Characterization and expression of a novel caspase gene: Evidence of the expansion of caspases in Crassostrea gigas.

Caspases are a group of cysteine-aspartate proteases involved in apoptosis and a variety of non-apoptotic processes. In this study, a novel caspase gene was cloned and its potential role in apoptosis was investigated. The caspase gene (CgCasp 3/7) has an open reading frame of 1626bp encoding 541 amino acids containing the conserved functional domains and motifs of effector caspases. Its amino acid sequence shows low identity with the other effector caspases of Crassostrea gigas and contains a unique long intersubunit linker (IL). The CgCasp 3/7 mRNA was expressed highly in oocytes and then decreased gradually after fertilization, indicating CgCasp 3/7 could function in oocyte apoptosis. In adult tissues, it is located primarily in the gills and hepatopancreas. We examined the mRNA expression of CgCasp 3/7 in gills of oysters immersed in ambient (17°C) or heated (27°C) seawater. The thermal stress stimulated mRNA expression of CgCasp 3/7 by 2.5- and 4.1-fold at 2h and 6h post-treatment, respectively, indicating CgCasp3/7 was involved in the early response to thermal stress. To examine the function of the IL, CgCasp 3/7 and CgCasp 3/7-T (with a truncated IL) were expressed using an in vitro translation system and their DEVDase activity was measured. Both proteins showed a significantly higher level of DEVDase activity than control, but CgCasp3/7-T had lower DEVDase activity than CgCasp3/7, indicating CgCasp3/7 had DEVDase activity and the IL was required for maximal DEVDase activity. Our study adds to the complexity of caspases in C. gigas.