Functional characterization of Cystatin C in orange-spotted grouper, Epinephelus coioides

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.