Conserved function of Pacific cod Caspase-3 in apoptosis

Apoptosis plays a significant role in the cellular immune responses against infections, especially those related to viruses. Across various species, Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. However, its role remains relatively unknown in cod fish. In this study, we aimed to reveal the role of Pacific cod Caspase-3 (GmCasp3) in apoptosis and its evolutionary position. Our results showed that the GmCasp3 cDNA contains an open reading frame of 864 nucleotides; that codes for 287 amino acids long protein with a molecular weight of 32.03 kDa. The sequence alignments and 3-D model indicated that GmCasp3 contained highly conserved domains, such as "QACRG", "GSWFI" and "HG" active sites, however, the phylogenetic tree analysis revealed that both GmCasp3 and Atlantic cod caspase-3 clustered together are far from the high vertebrate branch, indicating they are at a lower position in vertebrate evolution. Red fluorescent labeling vector pDsRed2-C1-GmCasp3 was constructed and it was transfected into EPC cell lines. The result showed that GmCasp3 protein was distribute in the protoplasm and expressed in apoptotic cell debris. Moreover, the GmCasp3 enzyme activity increased with the increased post-transfection analysis time, while the genome DNA was visibly fragmented at 36 h post transfection. Flow cytometry analysis showed that the proportion of apoptosis cells increased from 12 h to 24 h post transfection. In conclusion, the conserved functions of GmCasp3 in apoptosis indicated that Pacific cod has the similar apoptotic characteristics as other animals.