Functional Analyses Demonstrate that Common Polymorphisms in C3, Factor B and Factor H Collaborate to Determine Systemic Complement Activity and Risk for AMD

Abstract

Purpose: : Genetic studies have associated common variations in the complement alternative pathway (AP) proteins C3, factor B (fB) and factor H (fH) with age-related macular degeneration (AMD). We demonstrated that the fB_32Q variant forms a C3 convertase less efficiently than the fB_32R, leading to decreased amplification of the AP, while fH_62I is a better cofactor for factor I-mediated inactivation of C3b than fH_62V. These observations explain their protective effects. Here, we perform a functional analysis of the C3_R102G variation and investigate how common variation in all three complement proteins combine to affect disease risk.

Methods: : C3, fB and fH variants were purified from plasma of homozygous individuals. Surface plasmon resonance was used to study differences in ligand binding, and haemolysis assays were designed to reveal individual and combined functional differences of complement activating and control proteins.

Results: : C3_102G activates the AP more efficiently in whole serum and increases target cell lysis. fB binding kinetics and convertase stability were identical, but native and recombinant fH bound better to C3b_102R resulting in significantly decreased cofactor activity. Variants were tested in combination; convertase formed from C3_102G and fB_32R had three-fold lytic activity compared to C3_102R and fB_32Q and convertase formed from C3_102G and controlled by fH_62V showed two-fold increased lysis compared to C3_102R and fH_62I. Combining disease ‘risk’ variants (C3_102G, fB_32R, fH_62V) yielded six-fold higher AP activity compared to ‘protective’ variants (C3_102R, fB_32Q, fH_62I; P<0.0001).

Conclusions: : We demonstrate here the molecular basis for association of C3_102R with AMD, and show that AMD ‘risk’ variants collaborate to enhance AP cycling, while ‘protective’ variants reduce cycling. These data introduce the concept of a functional complotype (combination of polymorphisms) defining complement activity in an individual, and influencing susceptibility to AP-driven disease.

Keywords: age-related macular degeneration • inflammation • protein structure/function