Pao, P-J; (2017) Effects of zinc and the complement factor H Tyr402His polymorphism on age-related macular degeneration. Doctoral thesis , UCL (University College London).

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Abstract

Factor H (FH) is a major regulator of the complement alternative pathway of innate immunity. FH is comprised of 20 short complement regulator (SCR) domains. The Tyr402His polymorphism in SCR-7 of FH is genetically associated with age-related macular degeneration (AMD), characterised by the presence of sub-retinal pigment epithelial (RPE) deposits in which FH, zinc and heparin were present. The Tyr402His polymorphism is proximate to zinc-binding and heparin-binding sites in SCR-6/8. To investigate the joint effect of zinc and heparin on FH, the oligomerisation of full-length FH was studied using size distribution c(s) analyses in analytical ultracentrifugation (AUC). With both zinc and heparin, FHHis402 showed a notably increased number of peaks with higher sedimentation coefficients (s20,w), compared to those of FHTyr402. Integration of the c(s) plots showed that FHHis402 formed 40% greater oligomers than FHTyr402. Control experiments with the SCR-6/8His402 fragment showed 80% greater oligomer formation than for SCR-6/8Tyr402 in the presence of both zinc and heparin, in agreement with the results for full-length FH. A daily supplementation of 80 mg zinc is recommended to AMD patients. The effects of zinc on human primary RPE cells were studied by culturing these with µM levels zinc in the medium. Cultures with zinc resembled the native tissue more than those without zinc, as demonstrated by the morphological characteristics of polarisation. The secretion profiles of the RPE cultures showed that FH was significantly upregulated towards the apical side when zinc was added. Treating the cells with FHHis402- and FHTyr402-genotyped human sera showed complement activation through observation of the membrane attack complex beneath the RPE. While immunolabelling revealed the presence of individual proteins, including FH, apolipoprotein E, C3 and the membrane attack complex, no deposits were formed in the cell cultures. These biochemical and cell biology results were rationalised by a FH allotype-based AMD model for the development of sub-RPE deposits and the effectiveness of zinc supplementation on reducing disease progression.

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