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Among molluscs, the shell biomineralization process is controlled by a set of extracellular macromolecular components secreted by the calcifying mantle. In spite of several studies, these components are mainly known in bivalves from only few members of pteriomorph groups. In the present case, we investigated the biochemical properties of the aragonitic shell of the freshwater bivalve Unio pictorum (Paleoheterodonta, Unionoida). Analysis of the amino acid composition reveals a high amount of glycine, aspartate and alanine in the acid-soluble extract, whereas the acid-insoluble one is rich in alanine and glycine. Monosaccharidic analysis indicates that the insoluble matrix comprises a high amount of glucosamine. Furthermore, a high ratio of the carbohydrates of the soluble matrix is sulfated. Electrophoretic analysis of the acid-soluble matrix revealed discrete bands. Stains-All, Alcian Blue, periodic acid/Schiff and autoradiography with 45Ca after electrophoretic separation revealed three major polyanionic calcium-binding glycoproteins, which exhibit an apparent molecular mass of 95, 50 and 29 kDa, respectively. Two-dimensional gel electrophoresis shows that these bands, provisionally named P95, P50 and P29, are composed of numerous isoforms, the majority of which have acidic isoelectric points. Chemical deglycosylation of the matrix with trifluoromethanesulfonic acid induces a drastic shift of both the apparent molecular mass and the isoelectric point of these matrix components. This treatment induces also a modification of the shape of CaCO3 crystals grown in vitro and a loss of the calcium-binding ability of two of the main matrix proteins (P95 and P50). Our findings strongly suggest that post-translational modifications display important functions in mollusc shell calcification.
Among molluscs, the shell biomineralization is a matrix-mediated process, performed extracellularly. This matrix is a complex mixture of proteins, glycoproteins, polysaccharides and lipids, which are secreted by the mantle calcifying epithelium, together with the mineral precursors . All these components are released in the extrapallial space, where they are supposed to self-assemble in an orderly manner. The matrix may have several functions: it organizes spatially a 3D framework, concentrates locally mineral ions above the supersaturation threshold, catalyzes mineral precipitation, nucleates crystals, determines the polymorph at crystal lattice scale, controls the crystals shape by stereo-specific adsorptions and, finally, inhibits crystal growth . In addition, the matrix is suspected to be involved in cell signalling with the calcifying epithelium .
During the last decade, several macromolecules have been characterized from the molluscan shells . One drawback of these previous studies on matrix is that they focused mainly on protein components. To date, there are limited data available dealing with other macromolecular components (i.e. sugars and lipids) [5,6]. Another point worthy of note is the scarcity of tackled biological models. For example, in bivalves, most of the findings were obtained from seven genera only, all belonging to the pteriomorph subclass. In particular, due to economical purposes, the pearl oyster Pinctada sp. constitutes the prominent model for studying the formation of mother-of-pearl.
For many reasons, paleoheterodont bivalves represent fascinating models. This small subclass, which comprises unionoida and trigonioida, is traditionally positioned between pteriomorphs and the ‘modern’ heterodont bivalves . Most of them are freshwater species, which are observed worldwide. Until now, they have been often used in environmental pollution studies, notably as metal accumulation indicators [8,9]. If microsocopic observations have been performed describing the structure of the shell of some unionoida bivalves [10,11], very little is known about the organic components of the shell, especially at the molecular level [8,12–14].
Like nacro-prismatic pteriomorph bivalves (including Mytiloida and Pterioida), paleoheterodont bivalves exhibit a rather ‘primitive’ shell texture, comprising an outer prismatic shell layer (not always present among some species) and an inner one made of the typical brick-wall nacre. Unlike most pteriomorphs (but like some gastropods and cephalopods), the prismatic shell layer of paleoheterodonts is entirely aragonitic . Moreover, the shell hinge of paleoheterodonts is rather ‘modern’ and similar to that of heterodont bivalves. Thus, paleoheterodont bivalves represent a case of complex ‘mosaic’ evolution because they exhibit both primitive and derived characters .
One key question is whether the macromolecules that constitute the shell organic matrix are similar to those found in the aragonitic layers of pteriomorph bivalves. In this paper, we present the first biochemical characterization of the shell matrix of the freshwater unionoida bivalve Unio pictorum. We focus our attention on the nacreous layer, notably on its saccharidic composition, and underline the importance of the role of glycosyl moiety in the nacre mineralization process.
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- Experimental procedures
In the present paper, we have characterized biochemically the calcifying matrix associated with the nacreous shell layer of the paleoheterodont freshwater bivalve U. pictorum. As regards the decalcification method used, the matrix comprises two fractions, one acetic acid-soluble and the other, acetic acid-insoluble. The amino acid compositions of both fractions are typical of soluble and insoluble nacre fractions, respectively. The ASM is fairly acidic. This finding corroborates previous data obtained from the ASM extracted from nacre . The amounts of Asx and Glx residues are significantly lower than in calcitic microstructures [25,26]. The AIM exhibits the signature of hydrophobic proteins (enrichment in Gly and Ala), which belong to the ‘silk fibroin-like’ group [27,28]. Interestingly, the amino acid composition of the AIM of U. pictorum resembles that reported by Weiner et al.  on Neotrigonia margaritacea, another paleoheterodont bivalve. In particular, the same decreasing order of major amino acids is observed: Gly, Ala, Ser and Asx. Similar results were obtained with the insoluble nacreous matrices of the edible mussel, Mytilus edulis, of the cephalopod, Nautilus pompilius, and of the freshwater mussel, Anodonta cygnea, this latter being closely related to U. pictorum.
However, the distinction between a soluble and insoluble matrix is purely technical. Recent findings on nacre from pteriomorph bivalves have suggested that the so-called ‘insoluble’ matrix may be secreted as an aqueous gel , in which each aragonitic nacre tablet is crystallized from transient amorphous calcium carbonate , growing centrifugally from a nucleation centre, as previously suggested by Nakahara . This gel also comprises acidic glycoproteins, which may surround the growing nacre tablets or be occluded within them . The complex formed by the gel and the acidic glycoproteins is itself embedded between chitin layers, which display a framework function . We reasonably assume that this model can be extrapolated to paleoheterodont bivalves, such as U. pictorum. Indeed, pteriomorph and paleoheterodont nacres exhibit a similar ‘brick wall’ structure in cross-section [11,35]. Furthermore, they present similarities at the crystallographic level . Finally, our biochemical data corroborate those obtained from pteriomorph matrices. First, even by considering that all Asx and Glx residues are in their acidic form, both nacre ASMs exhibit much lower acidic amino acid compositions (Asx + Glx < 22%) than calcite-associated matrices [8,25–27,29,37]. Second, both exhibit a lower capacity than calcite-associated matrices to inhibit calcium carbonate precipitation in the pH-metric and CaCO3 precipitation interference tests [26,38,39]. Third, as regards the saccharidic composition of the shell matrices of U. pictorum, high amounts of glucosamine are detected in both extracts. With our analytical technique involving trifluoroacetic acid hydrolysis, we cannot distinguish glucosamine from its N-acetylated form, which is the monomer of chitin. Chitin has been detected in the insoluble shell matrices of diverse groups of molluscs, such as pteriomorph bivalves [14,27,40,41], and is proposed to be involved in the 3D structuration of the matrix . In our analyses, glucosamine is two-fold more concentrated in the AIM than in the ASM. This strongly suggests that a large amount of the detected glucosamine originates from the partial hydrolysis of chitin by trifluoroacetic acid.
In the present paper, we have unequivocally demonstrated that the major proteins of the ASM (P95, P50 and P29) are heavily glycosylated. The sugar moiety is partly composed of acidic sugars, in particular sulfated ones, which are responsible for the lowering of the pI of ASM components. Similarly, the nacre of the unionoida, Lamellidans marginalis, also exhibits a weakly acidic amino acid composition, a high amount of polysaccharide and the presence of sulfated groups . Previous studies have reported the occurrence of sulfated sugars in the mollusc matrix [41,42]. More recently, Marxen and Becker  observed that sulfate groups are quantitatively important in the ASM, but depleted in the AIM of the shell of the freshwater gastropod, Biomphalaria glabrata. We also demonstrated that the polysaccharide moiety shows a calcium-binding activity, which is dramatically altered after deglycosylation. There is a striking example among vertebrates where the acidic saccharidic moiety of a calcified tissue-associated glycoprotein exhibits a calcium-binding capacity . Calcium-binding activity due to saccharides is also known in the echinoderm skeletal matrix , and has been suspected among mollusc shell components . Although we did not measure the affinity of the matrix for calcium, we suspect that it is low, a fact in agreement with the function of the matrix, which temporarily sequesters calcium ions and releases them where required . Furthermore, we demonstrated that the saccharidic moiety of the shell glycoproteins modulates the shape of calcite crystals grown in vitro. In particular, the deglycosylation of the ASM leads to the formation of truncated corners without any microsteps. Interestingly, similar patterns were observed with an unglycosylated matrix extract of the pteriomorph mollusc, Atrina rigida, and similar microsteps patterns were observed with glycosylated proteins of different sources [48,49].
One central finding of our study is that the saccharidic moieties of the shell matrix play a key role, although this role is not yet understood. Most of the studies performed on molluscs to date have focused on the matrix proteinaceous components, and only a few reports deal with the characterization of carbohydrates, in particular studies by Simkiss , Crenshaw , Marxen & Becker  and Marxen et al. . The first sequence of an oligosaccharide covalently N-linked to the dermatopontin of the snail Biomphalaria glabrata was obtained only recently . Various functions have been proposed for ASM polysaccharides in CaCO3 mineralization. Acidic sugars, especially sulfated ones, may concentrate calcium ions at the vicinity of the acidic proteins, inducing crystal nucleation [52,53]. This was observed histochemically by Crenshaw and Ristedt , and confirmed recently by Nudelman et al. . Polysaccharides may also play a role in mineral surface recognition  and in polymorph selection . They also may be involved in water or ion entrapment within the hydrogel, and they may modify its viscosity at the nanoscale, as mucins and carrageenans can do [21,56]. Lastly, matrix polysaccharides may display an active role in mediating mantle cell–matrix interaction and cell signalling . Further experiments, including primary structure determination and in situ localization, are essential for improving our understanding of the function of these matrix glycoproteins in biomineralization.