Plumb M E, Sodetz J M
Department of Chemistry and Biochemistry and the School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA.
Biochemistry. 2000 Oct 24;39(42):13078-83. doi: 10.1021/bi001451z.
Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.
人C8是五种补体成分（C5b、C6、C7、C8和C9）之一，这些补体成分相互作用形成溶细胞性膜攻击复合物（MAC）。它是一种由三个亚基（C8α、C8β、C8γ）组成的寡聚蛋白，这三个亚基是不同基因的产物。在血清来源的C8中，它们以二硫键连接的C8α-γ二聚体形式排列，该二聚体与C8β非共价结合。在本研究中，确定了C8α上介导C8γ细胞内结合以形成C8α-γ的位点。通过对C8α及其结构同源物C8β、C6、C7和C9中的插入/缺失（indels）进行比较分析，确定C8α包含一个独特的插入序列（第159 - 175位氨基酸残基），其中包括与C8γ形成二硫键的半胱氨酸（Cys）164。将该序列整合到C8β中，并将所得构建体（iC8β）与C8γ共表达，产生了iC8β-γ，一种非典型的二硫键连接二聚体。在相关实验中，C8γ被证明与C8α和iC8β的突变形式非共价结合，这些突变形式中的半胱氨酸（Cys）164被替换为甘氨酸（Gly）164。此外，C8γ特异性结合到含有突变插入/缺失序列的固定合成肽上。这些结果共同表明：（a）C8γ与C8α的细胞内结合主要由C8α插入/缺失序列中的残基介导；（b）结合并不严格依赖于半胱氨酸（Cys）164；（c）C8γ必须含有与C8α插入/缺失序列互补的结合位点。
