Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis
Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis
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DOI：
10.1073/pnas.93.20.10996
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发表时间：
1996-10-01
影响因子：
11.1
通讯作者：
Pangburn, MK
中科院分区：
文献类型：
--
作者：
Sharma, AK;Pangburn, MK
人类补体因子H控制血浆中补体的自发激活，在区分宿主细胞和补体替代途径激活剂方面起着重要作用。在小鼠和人类中，该蛋白由20个同源的短一致重复序列(SCR)结构域组成，其大小表明C3b结合位点(SCR1-4)以外的部分结构具有重要的生物学作用。我们已经在杆状病毒系统中表达了H因子的全长cDNA，并证明了重组蛋白是完全有活性的，这种全长蛋白的突变体现在已经被制备、纯化，并检测了辅因子活性和与C3b和肝素的结合。结果表明：(I)H因子至少有三个结合C3b的位点，(Ii)这些位点中的一个位于SCR结构域1-4，如其他人所示，(Iii)第二个位点存在于6-10区，(Iv)第三个位点驻留在SCR 16-20区，和(V)在因子H中存在两个肝素结合位点，一个在Scr 13附近，另一个在Scr 6-10区域，功能分析表明只有位于Scr 1-4的第一个C3b位点表达因子I辅因子活性，三个C3b结合位点中的任何一个缺失的突变蛋白对绵羊红细胞上C3b的亲和力降低了6-8倍，表明这三个位点都有助于控制红细胞上的补体激活。对H因子上多个不同功能位点的鉴定阐明了该蛋白的许多迄今无法解释的行为，包括H因子与表面结合的C3b的异质性结合，胰酶切割的影响，以及补体激活对补体替代途径的激活物和非激活物的不同控制。
Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement, In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains, The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active, Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin, The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region, Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity, Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.