Chang C P, Hüsler T, Zhao J, Wiedmer T, Sims P J
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee 53233.
J Biol Chem. 1994 Oct 21;269(42):26424-30.
The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.
CD59抗原是一种质膜糖蛋白，作为补体C5b - 9复合物的抑制剂。这种抑制活性似乎与CD59在靶膜上C5b - 9复合物组装过程中与人类C8α链亚基以及人类C9的C9b结构域（氨基酸残基245 - 538）中新暴露位点高亲和力结合的能力有关（Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675 - 13680）。C9中CD59结合位点最初是通过对分离的C9b结构域有限消化产生的CD59结合肽进行N端测序来研究的。这些实验揭示了一个17 kDa的片段（从C9残基Thr - 320开始），它保留了对CD59的亲和力，这表明通过用小的C9衍生肽进行图谱分析来定位CD59结合位点的可能性。跨越整个C9b序列的肽在大肠杆菌中表达，然后用CD59进行检测。CD59特异性结合所有从C9残基359的N端开始且C端延伸超过残基411的肽。对于从残基359的C端开始或在残基411的N端被截断的各种C9衍生肽，几乎没有观察到CD59结合。针对C9残基320 - 411的亲和纯化抗体抑制CD59与C9的结合超过50%，并完全抑制其与分离的C9b结构域的结合。对于局限于C9b内假定铰链结构域（残基245 - 271）的肽，几乎没有检测到CD59的特异性结合。这些结果表明，CD59结合位点位于C9多肽的残基320和411之间，并表明该位点的亲和力主要由残基359 - 411决定。
