Molecular cloning and functional characterization of caspase 9 in large yellow croaker (Pseudosciaena crocea)

The genomic and cDNA sequences of a caspase 9 homologue were cloned from large yellow croaker (Pseudosciaena crocea). The large yellow croaker caspase 9 gene (Lyccasp9) consists of 10 exons and 9 introns, spanning 3836 nucleotides. The full-length cDNA of Lyccasp9 is 2595bp with an open reading frame of 1314bp encoding a polypeptide of 437 amino acids (aa), which includes a 90-aa caspase recruitment domain (CARD, residues 1-90), a 133-aa p20 domain (residues 171-303) with two putative caspase family histidine and cysteine active sites, as well as an 87-aa p10 domain (residues 348-435). Recombinant Lyccasp9 (rLyccasp9) demonstrated obvious proteolytic activity. However, when histidine(249) in the histidine active site was replaced by aspartic acid (D), or cysteine(299) in the cysteine active site was replaced by glycine (G), the mutated rLyccasp9 (rLyccasp9-Mut-His(249)-D or rLyccasp9-Mut-Cys(299)-G) displayed significantly decreased proteolytic activity. Moreover, the proteolytic activity of rLyccasp9-Mut-Cys(299)-G was lower than that of rLyccasp9-Mut-His(249)-D, indicating that both the histidine and cysteine active sites are essential in maintaining the Lyccasp9 proteolytic activity and that the latter is more important. The Lyccasp9 was constitutively expressed in all analyzed tissues, although expression levels varied from tissue to tissue. Real-time PCR analysis revealed that Lyccasp9 transcript in spleen and kidney was quickly increased and then slowly decreased after stimulation with either poly(I:C), a viral mimic, or inactivated trivalent bacterial vaccine. On the other hand, enzyme activities of caspase 9 were also increased in these two tissues post-stimulation, suggesting that the activation of the intrinsic apoptotic pathway may be involved in the immune response induced by poly(I:C) or bacterial vaccine in large yellow croaker.