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.
Highlights
The shell of mollusks represents a major biological innovation that largely contributed to the great evolutionary and ecological success of the phylum throughout Phanerozoic times
With Alcian blue staining at low pH (1.0), which detects sulfated polysaccharides and glycosaminoglycans, significant discrete signals were visible at 25 and 50 kDa in acid-soluble matrix (ASM) and at 70 kDa in acid-insoluble matrix (AIM) (Figure 1C), in spite of the intense smearing staining in all extracts
We suggest that the combination of chitinbinding domain(s) (ChBD) and T-rich low-complexity regions (LCRs) in some shell matrix proteins (SMPs) sequences may be an essential requisite to form a molecular bridge between chitin and calcium carbonate
Summary
The shell of mollusks represents a major biological innovation that largely contributed to the great evolutionary and ecological success of the phylum throughout Phanerozoic times. Beside complex genetic equipment involved in development, the formation of mollusk shell is a highly regulated process that requires a large set of macromolecules, i.e., proteins, polysaccharides and lipids, secreted from the dorsal mantle tissue, and which self-assemble into an organic matrix, the framework for shell mineralization These macromolecules – in particular shell matrix proteins, defined here as SMPs - play crucial role in nucleation and growth of calcium carbonate crystal. To render the situation more complex, it has been shown that larval shell SMP repertoires are almost entirely different from that of adult shells (Zhao et al, 2018) Beside this diversity, some common characteristics can be identified: they include the presence of shared functional domains and the abundance of low complexity regions, referred to as LCRs (Kocot et al, 2016; Marie et al, 2017). In order to draw a general view of biomineralization process and its evolutionary origin, it is essential to compile a “dictionary” of SMPs that is identified from diverse range of taxa
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