The ‘Shellome’ of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization

Abstract

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules – in particular proteins – that are essential for molluscan shell formation.

Variation of Gene Expression in the Endemic Dinaric Karst Cave-Dwelling Bivalve Mollusk Congeria kusceri during the Summer Season

“…Several protease inhibitors belonging to five distinct families, i.e., kazal-type, kunitztype, tissue inhibitor of metalloprotease (TIMP), whey acidic protein (WAP), and alpha-2macroglobulins (A2M), were upregulated at T1. As previously noted by other authors [79], protease inhibitors are a recurrent theme in shell biomineralization proteomes, where they are believed to prevent the premature degradation of extracellular matrix proteins by the numerous proteolytic enzymes present in the extracellular space. Among these five protease inhibitor families, the most represented one among the DEGs detected at T1, with 11 out of 12 upregulated members, was undoubtedly A2M.…”

Expression of Clementine Asp-Rich Proteins (CcASP-RICH) in Tobacco Plants Interferes with the Mechanism of Pollen Tube Growth

“…The encoded proteins are acidic because aspartic amino acids predominate (i.e., ASP-RICH). A putative role of acidic proteins has been suggested in clams where they are key components of the calcification process, possibly being involved in nucleation, inhibition, and orientation of crystal growth [ 18 ]. This is because Asp-/Glu or poly-Asp/poly-Glu-rich domains can bind high amounts of Ca 2+ [ 18 ].…”

“…A putative role of acidic proteins has been suggested in clams where they are key components of the calcification process, possibly being involved in nucleation, inhibition, and orientation of crystal growth [ 18 ]. This is because Asp-/Glu or poly-Asp/poly-Glu-rich domains can bind high amounts of Ca 2+ [ 18 ]. The finding that CcASP-RICH can bind calcium ions is partly supported by biochemical evidence previously obtained using the Stains-All dye [ 17 ], which showed that CcASP-RICH can exhibit calmodulin-like behavior and thus bind calcium ions.…”

Multi-omic insights into the formation and evolution of a novel shell microstructure in oysters

“…Major proteins and the best matches of minor proteins were identified as SMPs of C. nippona in this study. Furthermore, SMPs of the other six molluscs including C. gigas [ 30 ], Atrina pectinata [ 15 ], Tridacna crocea [ 121 ], P. fucata [ 30 ], Lottia gigantea [ 122 ], and N. pompilius [ 49 ] were download and used for comparative analysis of shell proteomes. Functional domain annotations of SMPs were performed by searching against various databases, including SMART, CDD, Pfam, PROSITE patterns, PROSITE profiles, and SUPERFAMILY, using InterProScan (v 5.52–86.0) [ 101 ].…”