Identification of cvSI-3 and evidence for the wide distribution and active evolution of the I84 family of protease inhibitors in mollusks

Protease inhibitors are an extremely diverse group of proteins that control the proteolytic activities of proteases and play a crucial role in biological processes including host defenses. The I84 family of protease inhibitors in the MEROPS database currently consists of cvSI-1 and cvSI-2, two novel serine protease inhibitors purified and characterized from the eastern oyster Crassostrea virginica plasma and believed to play a role in host defense and disease resistance. In the present study, a third member of I84 family, named cvSI-3, was identified from C. virginica by cDNA cloning and sequencing. The full cvSI-3 cDNA was composed of 342 bp including a 255 bp open reading frame (ORF) that encodes an 84-amino acid peptide. The mature cvSI-3 molecule was predicted to have 68 amino acid residues after removal of a 16-amino acid signal peptide, with a calculated molecular mass of 7724.5 Da and a theoretical isoelectric point (pI) of 6.28. CvSI-3 amino acid sequence shared 41% identity with cvSI-2 and 37% identity with cvSI-1, which included 12 conserved cysteines. Quantitative real-time PCR determined that cvSI-3 gene expressed primarily in oyster digestive glands. Real-time PCR also detected that cvSI-1, cvSI-2 and cvSI-3 expression levels in digestive glands varied significantly, with cvSI-2 showing the highest expression level and cvSI-3 the lowest. Additionally, a significant correlation was detected between cvSI-2 and cvSI-3 mRNAs levels. Searches into sequence databases using cvSI-1, cvSI-2 and cvSI-3 as queries retrieved ESTs suggesting the possible existence of at least 9 more I84 family members in eastern oysters and of I84 family protease inhibitors in various bivalve and gastropod species. Moreover, orthologs of all C. virginica I84 family members or potential member genes were found to be present in the C. gigas genome, and their distributions among species provided important information about the evolution of the I84 family of protease inhibitors. It appears that the I84 family of protease inhibitors is widely distributed and actively evolving in the Phylum Mollusca.