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Calcified structures of sea urchins are biocomposite materials that comprise a minor fraction of organic macromolecules, such as proteins, glycoproteins and polysaccharides. These macromolecules are thought to collectively regulate mineral deposition during the process of calcification. When occluded, they modify the properties of the mineral. In the present study, the organic matrices (both soluble and insoluble in acetic acid) of spines and tests from the Mediterranean black sea urchin Arbacia lixula were extracted and characterized, in order to determine whether they exhibit similar biochemical signatures. Bulk characterizations were performed by mono-dimensional SDS/PAGE, FT-IR spectroscopy, and an in vitro crystallization assay. We concentrated our efforts on characterization of the sugar moieties. To this end, we determined the monosaccharide content of the soluble and insoluble organic matrices of A. lixula spines and tests by HPAE-PAD, together with their respective lectin-binding profiles via enzyme-linked lectin assay. Finally, we performed in situ localization of N-acetyl glucosamine-containing saccharides on spines and tests using gold-conjugated wheatgerm agglutinin. Our data show that the test and spine matrices exhibit different biochemical signatures with regard to their saccharidic fraction, suggesting that future studies should analyse the regulation of mineral deposition by the matrix in these two mineralized structures in detail. This study re-emphasizes the importance of non-protein moieties, i.e. sugars, in calcium carbonate systems, and highlights the need to clearly identify their function in the biomineralization process., (© 2015 FEBS.)

Proteins that are occluded within the molluscan shell, the so-called shell matrix proteins (SMPs), are an assemblage of biomolecules attractive to study for several reasons. They increase the fracture resistance of the shell by several orders of magnitude, determine the polymorph of CaCO(3) deposited, and regulate crystal nucleation, growth initiation and termination. In addition, they are thought to control the shell microstructures. Understanding how these proteins have evolved is also likely to provide deep insight into events that supported the diversification and expansion of metazoan life during the Cambrian radiation 543 million years ago. Here, we present an analysis of SMPs isolated form the CaCO(3) shell of the limpet Lottia gigantea, a gastropod that constructs an aragonitic cross-lamellar shell. We identified 39 SMPs by combining proteomic analysis with genomic and transcriptomic database interrogations. Among these proteins are various low-complexity domain-containing proteins, enzymes such as peroxidases, carbonic anhydrases and chitinases, acidic calcium-binding proteins and protease inhibitors. This list is likely to contain the most abundant SMPs of the shell matrix. It reveals the presence of both highly conserved and lineage-specific biomineralizing proteins. This mosaic evolutionary pattern suggests that there may be an ancestral molluscan SMP set upon which different conchiferan lineages have elaborated to produce the diversity of shell microstructures we observe nowadays., (© 2012 The Authors Journal compilation © 2012 FEBS.)

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.