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Brachiopods are a phylum of marine invertebrates that have an external bivalved shell to protect their living tissues. With few exceptions, this biomineralized structure is composed of calcite, mixed together with a minor organic fraction, comprising secreted proteins that become occluded in the shell structure, once formed. This organic matrix is thought to display several functions, in particular, to control mineral deposition and to regulate crystallite shapes. Thus, identifying the primary structure of matrix proteins is a prerequisite for generating bioinspired materials with tailored properties. In this study, we employed a proteomic approach to identify numerous peptides that constitute the shell proteins, in three rhynchonellid brachiopods from different localities. Our results suggest that the shell protein repertoires identified thus far, differ from that of better known calcifying metazoans, such as molluscs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.)

Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks., (© 2012 WILEY PERIODICALS, INC.)

In mollusks, the shell mineralization process is controlled by an array of proteins, glycoproteins and polysaccharides that collectively constitute the shell matrix. In spite of numerous researches, the shell protein content of a limited number of model species has been investigated. This paper presents biochemical data on the common edible land snail Helix aspersa maxima, a model organism for ecotoxicological purposes, which has however been poorly investigated from a biomineralization viewpoint. The shell matrix of this species was extracted and analyzed biochemically for functional in vitro inhibition assay, for amino acid and monosaccharides compositions. The matrix was further analyzed on 1 and 2D gels and short partial protein sequences were obtained from 2D gel spots. Serological comparisons were established with a set of heterologous antibodies, two of which were subsequently used for subsequent immunogold localization of matrix components. Our data suggest that the shell matrix of Helix aspersa maxima may differ widely from the shell secretory repertoire of the marine mollusks studied so far, such as the gastropod Haliotis or the pearl oyster Pinctada. In particular, most of the biochemical properties generally attributed to soluble shell matrices, such as calcium-binding capability, or the capacity to interfere in vitro with the precipitation of calcium carbonate or to inhibit the precipitation of calcium carbonate, were not recorded with this matrix. This drastic change in the biochemical properties of the landsnail shell matrix puts into question the existence of a unique molecular model for molluscan shell formation, and may be related to terrestrialisation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The shell of the Pacific oyster Crassostrea gigas is composed of more than 99% CaCO₃ and of around 0.5% of occluded organic matrix. According to classical views, this matrix is supposed to regulate the shell mineral deposition. In this study, we developed one of the first proteomic approaches applied to mollusk shell in order to characterise the calcifying matrix proteins. The insoluble organic matrix, purified after demineralisation of the shell powder, was digested with trypsin enzyme, and separated on nano-LC, prior to nanospray quadrupole/time-of-flight analysis. MS/MS spectra were searched against the above 220,000 EST sequences available in the public database for Crassostrea. Using this approach, we were able to identify partial or full-length sequence transcripts that encode eight novel shell matrix proteins.

The shell of the Manila clam Venerupis philippinarum is composed of more than 99% calcium carbonate and of a small amount of organic matrix (around 0.2%). In this study, we developed one of the first proteomic approaches applied to mollusc shell in order to characterise the matrix proteins that are believed to be essential for the formation of the biomineral. The insoluble organic matrix, purified after demineralisation of the shell powder with cold acetic acid (5%), was digested with trypsin enzyme and then separated on nano-LC prior to nanospray/quadrupole time-of-flight analysis. MS/MS spectra were searched against the above 11,000 EST sequences available on the NCBI public database for Venerupis. Using this approach, we were able to identify partial or full-length sequence transcripts that encode for shell matrix proteins. These include three novel shell proteins whose sequences do not present any homologous proteins or already described domains, two putative protease inhibitor proteins containing Kazal-type domains, and a putative Ca(2+)-binding protein containing two EF-hand domains. Biomineral formation and evolutionary implications are discussed.