A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein
A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein
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DOI：
10.1002/eji.200323541
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发表时间：
2003-04-01
影响因子：
5.4
通讯作者：
Gordon, DL
中科院分区：
文献类型：
--
作者：
Giannakis, E;Jokiranta, TS;Gordon, DL
补体抑制因子H（fH）通过其第七短共有重复序列（SCR）结构域与包括肝素、链球菌M蛋白和C反应蛋白（CRIP）在内的多种配体相互作用。本研究的目的是定位这些相互作用所需的SCR 7中的残基。我们最初使用fH SCR 15-16和牛痘对照蛋白SCR 3-4的平均NMR结构作为模板建立了fH SCR 6-7的同源性模型。模型表面的静电势证明了SCR 7上的三个带正电荷的残基簇的共定位，标记的位点A（R369和K370）、位点B（R386和K387）和位点C（K392）。定位于SCR 7之前的接头区和SCR 7中“高变环”的末端的这些残基在含有SCR 1-7的fH构建体中被不带电荷的丙氨酸残基系统地置换。将所得蛋白在甲基营养型酵母巴斯德毕赤酵母中表达。通过ELISA分析，我们证明：第一，取代位点A抑制肝素和CRP结合;第二，取代位点B抑制与肝素、CRP和M蛋白的结合;第三，取代位点C仅明显抑制肝素结合。
The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRIP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.