Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
PLoS One. 2012;7(9):e44884. doi: 10.1371/journal.pone.0044884. Epub 2012 Sep 18.
ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation.
ISG15 是一种泛素样蛋白，可被干扰素迅速诱导。与泛素一样，ISG15 以一种称为 ISGylation 的过程与靶蛋白形成共价缀合物，在哺乳动物中，该过程被认为在抗病毒免疫中发挥作用。与哺乳动物的 ISG15 不同，鱼类 ISG15 的功能尚不清楚。在这项研究中，我们鉴定并分析了舌鳎（Cynoglossus semilaevis）ISG15 同源物 CsISG15 的功能。CsISG15 由 162 个残基组成，具有两个串联的泛素样结构域和所有已知 ISG15 中都存在的高度保守的 LRGG 基序。CsISG15 在广泛的组织中表达，并在肾脏和脾脏中被病毒和细菌感染上调。用原头肾（HK）淋巴细胞进行的体外研究表明，巨型病毒感染导致 CsISG15 表达的诱导和 CsISG15 蛋白的细胞外释放。纯化的重组 CsISG15（rCsISG15）激活 HK 巨噬细胞并增强 HK 淋巴细胞中免疫基因的表达，但当保守的 LRGG 序列突变为 LAAG 时，这两种作用均显著降低。进一步的研究表明，rCsISG15 在 HK 淋巴细胞感染巨型病毒期间的存在降低了细胞内病毒载量，而 CsISG15 的抗体阻断增强了病毒感染。同样，通过 RNAi 干扰 CsISG15 的表达促进了病毒感染。总之，这些结果表明，作为一种鱼类 ISG15，CsISG15 促进抗病毒免疫反应，并且与哺乳动物的 ISG15 不同，CsISG15 以未缀合的细胞外细胞因子的形式发挥其免疫调节作用。此外，这些结果还表明 LRGG 基序除了在蛋白质缀合中的作用外还有其他作用。
