Functional Analysis of the Y402H Variant of Complement Factor H: Implications for Risk of Age–Related Macular Degeneration.

Abstract

Purpose: : To assess the effect of the Tyrosine–402 to Histidine–402 polymorphism (Y402H) on both complement factor H and factor–H–like protein 1 (FHL–1) cofactor activity. Factor H, a 150 kDa plasma protein, is the major soluble inhibitor of the alternative complement pathway and is structurally composed of 20 short consensus repeats (SCRs). FHL–1 is a 42 kDa plasma protein, encoded by an alternatively spliced transcript of the Factor H gene, that is identical with the first seven SCRs of factor H plus 4 amino acids at its C–terminus. The Y402H variant, present in both proteins, was shown to dramatically increase the risk for developing age–related macular degeneration (AMD). The cofactor activity facilitates C3b inactivation by the protease factor I, releasing C3c. An assay was developed to determine if this function of factor H and FHL–1 was related to the incidence of AMD.

Methods: : Purified factor H and plasma (containing factor H and FHL–1) from 4 groups of patients, normal Grade 1 AMD homozygous for Y402H or Y402 and Grade 5 exudative AMD Y402H or Y402 homozygotes, were used in a functional C3c–release assay, which quantitates the inactivation of cell–bound radiolabeled C3b by human factor H, and factor I.

Results: : A functional assay for the cofactor activity of factor H was developed. It showed no statistically significant difference in cofactor activity of factor H with FHL–1 with respect to either the Y402H polymorphism, or the incidence of advanced Grade 5 AMD.

Conclusions: : A functional assay for factor H and FHL–1 cofactor activity was developed. It can be used with plasma as a source of factor H and FHL–1. It is reliable, reproducible, requires no more than 0.25ug of protein and allows for a high sample throughput. We have shown that the increase in risk of developing AMD associated with the presence of the common coding variant Y402H does not appear to be due to the effectiveness of factor H as a cofactor for factor I cleavage of C3b. This assay can be used to measure whether modulation of cofactor activity by known Factor H ligands (e.g. heparan sulfate) is altered by the Y402H coding variant.

Keywords: age-related macular degeneration • immunomodulation/immunoregulation • protein purification and characterization