Molecular characterization, antiviral activity, and UV-B damage responses of Caspase-9 from Amphiprion clarkii.

Apoptosis plays a vital role in maintaining cellular homeostasis in multicellular organisms. Caspase-9 (casp-9) is one of the major initiator caspases that induces apoptosis by activating downstream intrinsic apoptosis pathway genes. Here, we isolated the cDNA sequence (1992 bp) of caspase-9 from Amphiprion clarkii (Accasp-9) that consists of a 1305 bp coding region and encodes a 434 aa protein. In silico analysis showed that Accasp-9 has a theoretical isoelectric point of 5.81 and a molecular weight of 48.45 kDa. Multiple sequence alignment revealed that the CARD domain is located at the N-terminus, whereas the large P-20 and small P-10 domains are located at the C-terminus. Moreover, a highly conserved pentapeptide active site ( ²⁹⁶ QACGG ³⁰¹ ), as well as histidine and cysteine active sites, are also retained at the C-terminus. In phylogenetic analysis, Accasp-9 formed a clade with casp-9 from different species, distinct from other caspases. Accasp-9 was highly expressed in the gill and intestine compared with other tissues analyzed in healthy A. clarkii. Accasp-9 expression was significantly elevated in the blood after stimulation with Vibrio harveyi and polyinosinic:polycytidylic acid (poly I:C; 12-48 h), but not with lipopolysaccharide. The nucleoprotein expression of the viral hemorrhagic septicemia virus was significantly reduced in Accasp-9 overexpressed fathead minnow (FHM) cells compared with that in the control. In addition, other in vitro assays revealed that cell apoptosis was significantly elevated in poly I:C and UV-B-treated Accasp-9 transfected FHM cells. However, H ^248P or C ^298S mutated Accasp-9 significantly reduced apoptosis in UV-B irradiated cells. Collectively, our results show that Accasp-9 might play a defensive role against invading pathogens and UV-B radiation and H ²⁴⁸ and C ²⁹⁸ active residues are significantly involved in apoptosis in teleosts.
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