May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
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
Author Affiliations & Notes
  • F. Slingsby
    MRC Human Genetics Unit, Edinburgh, United Kingdom
  • X. Shu
    MRC Human Genetics Unit, Edinburgh, United Kingdom
  • A. Herbert
    School of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh Biomolecular NMR Unit, Edinburgh, United Kingdom
  • M. Lyon
    University of Manchester, Department of Medical Oncology, Manchester, United Kingdom
  • L. Mackay
    School of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh Biomolecular NMR Unit, Edinburgh, United Kingdom
  • J. Creanor
    School of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh Biomolecular NMR Unit, Edinburgh, United Kingdom
  • D. Uhrin
    School of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh Biomolecular NMR Unit, Edinburgh, United Kingdom
  • P. Barlow
    School of Chemistry and Biological Sciences, University of Edinburgh, Edinburgh Biomolecular NMR Unit, Edinburgh, United Kingdom
  • R. Sim
    Department of Biochemistry, University of Oxford, MRC Immunochemistry Unit, Oxford, United Kingdom
  • A. Wright
    MRC Human Genetics Unit, Edinburgh, United Kingdom
  • Footnotes
    Commercial Relationships  F. Slingsby, None; X. Shu, None; A. Herbert, None; M. Lyon, None; L. Mackay, None; J. Creanor, None; D. Uhrin, None; P. Barlow, None; R. Sim, None; A. Wright, None.
  • Footnotes
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Investigative Ophthalmology & Visual Science May 2008, Vol.49, 200. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: age-related macular degeneration • protein structure/function • retinal pigment epithelium 
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