Department of Structural and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, U.K.
Biochem J. 2012 Jun 15;444(3):417-28. doi: 10.1042/BJ20120183.
FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) values of 4-20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.
FH（因子 H）具有 20 个 SCR（短补体调节蛋白）结构域，是血清中补体的主要调节因子，该基因的缺陷与炎症性疾病有关。硫酸乙酰肝素是一种细胞表面糖胺聚糖，由磺酸化的 S 结构域和未磺酸化的 NA 结构域组成。为了阐明 FH 与糖胺聚糖结合的分子机制，我们使用 FH 和糖胺聚糖片段进行了超速离心、X 射线散射和表面等离子体共振实验。超速离心实验表明，FH 与纯化的肝素片段（相当于 S 结构域）形成了高达 63%的定义明确的寡聚体，并显示出约 0.5μM 的解离常数 K(d)。用肝素交联 SCR-7 和 SCR-20 的二价 FH 未改变结构，解释了 FH-肝素寡聚体的沉降系数。在存在肝素片段 18-36 个单糖单位的情况下，FH 的 X 射线回转半径 R(G)从 10.4nm 显著增加到 11.7nm，FH 的最大长度从 35nm 增加到 40nm，证实形成了大的紧凑寡聚体。固定化全长 FH 与肝素的表面等离子体共振实验得出 K(d)值为 1-3μM，SCR-6/8 和 SCR-16/20 片段的 K(d)值相似但较弱，为 4-20μM，证实了两个结合位点之间的协同作用。使用对应于大部分 NA 结构域的最小硫酸化硫酸乙酰肝素片段的实验表明，结合较弱，证明了 S 结构域在这种相互作用中的重要性。FH 与硫酸乙酰肝素结合的这种二价和协同模型为 FH 在宿主细胞表面的免疫功能提供了新的见解。
