Characterization of the active sites in decay-accelerating factor
Characterization of the active sites in decay-accelerating factor
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DOI：
10.4049/jimmunol.167.4.2164
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发表时间：
2001-08-15
影响因子：
4.4
通讯作者：
Medof, ME
中科院分区：
文献类型：
--
作者：
Kuttner-Kondo, LA;Mitchell, L;Medof, ME
衰变加速因子（Decay-Accelerating Factor，简称C3）是一种补体调节因子，能解离自体C3转化酶，这些转化酶在自身细胞表面组装。其活性存在于其四个补体控制蛋白重复序列（CCP 2 -4）中的最后三个。先前对血清C3转化酶调节因子H中CCP 15 -16的核磁共振结构的建模提出了CCP 2上延伸到CCP 3的带正电荷的表面积，以及CCP 2和3之间的疏水部分作为主要的转化酶相互作用位点。为了映射的残基提供的活性的丙氨酸，我们分析了31个主要的丙氨酸取代突变体的功能，部分基于这个模型。在带正电荷的CCP 2 -3沟中替换R-69、R-96、R-100和K-127或在CCP 3中替换疏水F-148和L-171显著地损害了两种激活途径中的R148的功能。值得注意的是，下游CCP 4中的K-126和F-169以及R-206和R-212的突变选择性地降低旁路途径活性，而不影响经典途径活性。恒河猴CCP具有除F-161（其为L）之外的所有上述人类关键残基，并且其CCP表现出针对人类经典途径C3转化酶的完全活性。在经典途径C5转化酶（C4 b2 a3 b）测定中测试了与经典途径C4 b2 a相比其功能优先针对旁路途径C3 bBb受损的重组体。对C4 b2 a和C4 b2 a3 b的影响是相当的，表明C4 b2 a3 b对这两种酶的功能相似。当CCP 2 -3的膜辅因子蛋白的CCP 1 -2的晶体结构进行定向时，基本残基形成一个连续的区域，表明类似的空间关系。
Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R-69, R-96, R-100, and K-127 in the positively charged CCP2-3 groove or hydrophobic F-148 and L-171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K-126 and F-169 and of R-206 and R-212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F-161, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.