Heparan Sulfate in Human Bruch’s Membrane/Choroid Tissue Increases the Rate of Proteolytic Cleavage of C3b by factors H and I

Abstract

Purpose: : Recombinant fragments of complement factor H (CFH) spanning the region associated with risk of developing age-related macular degeneration (AMD), have been shown to differentially bind glycosaminoglycans (GAGs). These results raise the possibility that susceptibility to AMD may involve interaction of CFH-GAG on Bruch’s membrane. We investigated whether the native full-length disease-linked allotypic variant of CFH --namely V62/H402-- interacted with GAGs found in human Bruch’s membrane/choroid tissue, in such a way as to alter its functional role.

Methods: : Factor H was purified from plasma from individuals homozygous for the double variants: I62/Y402, V62/Y402 and V62/H402. The rate of proteolytic cleavage of C3b to iC3b by CFH and complement factor I (CFI) was measured when Bruch’s membrane/choroid tissue was introduced to the assay. The interaction of heparan sulfate with other complement components was also studied using hemolysis assays and a C3 convertase assay.

Results: : Human Bruch’s membrane/choroid tissue increases the rate of proteolytic cleavage of C3b to iC3b in the presence of CFH and CFI but there was not a statistically significant difference between the rate increase measured using different allotypic variants (V62/H402, V62/Y402, and I62/Y402) of CFH. The cleavage of factor B to Bb, in the presence of C3 and factor D, and the subsequent cleavage of C3 to C3b was shown to be inhibited in the presence of heparan sulfate and heparan sulfate inhibited the alternative pathway of complement when measured using a hemolysis assay.

Keywords: age-related macular degeneration • proteoglycans/glycosaminoglycans • immunomodulation/immunoregulation