| Popis: |
The complement system is an integral component of innate immunity. Complement activation is tightly regulated to restrict effector functions to pathogens or altered host tissues. Complement-associated diseases are characterised by inappropriate activation on host surfaces, usually due to impaired regulation. Factor H (FH) is the major negative regulator of the alternative complement pathway. FH inhibits complement activation in plasma and on host surfaces. The FH protein family includes five additional proteins, termed factor H-related (FHR) proteins. FHRs compete with FH for binding to complement but, unlike FH, do not regulate complement activation. Consequently, the balance between FH and FHRs binding determines the degree of surface complement activation. My thesis examined the hypothesis that the interactions of both FH and FHRs with surface complement are influenced by additional interactions with surface carbohydrates. I explored the interactions of FHRs and FH to sialoglycoconjugates and glycosaminoglycans (GAGs) using microtiter plate-based binding assays and neoglycolipid-based microarrays. My results showed overlapping specificities of the FHRs and FH for binding to GAGs, and that the interactions correlated with the degree of GAG sulphation. Inhibition of FHR and FH surface binding by soluble GAGs demonstrated differences in affinities and the mode of binding among FHRs and FH. The binding profiles of the FHRs and FH to sialo-glycoconjugates display differential interaction based on the sequence and linkage of analysed sialo-glycoconjugates. FHRs displayed a binding preference to α2-3-sialyl over α2-6-sialyl glycans, whereas FH bound most strongly to a disialyl glycan with both α2-3 and α2-6-linked sialic acid. In a surface C3b-binding competition assay, the addition of surface HS altered the ratio of FH/FHR binding, displaying less surface bound FH. My work has elucidated the carbohydrate ligands for FH and FHRs and provided insight into the molecular basis of surface discrimination by this protein family. Open Access |
