Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.
Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.
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人类补体第八个成分的α亚基的功能域：γ链独特结合位点的识别和表征。
DOI：
10.1021/bi00338a019
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Sodetz,JM
中科院分区：
文献类型：
--
作者：
Brickner,A;Sodetz,JM
南卡罗来纳州大学化学系和医学院，哥伦比亚，南卡罗来纳州29208接收1985年1月17日摘要：纯化的人补体第八组分（C8）的y亚基用于表征其在C8内的相互作用位点，并用于探测膜结合的C5 b-8和C5 b-9复合物的超微结构。y的纯化是通过将二硫键连接的ay亚基与非共价结合的α链分离，并将前者进行有限的还原、烷基化和离子交换色谱来完成的。在混合时，纯化的α和γ表现出彼此的高亲和力，这通过它们在稀释浓度下和在过量血清白蛋白存在下形成非共价等摩尔复合物的能力来证明。纯化的γ还表现出对C8 '的亲和力，C8'是一种先前描述的衍生物，其在功能上类似于C8，尽管其仅由α和β组成。这些结果表明，a具有与y相互作用的特异性位点，并且该位点在分离的亚基中被保留。此外，当a与ß关联时，该网站仍然可以访问。在相关的实验中，发现y与膜结合的C5 b-8 '和C5 b-（8'）9复合物特异性结合。这些结果表明，γ相互作用的位点在C5 b-8 '中的α上仍然是可接近的，并且在C5 b-（8'）9中没有被C9屏蔽。结论C8的y亚基位于膜结合的C5 b-8和C5 b-9的表面。补体的溶细胞C5 b-9复合物通过C5 b、C6、C7、C8和C9的顺序结合在靶膜上形成（Bhakdi & Tranum-Jensen，1983; Podack & Tschopp，1984）。在这些蛋白质中，C8是结构上最复杂的，因为它由三个非结构性亚基组成：α（MT 64000）、β（MT 64000）和γ（Mr 22000）（Kolb & Miiller-Eberhard，1976; Steckel et al.，1980年）。这些排列为二硫键连接的ay二聚体，其非共价地与circ缔合。最近的研究旨在描绘C8内的结构-功能关系，揭示了不同功能域的存在，这些功能域促进亚基之间以及与溶细胞复合物组分之间的相互作用。α亚基含有至少两个这样的结构域。其中一种与C5 b-7上的C8结合位点特异性相互作用，从而介导C8掺入该中间复合物中（Monahan & Sodetz，1980）。与第一个结构域不同的第二个结构域促进了α与γ的结合（Monahan & Sodetz，1981）。至少有三个功能域也被确定在一个。一个介导的非共价相互作用之间的α和α，如由事实证明，单独的α可以与等摩尔量的α。这种结合产生C8 '，1，一种缺乏γ但功能上类似于C8的衍生物（Brickner和Sodetz，1984）。已经显示α的第二结构域在C5 b-8在靶膜上组装期间直接插入脂质双层中（Steckel等人，1983年）。本期的前一篇论文描述了能够结合等摩尔量C9的第三个结构域（Stewart & Sodetz，1985）。该结构域在将C9整合到扩张性C5 b-8复合物中具有直接作用。与α和β相比，γ似乎在t中没有直接作用。这项研究得到了NIH Grant AI-16856和美国心脏协会授予JMS的既定研究者奖82-121的支持。
Department of Chemistry and School of Medicine, University of South Carolina, Columbia, South Carolina 29208 Received January 17, 1985 abstract: The purified y subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of y was accomplished by separating the disulfide-linked ay subunit from the noncovalently associated ß chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified a and y exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrationsand in the presence of excess serum albumin. Purified y also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only a and ß. These results indicate that a possesses a specific site for interaction with y and that this site is preserved in the isolated subunit. Furthermore, thissite remains accessible when a is associated with ß. In related experiments, y was found to specifically associate with membrane-bound C5b-8'and C5b-(8') 9 complexes. These results indicate that the site for y interaction remains accessible on a in C5b-8'and is not shielded by C9 within C5b-(8') 9. It is concluded that the y subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9. e cytolytic C5b-9 complex of complement is formed on target membranes by sequential association of C5b, C6, C7, C8, and C9 (Bhakdi & Tranum-Jensen, 1983; Podack & Tschopp, 1984). Among these proteins, C8 is the most structurally complex in that it is composed of three noniden-tical subunits: a (MT 64000), ß (MT 64000), and y (Mr 22000)(Kolb & Miiller-Eberhard, 1976; Steckel et al., 1980). These are arranged as a disulfide-linked ay dimer that is noncovalently associated with ß. Recent studies aimed at delineating structure-function re-lationships within C8 revealed the existence of distinct func-tional domains that facilitate subunit interactions with each other and with constituents of the cytolytic complex. The ß subunit contains at least two such domains. One interacts specifically with theC8 binding site on C5b-7 and thereby mediates incorporation of C8 into this intermediate complex (Monahan & Sodetz, 1980). A second domain that is distinct from the first facilitates association of ß with ay (Monahan & Sodetz, 1981). At least three functional domainshave also been identified in a. One mediates the noncovalent interaction between ay and ß, as evidenced by the fact that a alone can associate with an equimolar amount of ß. This association yields C8', 1 a derivative that is lacking y yet is functionally similar to C8 (Brickner & Sodetz, 1984). A second domain of a has been shown to insert directly into the lipid bilayer during assembly of C5b-8 on target membranes (Steckel et al., 1983). The preceding paper in this issue describes a third domain on a that is capable of binding an equimolar amount of C9 (Stewart & Sodetz, 1985). This domain has a direct role in incorporating C9 into the precursive C5b-8 complex. In contrast to a and ß, y appears to have no direct role in tThis research was supported by NIH Grant AI-16856 and by Established Investigator Award 82-121 to JMS from the American Heart Association.