College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
College of Fishery, Guangdong Ocean University, Zhanjiang, 524088, China.
Dev Comp Immunol. 2019 Jul;96:37-46. doi: 10.1016/j.dci.2019.02.015. Epub 2019 Feb 27.
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.
半胱氨酸蛋白酶抑制剂 C 是一种内源性半胱氨酸蛋白酶抑制剂，广泛存在于生物体内。哺乳动物的几项研究表明，Cystatin C 在免疫防御微生物方面发挥着关键作用。众所周知，一些鱼类 Cystatin C 在炎症反应中具有重要的免疫调节功能。然而，鱼类 Cystatin C 在病毒感染中的功能及其潜在的分子机制仍有待阐明。本研究从卵形鲷（Epinephelus coioides）中鉴定出一种 Cystatin C 基因，命名为 Ec-CysC。Ec-CysC cDNA 全长 817bp，开放阅读框（ORF）长 387bp，编码 129 个氨基酸（aa）的蛋白质，包括 18 个 aa 的信号肽和 111 个 aa 的成熟多肽。Ec-CysC 的推导氨基酸在 N 端区域含有 Glycine，中间有 QVVAG 基序，C 端附近有 PW 基序，这三个保守结构域在鱼类 Cystatin C 中共享。Ec-CysC 基因的转录分析表明，它在包括肝脏、脾脏、肾脏、大脑、肠、心脏、皮肤、肌肉、鳍、胃、鳃和头肾在内的 12 种检查组织中均有表达。进一步在脾脏中检测 Ec-CysC 对新加坡石斑鱼虹彩病毒（SGIV）刺激的反应，感染后 12h 时 Ec-CysC 的相对表达显著上调。亚细胞定位实验表明 Ec-CysC 在卵形鲷脾脏（GS）细胞中主要分布在细胞质中。在体外，GS 细胞中 Ec-CysC 的过表达显著降低了病毒基因 ORF162、ORF049 和 ORF072 的表达。同时，我们发现 SGIV 感染时过表达 Ec-CysC 导致促炎细胞因子 TNF-α、IL-1β 和 IL8 的表达呈上升趋势。此外，过表达 Ec-CysC 也削弱了脂鲦（FHM）细胞中 SGIV 诱导的细胞凋亡和 Caspase-3 活性。这些结果表明 Ec-CysC 可能更深地参与了鱼类的免疫防御，并在 SGIV 诱导的炎症和凋亡中发挥了重要作用。
