Novel glycomic approaches to unravel protein- carbohydrate interactions in complement-associated kidney disease
File(s)
Author(s)
Gyapon Quast, Frederick
Type
Thesis
Abstract
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.
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.
Version
Open Access
Date Issued
2016-11
Date Awarded
2017-11
Advisor
Pickering, Matthew
Feizi, Ten
Sponsor
Wellcome Trust (London, England)
Grant Number
WMIT F28038
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)