College of Marine Sciences, South China Agricultural University, Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
College of Marine Sciences, South China Agricultural University, Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), 528478, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266000, China.
Dev Comp Immunol. 2022 Nov;136:104502. doi: 10.1016/j.dci.2022.104502. Epub 2022 Aug 5.
Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.
半胱氨酸蛋白酶抑制剂 A（CyA）是一种广泛研究的免疫防御和癌症治疗的抑制剂。然而，CyA 的功能及其在鱼类病毒感染中的潜在分子机制尚不清楚。在我们的研究中，我们从橙色斑点石斑鱼（Ec-CyA）中克隆了 CyA 同源物的开放阅读框（ORF），该 ORF 由 303 个核苷酸组成，编码一个 101 个氨基酸的蛋白质。Ec-CyA 包含两个保守序列，包含一个 N 端甘氨酸片段和一个 QXVXG 序列（48aa-52aa），没有信号肽。组织分布分析表明，Ec-CyA 在脾脏和头肾中高度表达。此外，进一步的分析表明，Ec-CyA 在石斑鱼脾脏（GS）细胞中模拟 SGIV 时表达增加。亚细胞定位分析表明，Ec-CyA 主要分布在 GS 细胞的细胞质中。过表达 Ec-CyA 促进了病毒基因 MCP、VP19 和 LITAF 的 mRNA 水平。同时，促进了脂肪头鲦（FHM）细胞中 SGIV 诱导的细胞凋亡，以及 caspase-3/7、caspase-9 的激活。此外，Ec-CyA 过表达下调了干扰素（IFN）相关分子的表达，包括 ISG15、IFN、IRF3、MAVS、MyD88、TRAF6，并上调了促炎因子，如 IL-1β、IL-8 和 TNF-α。同时，Ec-CyA 过表达抑制了 IFN 和 ISRE 启动子的活性，但通过荧光素酶报告基因检测诱导了 NF-κB 启动子的活性。总之，我们的研究结果表明，Ec-CyA 参与了先天免疫反应，并在 DNA 病毒感染中发挥了关键作用。
