Molecular cloning, characterization and expression analysis of caspase-6 in puffer fish (Takifugu obscurus)

Abstract

Caspases are a family of proteases involved in many important biological processes, including inflammation, cell differentiation and apoptosis. Among them, caspase-6 plays an important role in the execution phase of the apoptotic death cascade. In this study, the full-length of puffer fish caspase-6 (Pfcasp-6) was cloned and characterized, containing an open reading frame of 912 bp, a 5′-UTR of 168 bp and a 3′-UTR of 632 bp with a poly (A) tail. The Pfcasp-6 encoded a polypeptide of 303 amino acids with an estimated molecular mass of 34.09 kDa and a predicted isoelectric point of 5.35. Phylogenetic analysis indicated that Pfcasp-6 was highly homology to that of other teleostean species. Spatial mRNA expression analysis revealed that the ubiquitous expression of Pfcasp-6 in all examined tissues, with high expression in skin, followed by gill, intestine, kidney, blood, heart and spleen. After the challenges of high temperatures and Aeromonas hydrophila, the transcript expressions of Pfcasp-6 were significantly up-regulated in blood and spleen. In addition, cellular localization analysis indicated that the distribution of Pfcasp-6 was in the cytoplasm and nucleus. Overexpression of Pfcasp-6 was able to trigger a significant level of cell death in HeLa cells, suggesting its putative role in cell apoptosis. Taken together, the results of this study suggested that Pfcasp-6 might be an important component in the caspases cascade, and was likely to get involved in the response to different stress in puffer fish.