Abstract
Purpose:
A number of genetic alterations are associated with an increased risk of developing age related macular degeneration (AMD). Many of these risk mutations and polymorphisms arise in genes involved in the complement system, part of a host’s innate immune system. While much work has focused on the Y402H polymorphism in the main complement regulator factor H (CFH), increasing evidence suggests this protein does not work alone in protecting the Bruch’s membrane in the human eye. Factor H-like protein 1 (FHL-1) arises from an alternative splice variation of the cfh gene and retains all of the regulatory activity of CFH: FHL-1 is also subject to the Y402H polymorphism. Furthermore, 5 factor H-related (FHR) proteins also exist and share varying degrees of homology with CFH. Changes in the FHR genes are also associated with AMD, where complete gene deletions for FHR1 and 3 are believed to be protective.
Methods:
Here, we use immunohistochemistry and targeted mass spectrometry for analysis of CFH, FHL-1 and FHR isoforms on human macular sections and isolated Bruch’s membrane. Furthermore, rtPCR of human retinal pigment epithelial (RPE) cells identifies which of these complement regulators are transcribed locally. Surface plasmon resonance and solid phase plate assays are used to investigate the binding properties of FHL-1 and the FHR proteins to specific ligands.
Results:
Using specific antibodies, we identify the significant presence of FHL-1 on the Bruch’s membrane. This is converse to the understanding that CFH is the major regulator of complement in this region of the eye. In this regard, we demonstrate local expression of FHL-1 and the fact that the full length CFH protein is not able to diffuse through the Bruch’s membrane from human sera. The presence of FHR proteins also adds weight to the hypothesis that they may well compete with CFH, or indeed FHL-1, to binding sites on the Bruch’s membrane.
Conclusions:
The interplay between CFH, FHL-1 and the FHR proteins represents a novel mechanism to ensure immune homeostasis. An imbalance in this fine regulation based on genetics and driven via environmental or age-related factors, will lead to inappropriate complement activation and local inflammation. A proper understanding of the FHL-1 and FHR proteins is necessary if we are to treat this devastating disease.
Keywords: 412 age-related macular degeneration •
555 immunomodulation/immunoregulation •
519 extracellular matrix