IDENTITY OF THE SEGMENT OF HUMAN-COMPLEMENT C8 RECOGNIZED BY COMPLEMENT REGULATORY PROTEIN CD59
IDENTITY OF THE SEGMENT OF HUMAN-COMPLEMENT C8 RECOGNIZED BY COMPLEMENT REGULATORY PROTEIN CD59
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DOI：
10.1074/jbc.270.34.19723
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发表时间：
1995-08-25
影响因子：
4.8
通讯作者：
SIMS, PJ
中科院分区：
文献类型：
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作者：
LOCKERT, DH;KAUFMAN, KM;SIMS, PJ
CD59抗原是一种膜糖蛋白抑制C5b-9膜攻击的活动复杂(MAC),从而保护人类细胞裂解的人类补,CD59的抑制作用源于其能力与C8和C9组件交互的MAC,防止组装membrane-inserted C9聚合物,MAC-inhibitory CD59 species-selective和活动是最有效的C8和C9源自人类或其他灵长类动物的血浆,兔子CS和C9,为了鉴定CD59识别的人C8片段，我们在大肠杆菌中表达了含有人或兔C8序列的重组肽，并对其进行了纯化。研究发现CD59特异性地与人类CS α -亚基334-385残基对应的肽结合，并且在Cys（345）和Cys（369）之间需要一个二硫键。兔C8 α的相应序列（残基334-386）与NO特异性结合。为了获得CD59选择性识别人C8 α片段的功能证据，我们将C8 α cDNA的人/兔嵌合体以及编码C8 β和C8 γ链的cDNA共转染COS-7细胞，制备了重组C8蛋白。用CD59重组靶细胞分析了嵌合C8形成的MAC的溶血活性。这些实验证实，CD59识别了一个构象敏感的表位，该表位位于人类C8 α残基320-415的内部片段内。我们的数据还表明，CD59与人类C8 α片段的最佳相互作用受C8 α和人类C8 β的n端侧翼序列的影响，但不受CS γ的影响。
CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement, The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer, MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma, Rabbit CS and C9, which can substitute for human CS and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59, In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human CS alpha-subunit, and to require a disulfide bond between Cys(345) and Cys(369). NO specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415, Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by CS gamma.