Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

M Keith Wyatt,Robert N Fariss,Cynthia Jaworski,Graeme Wistow,Sanghamitra Mishra,Jen-Yue Tsai,Steven L Bernstein,Maria Campos,Olaf Strauß

doi:10.1371/journal.pone.0068088

M Keith Wyatt, Robert N Fariss + Show 7 more

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https://doi.org/10.1371/journal.pone.0068088

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Abstract

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.

Highlights

Age related macular degeneration (AMD) is a major cause of vision loss in aging western populations [1]
Yeast 2-Hybrid (Y2H) Screening Yeast 2-hybrid screens provide a method for searching for potential interactions of a protein domain with the expressed proteome of a target tissue
Baits were made for complement factor H (CFH) short consensus repeat domain 7 (SCR7), containing either Y or H at position 402 and for the equivalent domain of the closely related CFHR3 gene and were independently screened against the retinal pigment epithelium (RPE)/choroid library

Summary

Introduction

Age related macular degeneration (AMD) is a major cause of vision loss in aging western populations [1]. A recent study has shown loss of activity of the enzyme Dicer and toxicity from accumulating Alu RNA in the terminal stage of AMD [11], many different upstream events may lead to this final outcome. AMD is generally associated with pathological changes in the retinal pigment epithelium (RPE) and Bruch’s membrane (a collagen-rich extracellular matrix between the RPE and choroidal vasculature) including formation of plaque-like deposits called drusen [12]. Proteomics and immuno-labeling studies have shown that drusen of all kinds contain a number of proteins, including several components of the complement system, and cholesterol-rich lipids [6,14,15,16]

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