Abstract
Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.
Highlights
The complement system is an enzymatic cascade in the innate immunity which acts against damaged cells or invading pathogens before they can cause infection
The dissociation constants KD values for dimer formation ranged between 8–28 μM [45], Complement Factor H (CFH) dimers are expected to co-exist with CFH monomers at physiological conditions in serum
It was unlikely that the dimerization site would reside on either SCR19 or short complement regulator (SCR)-20, because of the functional interaction of SCR-19 and SCR-20 with the C3d fragment of complement C3b and sialic acid as reported in crystal structures [47,48,49,50,51] (PDB codes 3OXU, 2XQW, 4ONT, 4ZH1, 5NBQ)
Summary
The complement system is an enzymatic cascade in the innate immunity which acts against damaged cells or invading pathogens before they can cause infection. In the alternative pathway of complement activation, non-active complement C3 is spontaneously hydrolyzed in a tickover mechanism to C3u [ known as C3(H2O)], which is conformationally similar to active C3b. C3u leads to the amplification of C3 cleavage through the C3 convertase, which hydrolyses C3 to form active C3b. C3b binds to exposed cell surfaces, targeting them for immune destruction. Complement Factor H (CFH) prevents complementmediated host cell destruction through the interaction of its Cterminus with surface-bound C3b on anionic host cell surfaces [1, 2]. CFH acts as a cofactor for Factor I which cleaves C3b to inactive iC3b [3]
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