Molecular role of metal-induced complement protein aggregation in age-related macular degeneration

Our immune system is vital to protect us from all types of bacterial, fungal and viral infections. There are different types of immune defence in the body, and that relevant to this proposal is called ?innate immunity? in which blood proteins called ?complement? acts to destroy invading foreign bacteria. While highly effective against bacteria, complement requires tight control so that it does not attack the body?s own cells. This control is achieved by a series of regulatory proteins, and Factor H is one such protein. In recent years, Factor H has been implicated in age-related macular degeneration, the most common cause of blindness in old age. The disease, which results in loss of vision in the centre of the eye, is caused by the build up of deposits on the retina at the back of the eye called ?drusen?. Many people have a version of Factor H that leads to a high risk of developing age-related macular degeneration. Even though we know Factor H is involved in the disease, we do not understand the reasons why. However, we have made much recent progress in understanding the functioning of Factor H. We discovered that Factor H has a tendency to clump together (or form self-aggregates) when present in large amounts, and this tendency is strongly augmented in the presence of the metal zinc. Zinc is known to inhibit Factor H regulation. Interestingly, very large amounts of zinc have been reported in the drusen of patients. While zinc-Factor H interactions may not be the only reason for drusen formation, the potential involvement of zinc in this process constitutes a very plausible idea for investigation. We wish to determine the molecular basis for the zinc-mediated aggregation (oligomer formation) of Factor H by making modified forms of Factor H and testing their effect on zinc binding. If successful, we will have unravelled the first one of the molecular mechanisms that lead to drusen formation, and this will open the way for the development of novel therapeutic strategies for the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) accounts for 50% of blind registration in the elderly in the UK. AMD involves the appearance of drusen (?deposits?) on the retina. Drusen contain about 120 proteins including complement proteins such as Complement Factor H (CFH), and cross-linked lipids. CFH is a major complement regulator, and is comprised of 20 small short complement regulator (SCR) domains. A genetic polymorphism leading to a Y402H mutation in CFH is a major risk factor for AMD. However, the molecular mechanism whereby CFH is implicated in drusen growth is unclear. Recently we discovered that serum CFH self-associates, both when unliganded and in the presence of zinc. Metals such as zinc are present at 2-3 mM in drusen, and our recent work shows these amounts are 100-fold greater than needed to cause uncontrolled CFH self-aggregation. We will therefore test the novel hypotheses that either (1) the self-association of CFH is the trigger for drusen formation, or (2) the inhibition of CFH activity will promote inflammatory attack, leading to drusen formation. These ideas are radically different from the classic view that the malfunctioning of CFH causes a failure of complement regulation and results in inflammation. First, we will generate recombinant CFH fragments that will be tested with homozygous (Y402 or H402) CFH by analytical ultracentrifugation, X-ray scattering and surface plasmon resonance (Biacore) methods. This will identify which SCR domains are responsible for CFH self-association. Once the key SCR domains have been identified, mutagenesis will identify the residues responsible for CFH self-association. This work will inform the design of potential therapeutic compounds that will block CFH aggregation and will also lead to experiments by our collaborators at the Insitute of Ophthalmology to test the involvement of CFH in the cell biology of drusen formation. Examples will include the study of metal levels in drusen from genotyped AMD patients by X-ray fluorescence and zinc labelling to establish whether these correlate with CFH genotype, and the study of zinc and anionic oligosaccharides released from damaged retinal pigmented epithelium cells. The degree to which the CFH mutagenesis experiments are successful will show whether there is a simple explanation for the onset of AMD based on CFH polymorphism, and suggest novel therapeutic strategies to reduce its effects.