The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida)

Abstract

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 [1]. 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 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 45 Ca 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 CaCO 3 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.Abbreviations AIM, acid-insoluble matrix; ASM, acid-soluble matrix; BCA, bicinchoninic acid; CBB, Coomassie Brilliant Blue; DMB, 1,9-dimethylmethylene blue; IEF, isoelectric focusing; IPG, immobilized pH gradient; PAS, periodic acid ⁄ Schiff; TFMS, trifluoromethanesulfonic acid.

Evolution of Nacre: Biochemistry and Proteomics of the Shell Organic Matrix of the Cephalopod Nautilus macromphalus

“…This is in agreement with A C H T U N G T R E N N U N G previous works on organic compounds in other mollusk shells. [29,32,[36][37][38] Carboxylate (COO À ) and sulfate (SO 4 2À ) absorption bands are also present in both extracts, at around 1420 cm À1 and 1234 cm À1 , respectively. [38] On the other hand, ASM exhibits a strong carbohydrate absorption band around 1060 cm À1 , whereas for AIM this band is weaker.…”

“…This result is consistent with those previously obtained with other Nautilus species, [31] because the AIM exhibits the signature of hydrophobic extracts, which characterizes the "silk fibroin-like" proteins [10,27] found in the nacre of various mollusks. [37,39,40] The composition of the ASM is characterized by large amounts of Gly and Asx, which represent more than two-fifths of the total amino acids. If most of the Asx residues are in their acidic form, as previously suggested, [28] this implies that the ASM of Nautilus macromphalus nacre is acidic, an assumption that is further confirmed on 2D gels.…”

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

“…[28] Peaks characteristic of sugar moieties are observed in the 1200-900 cm À1 range and at 1105, 1060, 1001, and 965 cm À1 , and can be attributed in particular to CÀCÀO and CÀOÀC stretchings. [7,29] Furthermore, the weak absorption at 1243 cm À1 might reflect the presence of sulfated groups. [30] The monosaccharide composition of Upsalin was determined after hydrolysis and is shown in Figure 8 B.…”

“…Upsalin has a predicted signal peptide with a cleavage site between the positions 16 and 17 and a putative transmembrane region (positions [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. The mature form of the protein consists of 109 aa residues and has a theoretical molecular weight of 12.3 kDa.…”

“…This process results in a stable and well-packed organomineral assembly, [2] in which proteins were recognized early as key components in shell formation [3] and in modulation of calcium carbonate crystal shapes. [4] Consequently, great effort has been put into their identification and the characterization of their primary structure, [1,[5][6][7] in order to elucidate the mechanisms by which proteins interact in shell formation, [8] and to use these proteins to generate tailored composite biomaterials. [9] Classically, shell proteins are retrieved by dissolving the mineral phase of the shell with weak acid.…”