Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges

Protease inhibitors play critical roles in numerous biological processes including host defense in all multicellular organisms. Eighty three evolutionary families of protease inhibitors are currently accommodated in the MEROPS database and the I84 family currently consists of 3 novel serine protease inhibitors from the eastern oyster Crassostrea virglnica. In this study, we identified 2 new I84 family members from the Chinese razor clam Sinonovacula constricts, scSI-1 and scSI-2, using cDNA cloning and sequencing. The scSI-1 cDNA consisted of 494 bp with a 282 bp ORF encoding a 93-amino acid polypeptide that was predicted to have a 19-amino acid signal peptide and a 74-residue mature protein with a calculated molecular mass of 8248.5 Da. The scSI-2 cDNA was 490 bp long with a 273 bp ORF encoding a 90-amino acid polypeptide that was predicted to have an 18-amino acid signal peptide and a 72-residue nature protein with a calculated molecular mass of 7528.4 Da. ScSI-1 and scSI-2 shared high sequence similarity with the 3 known members of I84 family and both expressed primarily in the clam digestive glands. Protease inhibitory activity in the clam plasma also exhibited the signature kinetic characteristics of the I84 members from the oyster. In addition, levels of scSI-1 and scSI-2 gene expression in digestive glands and the protease inhibitory activity in plasma elevated significantly in clams challenged by bacterial injections and Vibria harveyi was more effective than Staphylococcus epidermitlis in inducing the gene expression and plasma protease inhibitory activity. Moreover, drastic changes of salinity and temperature also caused significant changes in the gene expression and plasma activity. These results indicated that scSI-1 and scSI-2 represented 2 new members of the I84 family and they likely play a role in clam host defense against infections and in reactions against physiochemical stressors.