Genome-wide screening of potential shell matrix proteins with low complexity regions in molluscs

Molluscan shells are complex biominerals with remarkable mechanical properties and are formed through the precise regulation of organic matrices, which are largely composed of proteins. Over the past decade, high-throughput proteomic assays have yielded the identification of a large number of shell matrix proteins (SMPs). However, many SMPs have tandem repeats or low complexity regions (LCRs) in the amino acid sequence, which hinder their identification by traditional proteomics. To overcome this bias and uncover this "dark matter" of the shell proteome, we developed a complementary, genome-wide screening strategy to identify secreted proteins enriched in specific amino acids (e.g., Gly, Asp, Tyr). Our screen successfully identified tens of proteins with LCRs in each group, including two major families: shematrin-like proteins and extremely acidic proteins. Shematrin-like proteins, characterized by glycine-tyrosine repeats and a basic tail, were prevalent in bivalves and gastropods with calcified shells but exhibited low sequence homology, suggesting rapid evolution or convergent evolution. Extremely acidic proteins (pI <4.5) were also widely identified in various molluscan classes. Expression analysis showed high gene expression levels of both protein families during larval shell development and/or in biomineralizing tissues. Based on their complementary basic tail of shematrins and the negatively charged acidic proteins, we propose that these proteins interact electrostatically to mediate shell matrix assembly, facilitating the crystallization of amorphous calcium carbonate on chitin scaffolds. This study provides a critical genomic supplement to shell proteomics and reveals novel widespread classes of SMPs, thus advancing our understanding of molluscan biomineralization.