Nan, R.; (2010) Self-association of complement factor H in the presence and absence of metals. Doctoral thesis , UCL (University College London).

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Abstract

In the complement system of innate immunity, factor H (FH) is a major regulator of its activation, and is comprised of 20 short complement regulator (SCR) domains. FH is related to age-related macular degeneration (AMD) through a Tyr402His polymorphism, and occurs in drusen deposits that are a key feature of early AMD. High concentrations of zinc are also present in drusen, and the function of FH is inhibited by zinc. In this thesis, FH self-association and its interaction with zinc were investigated. Using X-ray solution scattering and analytical ultracentrifugation, pooled heterozygous FH in physiological conditions self-associated to form 10-15 % of dimer and higher oligomers. Titrations of FH with zinc induced uncontrolled oligomerisation of FH when the zinc concentration was above 20 micromolar, and this correlated with the reduction of FH activity. Structurally distinct large oligomers were also observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers, and Mg and Ca showed no change. These experiments were repeated for the native Tyr402 allotype and the disease-related His402 allotype of FH. X-ray scattering combined with constrained modelling showed that the homozygous Tyr402 and His402 allotypes of FH have indistinguishable foldedback structures in solution. Both homozygous allotypes of full-length FH exhibit similar self-association properties in solution, showing no dependence on heterozygosity. Surface plasmon resonance confirmed these results, but showed that the His402 allotype of SCR-6/8 self-associates more than the Tyr402 allotype. Zinc titrations of the two FH allotypes showed that each allotype formed similar oligomers with zinc. While the major interaction sites of FH with zinc were located within the SCR-6/8 fragment, the surface exposed His402 residue in SCR-6/8 showed no preferential interactions with zinc. Overall, these findings provide insight on the development of drusen deposits in Bruch’s membrane and the uncontrolled inflammation associated with AMD.

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