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F. Slingsby, X. Shu, A. Herbert, M. Lyon, L. Mackay, J. Creanor, D. Uhrin, P. Barlow, R. Sim, A. Wright; A Disease-Causing Mutation in C1QTNF5 Shows Altered Affinity for Complement Factor H and the Y402H Polymorphism, Which Is Associated With Increased Risk of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2008;49(13):200.
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Late-onset retinal macular degeneration (L-ORMD), an autosomal dominant disease with features that are similar to age-related macular degeneration (AMD), is caused by a S163R mutation in the C1QTNF5 gene product. The Y402H polymorphism in complement factor H (CFH) is associated with increased risk of AMD. An interaction between CFH and C1QTNF5 was investigated, with the aim of identifying biochemical pathways involved in both L-ORMD and AMD.
C1QTNF5 was expressed in mammalian cells and purified using Ni-NTA affinity chromatography. Full length CFH was obtained commercially or purified from human plasma, whilst its short consensus repeat (SCR) modules 7-8 were produced using a Pichia pastoris expression system and purified by SP-Sepharose ion exchange chromatography. Interactions between C1QTNF5, CFH and SCR 7-8 were investigated using plate binding assays and surface plasmon resonance (SPR). MALDI-TOF analysis of C1QTNF5 was also carried out.
Plate binding assays and SPR show an interaction between C1QTNF5 and CFH. CFH shows a higher affinity for mutant C1QTNF5 compared to wildtype, with the interaction kinetics suggesting a ‘two-state’ binding model involving a conformational change. CFH modules SCR7-8 402Y and 402H both interact with C1QTNF5, and show similar affinities for wildtype but altered affinities for mutant C1QTNF5. The SCR7-8 module interactions with C1QTNF5 suggest a 1:1 binding model. MALDI-TOF analysis of native C1QTNF5 showed that the wildtype protein has slightly greater mass (around 260Da). Analysis of trypsin digested C1QTNF5 also revealed differences between wildtype and mutant.
Full length CFH interacts with C1QTNF5 and shows a higher affinity for the 163R mutant. Mutant C1QTNF5 also shows higher affinity for SCR 7-8 402H. Differences between wild type and mutant C1QTNF5 shown by MALDI-TOF could be the result of altered post-translational modification, which may be responsible for the altered CFH binding.
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