Department of Bioscience, School of Life Science, Nanchang University, Nanchang, 330031, China.
Department of Bioscience, School of Life Science, Nanchang University, Nanchang, 330031, China.
Dev Comp Immunol. 2020 Nov;112:103781. doi: 10.1016/j.dci.2020.103781. Epub 2020 Jul 6.
GPATCH3, a protein with G-patch domain, is known to participate in innate immune response and organ development in mammals. However, there are few reports on GPATCH3 in fish. Here the cDNA sequence of GPATCH3 was cloned from Ctenopharyngodon idella (CiGPATCH3, MN149902) and was determined its character. A cDNA sequence of CiGPATCH3 is 1646 bp and contains an ORF of 1221 bp translating a protein of 407 amino acids. Phylogenetic analysis uncovered that CiGPATCH3 possesses a relatively high degree of homology with Cyprinus carpio GPATCH3. The mRNA level of CiGPATCH3 was increased following the intracellular stimulation of poly (I:C) into CIK cells. In vivo, over-expression of CiGPATCH3 can significantly up-regulate IFN 1 and ISG15 expression at mRNA and protein levels. To investigate the molecular mechanism by which GPATCH3 initiates the innate immune response in fish, co-IP experiments were performed to analyze the substrates of CiGPATCH3. The results showed that CiGPATCH3 directly interacted with CiSTING, but not with CiIRF3, CiIRF7, CiTBK1 or CiIPS-1. As compared with the single transfection of CO cells with either CiGPATCH3 or CiSTING, the expression of IFN 1 was more significantly up-regulated in cells under treatment with dual transfection of CiGPATCH3 and CiSTING. Knockdown of CiGPATCH3 inhibited STING-mediated IFN 1 expression in fish cells. Over-expression of CiGPATCH3 and CiSTING facilitated the phosphorylation and cytoplasmic-to-nuclear translocation of CiIRF7. These results explicitly showed that CiGPATCH3 up-regulates IFN 1 and ISG15 expression via the activation of STING-IRF7 signal axis in vivo.
GPATCH3 是一种具有 G-补丁结构域的蛋白质，已知其参与哺乳动物的先天免疫反应和器官发育。然而，鱼类中关于 GPATCH3 的报道很少。本研究从草鱼（CiGPATCH3，MN149902）中克隆了 GPATCH3 的 cDNA 序列，并确定了其特征。CiGPATCH3 的 cDNA 序列长 1646bp，含有一个 1221bp 的开放阅读框，编码一个 407 个氨基酸的蛋白质。系统进化分析表明，CiGPATCH3 与鲤鱼 GPATCH3 具有较高的同源性。在 CIK 细胞中，CiGPATCH3 的 mRNA 水平在 poly（I:C）的细胞内刺激下增加。在体内，过表达 CiGPATCH3 可以显著上调 IFN 1 和 ISG15 的表达水平。为了研究 GPATCH3 在鱼类中启动先天免疫反应的分子机制，进行了 co-IP 实验以分析 CiGPATCH3 的底物。结果表明，CiGPATCH3 直接与 CiSTING 相互作用，但不与 CiIRF3、CiIRF7、CiTBK1 或 CiIPS-1 相互作用。与单独转染 CO 细胞的 CiGPATCH3 或 CiSTING 相比，双重转染 CiGPATCH3 和 CiSTING 的细胞中 IFN 1 的表达更为显著上调。在鱼类细胞中敲低 CiGPATCH3 抑制了 STING 介导的 IFN 1 表达。过表达 CiGPATCH3 和 CiSTING 促进了 CiIRF7 的磷酸化和细胞质到核内的易位。这些结果明确表明，CiGPATCH3 通过激活体内 STING-IRF7 信号轴来上调 IFN 1 和 ISG15 的表达。
