Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agricultural University, Changsha 410128, China.
Int J Mol Sci. 2021 Nov 6;22(21):12011. doi: 10.3390/ijms222112011.
Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, Df, from grass carp () and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of Df was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. Df harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that Df shares the highest identity (96%) and similarity (98%) with Df from . Phylogenetic analysis indicated that Df and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the Df gene structure is characterized by four introns and five exons. The incubation of recombinant Df protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of Df in the AP in promoting C3 cleavage, similar to Dfs in mammals. Df mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of Df mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of Df and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp .
补体因子 D（Df）是一种丝氨酸蛋白酶，它通过促进补体成分 3（C3）的裂解，在哺乳动物中激活替代途径（AP），从而参与先天防御。然而，在硬骨鱼类中，Df 在 AP 中的功能机制以及对抗病原体感染的功能机制还远不清楚。在本研究中，我们从草鱼中克隆和鉴定了 Df 基因，并分析了其在促进 C3 裂解以及草鱼呼肠孤病毒（GCRV）感染后表达变化中的作用。Df 的开放阅读框为 753bp，编码 250 个氨基酸，分子量为 27.06kDa。Df 含有一个保守的 Tryp_SPc 结构域，其中三个保守残基代表催化三联体，三个保守结合位点位于底物特异性口袋中。序列比对表明，Df 与来自 的 Df 具有最高的同源性（96%）和相似性（98%）。系统进化分析表明，Df 和其他鱼类的 Dfs 形成了一个独特的进化分支。与大多数来自其他脊椎动物的 Dfs 相似，Df 基因结构的特点是有四个内含子和五个外显子。重组 Df 蛋白与草鱼血清孵育后，显著增加了 C3b 含量，表明 Df 在 AP 中促进 C3 裂解的保守功能与哺乳动物的 Dfs 相似。在草鱼的各种组织中均广泛检测到 Df mRNA 的表达，并且在肝脏、脾脏和肠道中的表达水平相对较高。在 GCRV 感染的 168 小时期间，在肝脏、脾脏和肠道中的 Df mRNA 表达水平在 144 和 168 小时保持高水平，表明在 GCRV 感染的后期 AP 活性增强。综上所述，这些结果揭示了 Df 的保守结构和功能及其在 GCRV 感染后的独特表达模式，为研究 Df 和 AP 在草鱼 GCRV 感染中的作用提供了关键依据。
