was analyzed for its amino acid com- position and was compared with those of the nacre ASM, which was obtained previously for the same spe- cies [34] (Table 1). The six dominant amino acid resi- dues are Asx (18%), Gly (17%), Thr (11%), Ala (9%), Glx (8%) and Pro (8%). By comparison with the Fig. 1. Purification and characterization of N63. (A) 12% SDS ⁄ PAGE of ASM (and of N63) after its purification by preparative electrophoresis. The gel was stained with silver nitrate. The apparent molecular weights of the molecular markers (MM) are indicated on the left. (B) Infrared spectra of the ASM (gray line) and the purified N63 (black line). B. Marie et al. Nautilin-63, a novel shell nacre protein FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2119 amino acid composition of the bulk matrix, N63 is strongly enriched in Thr and Pro residues but depleted in Gly and Asx residues. A search based on the simi- larity of amino acid composition (AACompIdent: http://expasy.org/tools/aacomp/) did not produce any significant hits. Monosaccharide composition of N63 The purified N63 was analyzed for monosaccharide composition, which was subsequently compared with that of the whole ASM [34] (Table 2). We note that, similar to ASM, N63 contains a high amount of mono- saccharide fraction (total of 275 ngÆlg )1 ). The five dominant monosaccharides are glucose (22%), galac- tose (17%), glucosamine (17%), glucuronic acid (15%) and galactosamine (12%). By comparison with the monosaccharide composition of the bulk matrix, N63 appears strongly enriched in glucosamine and galactos- amine but also strongly depleted in glucose. Interest- ingly, for both samples, we noted an unknown peak on high performance anion exchange-pulsed amperometric detection (HPAE-PAD) chromatograms, which eluted in the ‘acidic monosaccharides’ area, near the expected galacturonic acid peak [36,37]. The identity of this peak needs to be investigated further. Chemical deglycosylation of N63 In a previous study [34], the periodic acid–Schiff and Alcian blue staining on SDS ⁄ PAGE suggested that N63 is an acidic glycoprotein. To confirm this finding, the nacre ASM was chemically deglycosylated with tri- fluoromethanesulfonic acid (TFMS) at 0 °C. The ASM and the deglycosylated-ASM were compared on SDS ⁄ PAGE gels with double Coomassie brillant blue (CBB) ⁄ silver and Alcian blue staining (Fig. 2A). This Table 1. Composition of the nacre ASM and purified N63: amino acid composition. Data are presented as the molar percentage of total amino acids for each extract. Note that Asx = Asn + Asp and Glx = Gln + Glu. Cysteine residues were quantified after oxidation. Tryptophan residues were not detected (ND) as a result of the hydrolysis conditions. Amino acid % of total amino acids ASM N63 Asx 20.8 18.0 Glx 7.5 8.2 Ser 6.8 8.6 His 2.1 1.3 Gly 21.4 16.8 Thr 4.8 10.6 Ala 7.3 8.7 Arg 3.1 2.2 Tyr 2.9 1.1 Cys 1.1 ND Val 3.1 4.4 Met 0.7 0.4 Phe 2.7 1.9 Ile 2.3 2.6 Leu 3.2 4.3 Lys 3.9 2.9 Pro 6.4 8.1 Trp ND ND Table 2. Composition of the nacre ASM and purified N63: mono- saccharidic composition. The composition of neutral sugars is obtained by HPAE-PAD. Data are represented as ngÆlg )1 of the total matrix and as a percentage of the total identified carbohydrate compounds. ND, not detected. Monosaccharide ngÆlg )1 of matrix (% of total) ASM N63 Fucose 15.1 (6) 21.8 (8) Rhamnose 13.3 (5) 11.6 (4) Arabinose ND ND Galactose 43 (17) 46.0 (17) Glucose 79.4 (31) 60.3 (22) Mannose 9.2 (4) 10.4 (4) Xylose 2.0 (1) 2.3 (1) Galactosamine 20.6 (8) 33.7 (12) Glucosamine 33.5 (13) 47.5 (17) Galacturonic acid a ND ND Glucuronic acid 37.0 (15) 41.4 (15) Total 253.1 (100) 275 (100) a An unattributed band was observed around the galacturonic acid band. Fig. 2. Glycosylation (A) and chitin-binding (B) characterizations of N63 by SDS ⁄ PAGE. (A) 12% SDS ⁄ PAGE of ASM and deglycosylat- ed-ASM (Deg-ASM) stained with silver nitrate + CBB (left) and with Alcian blue (right). (B) Chitin-binding ability of N63 (top) and BSA (down, negative control) on 12% SDS ⁄ PAGE stained with silver nitrate. Lane 1, water wash; lane 2, 0.2 M NaCl wash; lane 3, extract with LBS. For both proteins, the same volume of solution was loaded on the gel. Nautilin-63, a novel shell nacre protein B. Marie et al. 2120 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS double staining allowed visualization of most of the macromolecular compounds of the ASM on the same gel. N63 exhibits an important shift of approximately 10 kDa, which represents a loss of apparent molecular weight of 14%. This shift is primarily the result of the removal of covalently bound polysaccharides. Interest- ingly, the positive Alcian blue staining, observed for the N63 glycoprotein, was completely lost after degly- cosylation. Because we used Alcian blue under low pH conditions, this result confirms that an important part of the polyanionic properties of N63 is a result of the acidic glycosyl moieties [38,39]. Chitin-binding capability of N63 Framework proteins of the organic nacre matrix are hypothesized to interact with chitin [19,20]. However, this property has never been tested previously on this type of matrix. The putative chitin-binding ability of N63 was examined consequently (Fig. 2B). The nacre ASM was incubated in solution with powdered chitin, and the insoluble mixture was successively washed with distilled water, saline and finally with hot denaturing Laemmli sample buffer (LSB) [40]. Each washed sam- ple was analyzed by SDS ⁄ PAGE, stained with silver nitrate. BSA, used as a negative control, was com- pletely washed out with the successive water and saline treatments, with no band being detected in the LSB wash (Fig. 2B, bottom, lane 3). By contrast, a minor part of N63 was desorbed after the water and saline treatments (Fig. 2B, top, lanes 1 and 2) and the drastic LSB wash was required for complete N63 desorption from chitin (Fig. 2B, top, lane 3). This clearly suggests that N63 has a strong affinity for this insoluble poly- saccharide, and thus possesses a true chitin-binding ability. In vitro inhibition of CaCO 3 precipitation with N63 The effect of nacre ASM and purified N63 on the kinetics of CaCO 3 precipitation was determined by monitoring the pH decrease (Fig. 3). In the blank experiment (without sample), the pH decreased with- out any time lag (approximately 120 s), corresponding to the rapid precipitation of calcium carbonate. When samples were present in the solution, we observed a slight inhibition of CaCO 3 precipitation. First, the effect of the nacre matrix started to occur above 1 lg of the ASM and the delay of the reaction was dose- dependent. At approximately 50 lg of nacre ASM, a complete inhibition of the precipitation of calcium car- bonate was recorded. The observation of the inhibitory capacity of this matrix is consistent with previous stud- ies on the organic soluble matrix of nacre from differ- ent molluscs [24,41,42]. On the other hand, inhibition experiments performed with N63 demonstrate that it presents a five-fold lesser inhibition capacity than the total ASM. At 25 lg, the N63 inhibition curve can be superimposed to the 5 lg curve obtained with the total ASM. Taken together, our observations indicate that, if nacre ASM exhibits a moderate capacity of inhibi- tion of CaCO 3 precipitation, this effect is not a result of N63 because the latter presents only a weak inhibi- tion capacity, despite the fact that it carries sulfated (i.e. negatively charged) sugars. Interaction with CaCO 3 crystals precipitated in vitro The effect of purified N63 on the precipitation and morphology of calcium carbonate crystals grown in vitro was investigated by scanning electron micros- copy (SEM) (Fig. 4). When no protein is added, crys- tals exhibit the typical rhombohedral habitus of calcite with smooth crystal faces (Fig. 4A). In the presence of an increasing amount of purified N63 (0.1– 50 lgÆmL )1 ), the crystals produced appear mostly as polycrystalline aggregates with foliation and microsteps at the corners (Fig. 4B–F). At the highest concentra- tion (‡ 10 lgÆmL )1 ), the precipitated CaCO 3 crystals exhibit specific linear grooves at the edges of the poly- crystals (Fig. 4E,F). FTIR analysis of the calcium carbonate polymorph confirmed that these crystals were only made of calcite. Unexpectedly, at the highest concentrations of N63, no inhibition of crystal forma- tion occurs. Fig. 3. In vitro inhibition of CaCO 3 precipitation by nacre ASM and N63. The effects of different concentrations (1–50 lg) of purified N63 and whole ASM were monitored on the pH decrease induced by the in vitro precipitation of CaCO 3 in a CaCl 2 ⁄ NaHCO 3 solution [4]. Blank tests were performed in the absence of protein. B. Marie et al. Nautilin-63, a novel shell nacre protein FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2121 These results indicate that N63 interacts obviously with the precipitation of CaCO 3 because it induces drastic changes in the crystals morphology but does not (or very slightly) inhibit their formation, with the combination of these two effects in a shell matrix pro- tein being rather unusual. De novo sequencing of N63 Purified N63 was digested with trypsin before analysis by MS ⁄ MS. Peptide digests were loaded on a nanoLC column and analyzed by nanoESI-qQ-TOF. Because no genomic, nor transcriptomic data are available for Nautilus, the most intensive MS ⁄ MS peaks were manu- ally interpreted (de novo sequencing) after considering the complexity of the spectra and the numerous ion combinations. For N63, the sequence of 27 peptides, with lengths comprising between eight and 20 amino acids, was determined by de novo interpretation of their respective MS ⁄ MS spectra (Table 3). The partial protein sequences of N63 appear in the UniProt Knowledgebase under accession number P86702. Among them, two peptides (GPAAVVGVL ⁄ IGK and SFDSWL ⁄ ITK) present a sequence similar to two oth- ers previously obtained by de novo sequencing of the whole nacre ASM [34]. The MS ⁄ MS deduced sequences were individually submitted to a blastp search against Swiss-Prot nrdb using the EXPASY website (http: ⁄⁄expasy.org ⁄ ), and to a tblastn search against GenBank and the data- base for expressed sequence tags (EST) (dbEST) using the NCBI online tool (http://www.ncbi.nlm.nih.gov) (Table 3, central and right columns, respectively). Unexpectedly, we did not find any homology with already known mollusc shell proteins, and most of the observed hits concern only one unique peptide and are related to unknown putative proteins or to proteins that are not expected to be components of the mollusc shell matrices. These observations should be confirmed in future works by the use of complementary tech- niques. On the other hand, when the peptide GPAAVVGV- L ⁄ IGK was previously used for blast against an in-house database of mollusk shell matrix [34], we noted that it presents partial similarities with the sequence GPAAVPVAAG of mucoperlin, a shell matrix protein from the Pinna nobilis nacreous layer [24]. MS BLAST search The de novo-generated sequences of N63 were also sub- mitted to a MS blast database search, which enables the identification of the proteins or their assignment to a family of homologous proteins, considering all their internal peptide sequences together (Table 4). Sequence similarities were observed for several peptides of N63 A B C FE D Fig. 4. SEM micrographs of synthetic calcium carbonate crystals grown in vitro in the presence of N63 at increasing concentrations (lgÆmL )1 ). (A) Negative control without N63; (B) 0.1 lgÆmL )1 ; (C) 1 lgÆmL )1 ; (D) 5 lgÆmL )1 ; (E) 10 lgÆmL )1 ; (F) 50 lgÆmL )1 . Scale bars are 60, 20 and 2 lm, on the left, center and right, respectively. Nautilin-63, a novel shell nacre protein B. Marie et al. 2122 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS with vertebrate collagen XI, cuticle collagen from nem- atoda, and the spidroin-like protein of an arthropod. This sequence similarity with collagen-like proteins is partially supported by the fact that some peptides pres- ent Gxy repeats. Interestingly, we did not find any homology with already known mollusc shell proteins. Table 3. MS ⁄ MS derived sequences of N63 trypsic peptides. BLAST search results against Swiss-Prot and mollusc-restricted EST databases (taxid:6447) are presented in the central and right columns, respectively. The partial protein sequences of N63 appear in the UniProt Knowl- edgebase under accession number P86702. Alignment results of the BLAST searches are indicated on the peptide sequences: identical and synonymous amino acid positions are underlined or shown in bold for the BLAST search against Swiss-Prot and mollusc ESTs, respectively. Respective scores (similar amino acids ⁄ total amino acids) of both BLAST searches are indicated after the name of the matching proteins. The MS ⁄ MS technique does not allowed distinction between L and I residues, which exhibit identical masses. M+H + De novo sequence Expasy BLAST against Swiss-Prot NCBI TBLASTN against mollusc ESTs [sp.] 858.46 L ⁄ IPDL ⁄ IASSR – – 858.51 STL ⁄ IPVL ⁄ ITK – – 867.50 GPTGL ⁄ IL ⁄ IGPR – – 887.45 GPYGPL ⁄ IQR – – 949.48 FNL ⁄ IEL ⁄ ISAR – – 967.58 GPAAVVGVL ⁄ IGK – – 983.50 SFDSWL ⁄ ITK – – 1048.58 L ⁄ IGL ⁄ IPGPQGR – – 1078.54 PGPPGPGCR – – 1080.54 FAL ⁄ ISNQCL ⁄ IK – – 1177.65 L ⁄ IAVEFAGQSK – – 1227.61 FSSFL ⁄ IANEGKK – – 1260.53 EGPEGEEGPR – – 1262.53 TEFDGAYFAGGK – – 1356.81 FPVVGKPFPQL ⁄ IK – Unknown (Aplysia californica)(10 ⁄ 12) 1384.71 VFHAEPPFPTSR – Ubiquitin-like (Aplysia californica)(9 ⁄ 12) 1441.68 STYGPSGSQPGK – – 1512.88 KGVVTPFKGNQPL ⁄ IK – – 1528.68 FNDFL ⁄ IVESDSR – – 1567.67 CPPDDSSFER – – 1567.76 SPAVSGHSSPATL ⁄ INSR – – 1569.92 MKPAGFPGKGNGAPL ⁄ IK – Methyltransf. (Aristolochia californica)(9 ⁄ 16) 1740.93 NGL ⁄ IASDPLENL ⁄ IKNR – HEAT-containing (Euprymna scolopes)(11 ⁄ 14) 1748.85 L ⁄ IGSCFPDVL ⁄ IDEPPT – SWIRM-like (Euprymna scolopes)(10 ⁄ 14) 1774.85 S PFFTGPSGYTSDGL ⁄ INK Methylase a (11 ⁄ 17) – 1825.92 TPTVSKTL ⁄ IL ⁄ IL ⁄ ITAAGDPGP GAGK – Zona Pellucida (Lottia gigantea)(12 ⁄ 20) 2020.11 VL ⁄ IESSKTDL ⁄ IVAL ⁄ IQGEFQR – Unknown (Lottia gigantea)(13 ⁄ 18) a Methylase of Nitrosococcus oceani [Q3JDX6]. Table 4. Results of the MS BLAST search for the identification of N63 using de novo sequenced peptides. MS BLAST Identification Total score Best score Peptide matches Calculated mass (kDa) Phylum Swiss-Prot number Collagen a-1 (XI) 179 47 4 59 Vertebrata Q28083 Collagen a-1 (XI) 176 47 4 120 Vertebrata Q61245 Collagen a-2 (XI) 175 47 4 140 Vertebrata P13942 Cuticule collagen 178 60 4 33 Nematoda Q60LV9 Cuticule collagen 160 60 3 33 Nematoda Q23628 Cuticule collagen 102 58 2 28 Nematoda Q18536 Spidroin 2-like 119 50 3 10 Arthropoda Q9BIU5 Chitinase 3-like protein 96 58 2 43 Vertebrata Q8SPQ0 Chitinase 3-like protein 96 58 2 38 Vertebrata Q29411 B. Marie et al. Nautilin-63, a novel shell nacre protein FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2123 Immunolocalization of N63 Polyclonal antibodies raised against purified N63 were produced in rabbit. We checked the specificity of these antibodies by western blotting against both the whole acid-insoluble matrix (AIM) and ASM extracts (Fig. 5A,B). For these experiments, no immunological signal was observed for negative controls performed with pre-immune sera (data not shown). The antibody raised against the purified N63 showed a specific immunoreactivity for the 63 kDa band of the nacre ASM corresponding to N63. This observation demon- strates that the antibody recognizes exclusively N63 specific epitopes and that N63 protein is exclusively present in the ASM. To localize N63 directly in both the prismatic and the nacreous layers of the shell of N. macromphalus, the im- munogold technique was applied on shell cross-sections, followed by observation by SEM, as described previ- ously [43], using the antibodies raised against the puri- fied N63 (Fig. 5C–F). Although a low background was observed for negative control performed with pre- immune serum (Fig. 5C), the N63 antibodies exhibit a clear and specific signal on shell nacre (Fig. 5E,F), whereas very little signal is observed with the prismatic layer (Fig. 5D), testifying that N63 is specific of the nacreous layer. Immunolocalization on nacre cross-sec- tions (Fig. 5E,F) revealed that N63 is largely distrib- uted inside nacre tablets, and also in the inter-tablet matrix that separates nacre tablets of the same layer. Discussion In a previous study, we characterized the whole acid soluble matrix extracted from the nacre of the cephalo- pod N. macromphalus [34]. In particular, we obtained approximately 40 short sequences of different shell proteins, both extracted from the acid-soluble and from the acid-insoluble matrices. In the present study, we focus on one shell protein, which we named Nauti- lin-63 (N63), according to its apparent molecular weight on a 1D electrophoresis gel. N63 is an acidic shell matrix glycoprotein, which is unambiguously specific to the nacreous layer of N. mac- romphalus. N63 belongs to the acetic acid-soluble frac- tion, and to this fraction exclusively, because no signal was detected on western blotting (Fig. 6) and none of its sequenced peptides were found in the acetic acid- insoluble fraction in our previous study [34]. In vitro, N63 binds chitin, interacts with the shape of newly- grown calcite crystals but, apparently, has a very limited effect on the precipitation of calcium carbonate. Although its glucose-rich glycosyl moieties exhibits sul- fated groups, N63 does not bind calcium ions [34]. From the 27 peptidic sequences (of eight to 20 residues in length) obtained by de novo sequencing, only seven of them exhibit similarities with other putative molluscan proteins, which are not related to calcification. One pep- tide is partly similar to a short domain of mucoperlin, a bivalve shell protein. At least five obtained peptides have a collagen signature, characterized by Gxy triplets. Such a signature has already been found in a short domain of lustrin-A, a nacre protein of the abalone Haliotis rufescens [44]. By the immunogold technique, N63 appears to be particularly concentrated inside nacre tablets, as well as between them. The overall composition of all the obtained peptides of N63, taken together, is enriched in Gly and Pro AB C D F E Fig. 5. Immunodetection of N63 by western blotting (A–B) and the immunogold technique (C–F). (A) 12% SDS ⁄ PAGE of nacre AIM and ASM, stained with silver nitrate. (B) The nacre AIM and ASM were tested by western blotting and incubated with the polyclonal antibodies raised against purified N63. (C) SEM micrographs for the immunogold negative control performed on nacre without anti-N63 specific sera. (D–F) SEM micrographs of the immunogold technique with anti-N63 specific sera on prismatic (D) and nacreous (E,F) shell layers. Scale bars = 2 lm. Nautilin-63, a novel shell nacre protein B. Marie et al. 2124 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS residues, whereas the overall amino acid composition of the isolated protein, after its purification, is enriched in Asx, Gly and Thr residues, which constitutes a typi- cal shell protein signature. This apparent discrepancy between de novo sequencing data and the amino acid composition may be explained in different ways: first, the de novo sequences represent only approximately one half of the protein, thus giving a partial picture of its primary structure. Second, the de novo sequencing by MS presents technical limitations: on the one hand, it is ineffective on protein domains, which lack appro- priate cleavage sites (Arg and Lys in the case of trypsic digestion); on the other hand, the peptides resulting from the digestion are not detectable, if not ionized. Thus, the technique might introduce a bias in the rep- resentation of the analyzed peptides for the complete N63 sequence. In the present case, at the very least, it is likely that some of the nonsequenced peptides con- stituting the unknown part of N63 are enriched in Asx and Thr residues. What might be the role of N63 in the formation of nacre? Our biochemical data, when combined, give an unusual mosaic picture, which does not simply fit into the general structural framework given few years ago by Nudelman et al. [16] and Addadi et al. [19], and also recalled in the Introduction of the present study. First, several of the peptides determined by de novo sequencing are hydrophobic, and might suggest that N63 is part of the hydrogel where nascent nacre tablets grow. By contrast, N63 is a soluble and acidic protein, and is present not only around nacre tablets, but also inside the tablets. Second, because N63 contains sul- fated polysaccharides, it is tempting to assume that this protein is part of the nacre-nucleating complex (i.e. the central domain observed for each nacre tab- lets), whatever it is, either a central spot, as suggested by Crenshaw and Ristedt [15] or a central ring as reported by Nudelman et al. [16]. Once again, our immunogold labeling data do not support this hypoth- esis because we did not observe spots in the centre of nacre tablets, but did observe the peripheral distribu- tion of N63 around the tablets. Finally, we clearly demonstrate that N63 binds chitin in vitro. This may suggest that this protein is able to form macromolecu- lar complexes with chitin, which, in other words, means that it should be co-localized with chitin at the interlamellar interface. Obviously, our immunogold labeling data do not reveal such a location. The fact that N63 can, at the same time, strongly interact with the shape of CaCO 3 crystals precipitated in vitro without (or slightly) inhibiting their formation is puzzling. Indeed, we observed that, for many mol- lusc shell proteins, the ability to interact with CaCO 3 crystals and the capacity to inhibit the precipitation of CaCO 3 were often associated, as observed for P95 or for caspartin [24,26]. The present case constitutes the first report indicating that these two properties can be disconnected in a calcifying matrix protein. This unu- sual property could be somehow related to the fact that N63 does not bind calcium ions in solution as previously noted [34]. Taken together, these findings suggest that N63 exhibits ‘hybrid’ biochemical properties, some of which are usually found in framework matrix proteins (i.e. hydrophobicity, intertabular localization, ‘collagen sig- nature’, absence of calcium-binding, weak ability to inhibit CaCO 3 precipitation), whereas others are com- monly met in the soluble matrix components (i.e. solu- bility, acidity on 2D gels, enrichment in Asx residues, capacity to interact with CaCO 3 crystals). This clearly suggests that the models established in recent years for Nautilus nacre [16,19] must, in some ways, be refined, by taking in account proteins of ‘intermediate’ bio- chemical properties, such as N63. In the absence of a clear correlation between the structure of N63, its bio- chemical properties and a defined function, we suggest that N63 is a multifunctional protein that plays a key role in binding chitin, and thus in participating in the structuring of the organic framework, at the same time as finely interacting with the mineral phase. It is possi- ble that these two functions are displayed sequentially (chitin-binding, followed by mineral interaction). We are fully aware that trying to decipher the func- tion of one single component of the nacre matrix will not provide an explanation of the whole process of nacre fabrication. As highlighted in a previous study [16], ‘none of the components of the organic matrix functions in isolation. The organic matrix is a structural entity in which the assembly of all components is essential for the correct regulation of crystal nucleation, growth, mor- phology, and polymorph type’. However, we consider that the precise characterization of separate nacre mac- romolecular constituents will provide the complete bio- chemical framework required to precisely analyze the growth of nacre tablets. This framework constitutes the prerequisite for studying protein–protein and protein– polysaccharide interactions, and for any attempts aiming to understand the supramolecular chemistry that contributes to the emergence of nacre microstructure. Experimental procedures Shell preparation and nacre matrix extraction Fresh shells of the cephalopod N. macromphalus, 150 ⁄ 200 mm in length, were collected on the coast of New B. Marie et al. Nautilin-63, a novel shell nacre protein FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2125 Caledonia (Pacific). The external prismatic layer was removed by abrasion under cold water. The shells were mechanically crushed and fragments of the siphon were removed. The nacre fragments were immersed in 1% (v ⁄ v) NaOCl for 24 h to remove superficial contaminants, and then thoroughly rinsed with water. All of the subsequent extraction procedure was performed at 4 °C. The nacre powder (< 200 lm) was decalcified overnight in cold dilute acetic acid (5%, v ⁄ v) added by an automatic titrimeter (Titronic Universal; Schott Instruments GmbH, Mainz, Germany), until pH 4 was obtained. The solution was cen- trifuged at 3900 g for 30 min. The pellet, corresponding to the AIM, was rinsed six times with MilliQ water (Millipore Corp., Billerica, MA, USA) and finally freeze-dried. The supernatant comprising the ASM was filtered (5 lm) before being concentrated with an Amicon ultrafiltration system on a Millipore Ò membrane (YM10; 10 kDa cut-off). The concentrated solution (approximately 5–10 mL) was exten- sively dialyzed against MilliQ water (3 days, several water changes) before being freeze-dried and weighed. SDS ⁄ PAGE and gel staining procedures The separation of matrix components was performed under denaturing conditions by monodimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS ⁄ PAGE) containing 12% polyacrylamide (Mini-protean 3; Bio-Rad, Hercules, CA, USA). Protein concentration of the ASM was estimated by using the BCA-200 Protein Assay kit (Pierce, Rockford, IL, USA). The nacre matrices were directly suspended in LSB containing b-mercaptoethanol and heat-denatured [40]. One milligram of AIM was sus- pended in LBS, heat-denatured (10 min at 100 °C) and then centrifuged at 3900 g for 30 s. Twenty microlitres of the supernatant containing the Laemmli-soluble AIM were loaded onto gel. Fifty micrograms of ASM were loaded in each well. Because the classical CBB staining is often ineffi- cient at revealing all the proteins associated with calcified tissues, we chose to visualize proteins on the gel with both silver nitrate [45] and CBB R-250. Glycosylation of ASM macromolecules was studied qualitatively on denaturing mini-gels by Alcian Blue 8GX staining [39] at pH 1 for the detection of sulfated sugars [38]. Deglycosylation with TFMS Chemical deglycosylation of 5 mg of ASM was performed with 1.5 mL of TFMS ⁄ anisole (2 : 1, v ⁄ v) for 3 h, under a nitrogen atmosphere, with constant stirring [46]. The tem- perature was maintained at 0 °C, to preclude peptidic bond hydrolysis. After neutralization with 2 mL of 50% cold pyridine, the aqueous phase was extracted twice with diethyl ether and then extensively dialyzed against water (5 days) before being lyophilized. Fetuin was treated simi- larly and used as a positive control. All the deglycosylated extracts were analyzed on monodimensional SDS ⁄ PAGE followed by silver nitrate and Alcian blue staining. Chitin-binding ability A chitin-binding assay was performed in solution as described previously [47], with some modifications. One milligram of nacre ASM and 500 lg of BSA (used as negative control) were dissolved in 200 lL of water and incubated with 10 mg of chitin (C9752; Sigma-Aldrich, St Louis, MO, USA) for 2 h at 25 °C under constant stir- ring. Samples were centrifuged (13 000 g for 5 min) and the supernatants were taken away and preserved. The residues were then rinsed three times with 500 lL of distilled water, before washing with 300 lL of 0.2 m NaCl and centrifuga- tion. The precipitates were boiled with LSB for 10 min at 99 °C. Each supernatant and washing solution was ana- lyzed on SDS ⁄ PAGE under denaturing conditions. After electrophoresis, the gels were stained with silver nitrate [45]. Purification of N63 by preparative SDS ⁄ PAGE The nacre ASM was fractionated on a preparative 12% polyacrylamide gel under denaturing conditions as described previously [48]. Eighty fractions (5 mL each) were eluted from the preparative gel. Aliquots of the fractions were tested by SDS ⁄ PAGE with silver nitrate staining. Fractions containing the N63 protein were pooled, concentrated, thor- oughly dialyzed against MilliQ water and freeze-dried. Infrared analysis of N63 Infrared spectra were directly recorded on lyophilized sam- ples of nacre ASM and of purified N63 at a 2 cm )1 resolution on a FTIR spectrometer (Vector 22; Bruker, Ettlingen, Ger- many) equipped with a Specac Golden GateÔ ATR device in the wave number range 4000–500 cm )1 . For each extract, we obtained several spectra with a high reproducibility. Amino acid composition of N63 The amino acid composition of the purified N63 was determined by Eurosequence (Groningen, The Nether- lands). Freeze-dried samples were hydrolyzed with 5.7 m HCl in the gas phase for 1.5 h at 150 °C. The resulting hydrolysate was analyzed on an HP 1090 Aminoquant (Hewlett-Packard, Palo Alto, CA, USA) [49] by an auto- mated two-step precolumn derivatization with O-phthalal- dehyde for primary and N-(9-fluorenyl)methoxycarbonyl for secondary amino acids. Cysteine residues were quanti- fied after oxidation. The hydrolysis procedure does not allow the quantification of tryptophan residues. Experi- mentally determined amino acid values may deviate up to approximately 10%. For comparison, the amino acid Nautilin-63, a novel shell nacre protein B. Marie et al. 2126 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS [...]... 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York Velazquez-Castillo RR, Reyes-Gasga J, GarciaGutierrez DI & Jose-Yacaman M (2006) Crystal structure characterization of nautilus shell at different length scales Biomaterials 27, 4508–4517 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 Keith J, Stockwell S, Ball D, Remillard K, Kaplan D, Thannhauser . Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus Benjamin Marie 1,2 , Isabelle Zanella-Cle ´ on 3 , Marion. we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of