In corals, biocalcification is a major function that may be drastically affected by ocean acidification (OA). Scleractinian corals grow by building up aragonitic exoskeletons that provide support and protection for soft tissues. Although this process has been extensively studied, the molecular basis of biocalcification is poorly understood. Notably lacking is a comprehensive catalog of the skeleton-occluded proteins—the skeletal organic matrix proteins (SOMPs) that are thought to regulate the mineral deposition. Using a combination of proteomics and transcriptomics, we report the first survey of such proteins in the staghorn coral Acropora millepora. The organic matrix (OM) extracted from the coral skeleton was analyzed by mass spectrometry and bioinformatics, enabling the identification of 36 SOMPs. These results provide novel insights into the molecular basis of coral calcification and the macroevolution of metazoan calcifying systems, whereas establishing a platform for studying the impact of OA at molecular level. Besides secreted proteins, extracellular regions of transmembrane proteins are also present, suggesting a close control of aragonite deposition by the calicoblastic epithelium. In addition to the expected SOMPs (Asp/Glu-rich, galaxins), the skeletal repertoire included several proteins containing known extracellular matrix domains. From an evolutionary perspective, the number of coral-specific proteins is low, many SOMPs having counterparts in the noncalcifying cnidarians. Extending the comparison with the skeletal OM proteomes of other metazoans allowed the identification of a pool of functional domains shared between phyla. These data suggest that co-option and domain shuffling may be general mechanisms by which the trait of calcification has evolved.
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.
We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.The secretion of a shell by molluscs is a striking example of a selfassembling process performed outside living tissues. When a mollusc builds its shell, the calcifying epithelium of its mantle extrudes mineral ions, mainly calcium and bicarbonate. In addition, it secretes an extracellular matrix composed of proteins, glycoproteins, proteoglycans, and polysaccharides (1). This mixture is released into the extrapallial space, a microvolume delimited by the epithelium, the growing shell, and the leathery periostracum (2). In this supersaturated space, the released macromolecules interact with bicarbonate and calcium to form biocrystallites that self-aggregate in an orderly manner. The end product is a densely packed organomineral assembly, the shell, in which the mineral phase represents more than 95% by weight (3). As shell proteins exert a control on the biomineralization process, they can be used in the synthesis of biomimetic materials (4 -6). However, the resolution of the primary structure of molluscan shell proteins has been seriously impaired for decades by their polydispersity, their polyanionic properties, and finally their post-translational modifications (3, 7).Despite these obstacles, investigations on molluscan shell proteins have made significant advances in the last 6 years, with the characterization of 16 proteins and, for most of them, the identification of the corresponding transcript (8, 9). Among the different shell textures that molluscs use to produce their shell, mother-of-pearl has received the greatest deal of attention. Its intrinsic bea...
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.
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.
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.
The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.
The scleractinian coral Acropora millepora is one of the most studied species from the Great Barrier Reef. This species has been used to understand evolutionary, immune and developmental processes in cnidarians. It has also been subject of several ecological studies in order to elucidate reef responses to environmental changes such as temperature rise and ocean acidification (OA). In these contexts, several nucleic acid resources were made available. When combined to a recent proteomic analysis of the coral skeletal organic matrix (SOM), they enabled the identification of several skeletal matrix proteins, making A. millepora into an emerging model for biomineralization studies. Here we describe the skeletal microstructure of A. millepora skeleton, together with a functional and biochemical characterization of its occluded SOM that focuses on the protein and saccharidic moieties. The skeletal matrix proteins show a large range of isoelectric points, compositional patterns and signatures. Besides secreted proteins, there are a significant number of proteins with membrane attachment sites such as transmembrane domains and GPI anchors as well as proteins with integrin binding sites. These features show that the skeletal proteins must have strong adhesion properties in order to function in the calcifying space. Moreover this data suggest a molecular connection between the calcifying epithelium and the skeletal tissue during biocalcification. In terms of sugar moieties, the enrichment of the SOM in arabinose is striking, and the monosaccharide composition exhibits the same signature as that of mucus of acroporid corals. Finally, we observe that the interaction of the acetic acid soluble SOM on the morphology of in vitro grown CaCO3 crystals is very pronounced when compared with the calcifying matrices of some mollusks. In light of these results, we wish to commend Acropora millepora as a model for biocalcification studies in scleractinians, from molecular and structural viewpoints.
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