Calcium carbonate (CaCO ₃ ) biomineralizing organisms have played major roles in the history of life and the global carbon cycle during the past 541 Ma. Both marine diversification and mass extinctions reflect physiological responses to environmental changes through time. An integrated understanding of carbonate biomineralization is necessary to illuminate this evolutionary record and to understand how modern organisms will respond to 21st century global change. Biomineralization evolved independently but convergently across phyla, suggesting a unity of mechanism that transcends biological differences. In this review, we combine CaCO ₃ skeleton formation mechanisms with constraints from evolutionary history, omics, and a meta-analysis of isotopic data to develop a plausible model for CaCO ₃ biomineralization applicable to all phyla. The model provides a framework for understanding the environmental sensitivity of marine calcifiers, past mass extinctions, and resilience in 21st century acidifying oceans. Thus, it frames questions about the past, present, and future of CaCO ₃ biomineralizing organisms.

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.

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 formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.

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 ofCaCO₃deposited, 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 ofSMPs isolated form theCaCO₃shell of the limpetLottia gigantea, a gastropod that constructs an aragonitic cross‐lamellar shell. We identified 39SMPs 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 abundantSMPs 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 molluscanSMPset upon which different conchiferan lineages have elaborated to produce the diversity of shell microstructures we observe nowadays.Database: Novel protein sequences reported in this article have been deposited inSwiss‐Prot database under accession nos.B3A0P1–B3A0S4

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.