The complement system is a complex network of soluble and membrane-associated serum proteins that regulate immune response. Activation of the complement C5 generates C5a and C5b which generate chemoattractive effect on myeloid cells and initiate the membrane attack complex (MAC) assembly. However, the study of evolutionary process and systematic function of C5 are still limited. In this study, we performed an evolutionary analysis of C5. Phylogeny analysis indicated that C5 sequences underwent complete divergence in fish and non-fish vertebrate. It was found that codon usage bias improved and provided evolution evidence of C5 in species. Notably, the codon usage bias of grass carp was evolutionarily closer to the zebrafish genome compared with humans and stickleback. This suggested that the zebrafish cell line may provide an alternative environment for heterologous protein expression of grass carp. Sequence comparison showed a higher similarity between human and mouse, grass carp, and zebrafish. Moreover, selective pressure analysis revealed that the C5 genes in fish and non-fish vertebrates exhibited different evolutionary patterns. To study the function of C5, gene co–expression networks of human and zebrafish were built which revealed the complexity of C5 function networks in different species. The protein structure simulation of C5 indicated that grass carp and zebrafish are more similar than to human, however, differences between species in C5a proteins are extremely smaller. Spatial conformations of C5a–C5AR (CD88) protein complex were constructed, which showed that possible interaction may exist between C5a and CD88 proteins. Furthermore, the protein docking sites/residues were measured and calculated according to the minimum distance for all atoms from C5a and CD88 proteins. In summary, this study provides insights into the evolutionary history, function and potential regulatory mechanism of C5 in fish immune responses.

补体系统是调节免疫反应的可溶性和膜相关血清蛋白的复杂网络。补体 C5 的激活产生 C5a 和 C5b，它们对骨髓细胞产生化学吸引作用并启动膜攻击复合物 (MAC) 组装。然而，对C5进化过程和系统功能的研究仍然有限。在这项研究中，我们对 C5 进行了进化分析。系统发育分析表明，C5 序列在鱼类和非鱼类脊椎动物中经历了完全的分歧。发现密码子使用偏差得到改善，并提供了 C5 在物种中的进化证据。值得注意的是，与人类和棘鱼相比，草鱼的密码子使用偏差在进化上更接近斑马鱼基因组。这表明斑马鱼细胞系可能为草鱼的异源蛋白表达提供替代环境。序列比较表明人和小鼠、草鱼和斑马鱼之间的相似性更高。此外，选择压力分析表明，C5鱼类和非鱼类脊椎动物的基因表现出不同的进化模式。为了研究C5的功能，建立了人和斑马鱼的基因共表达网络，揭示了C5功能网络在不同物种中的复杂性。C5的蛋白质​​结构模拟表明，草鱼和斑马鱼与人类的相似度更高，但C5a蛋白的物种间差异极小。构建了C5a-C5AR（CD88）蛋白复合物的空间构象，表明C5a和CD88蛋白之间可能存在相互作用。此外，根据所有原子与 C5a 和 CD88 蛋白质的最小距离测量和计算蛋白质对接位点/残基。总之，这项研究提供了对进化历史的见解，