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Investigating a C1QTNF5 Mutation Associated with Macular Degeneration

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Investigating a C1QTNF5 Mutation Associated with Macular Degeneration Investigating a C1QTNF5 mutation associated with macular degeneration Fern Slingsby PhD The University of Edinburgh 2009 Declaration I declare that this thesis was composed by myself. The contributions of others to the work are clearly indicated. This work has not been submitted for any other degree or professional qualification. Fern Slingsby 2009 ii Acknowledgements I would like to thank Professor A. Wright and Dr X. Shu for giving me the opportunity to carry out this study. I would also like to thank all the members of the Wright Lab for their help and support over the last 3 years. In particular, I am grateful to Dr X. Shu for providing me with plasmids, cell lines and protocols, and Alan Lennon for his help with growing vast quantities of cell cultures. Thanks also to Kevin Chalmers, Brian Tulloch, Dafni Vlachantoni and Chloe Stanton, and HGU technical services whose technical support has been invaluable. Thanks to Dr M. Wear (University of Edinburgh) for training me with regard to SPR and ITC, Dr P. Perry (MRC Human Genetics Unit) for training and assistance on various microscopy techniques, and Dr J. Creanor for carrying out the MALDI-TOF MS analysis. I would like also to thank Prof. I Dransfield and Aisleen McColl (MRC Centre for Inflammation Research) to helping me with all aspects of the macrophage analysis, and to Shonna McCall for providing training on flow cytometry. Thanks to Dr A. Herbert, Prof. P. Barlow, Dr D.Uhrin and Henry Hocking (University of Edinburgh) for their discussion and advice. Particular thanks to Dr A. Herbert and Henry Hocking for allowing me to use their clones and purified proteins to investigate interactions with CFH. Professor R. Sim (University of Oxford) has provided me with valuable advice, as well as CFH with which to carry out assays, and I am also grateful to Prof. S. Lea (University of Oxford) for giving up her time to discuss my results and allowing me to spend time in her lab. I would therefore also like to thank Joe Ceasar (University of Oxford) for his assistance, it is just unfortunate we didn’t get the result we were hoping for! Finally I would like to thank my family, who have supported me in every way possible throughout this whole experience. iii Table of contents Abstract ...................................................................................................................... xv List of abbreviations.................................................................................................xvii List of figures...........................................................................................................xxii List of tables............................................................................................................ xxix 1 CHAPTER 1 : INTRODUCTION......................................................... 1 1.1 The retina and retinal pigment epithelium....................................................... 2 1.1.1 The structure of the retina .................................................................... 2 1.1.2 Bruch’s membrane ............................................................................... 6 1.1.3 The embryonic development of the RPE ............................................. 7 1.1.4 Transport mechanisms and growth factor secretion in the RPE .......... 8 1.1.5 The visual cycle.................................................................................... 9 1.1.6 RPE phagocytosis of rod outer segments........................................... 12 1.1.6.1 Receptors and ligands involved in ROS phagocytosis..................... 12 1.2 Age-related macular degeneration ................................................................. 16 1.2.1 General disease symptoms................................................................. 16 1.2.2 Deposits and changes in Bruch’s membrane...................................... 17 1.2.2.1 The composition of drusen............................................................... 19 1.2.2.2 Lipofuscin deposits.......................................................................... 20 1.2.2.3 Choroidal neovascularisation in AMD ............................................ 21 1.2.3 Oxidative damage in the RPE and AMD........................................... 21 1.2.4 Immune complexes and inflammation in the pathogenesis of AMD. 21 1.2.5 The role of phagocytes in AMD......................................................... 23 1.2.5.1 Macrophage recruitment in AMD.................................................... 23 1.3 Risk factors for AMD .................................................................................... 24 1.3.1 Genes showing association with AMD.............................................. 24 1.3.2 Identifying loci at 1q25-q31 and 10q26 associated with risk of AMD................................................................................................... 25 1.3.2.1 ARMS2/HTRA1 and risk of AMD.................................................. 25 1.3.2.2 The RCA gene cluster....................................................................... 26 iv 1.3.2.3 A complement factor H Y402H polymorphism and risk of AMD .. 26 1.3.2.4 Risk associated with other CFH SNPs and haplotypes.................... 26 1.3.2.5 CFH-like proteins and AMD risk..................................................... 27 1.3.2.6 Variants in Factor B and C2 are associated with risk of AMD........ 27 1.3.2.7 Variant in C3 is also associated with risk of AMD.......................... 28 1.4 The Complement System............................................................................... 28 1.4.1 Complement activation....................................................................... 28 1.4.2 Regulation of complement activation................................................. 30 1.5 Complement Factor H.................................................................................... 31 1.5.1 CFH co-factor activity with Factor I.................................................. 32 1.5.2 DAF activity of CFH.......................................................................... 32 1.5.3 CFH binding polyanions and host cells.............................................. 32 1.5.4 CFH interacts with CRP..................................................................... 33 1.5.5 CFH secondary structure and regulation of activity........................... 34 1.5.6 CFH-like proteins FHL-1 and CFHR-1-5.......................................... 34 1.5.6.1 CFHR1-5 functions.......................................................................... 34 1.5.6.2 FHL-1 function ................................................................................ 35 1.6 CFH and disease mechanisms in AMD ......................................................... 35 1.6.1 CFH expression and CFH localisation in the eye............................... 35 1.6.2 CFH expression by the RPE............................................................... 36 1.6.3 CFH knockout mouse and retinal phenotype..................................... 37 1.6.4 Effects of the Y402H polymorphism on CFH function ..................... 37 1.6.5 CFH mutations and kidney disease.................................................... 39 1.7 Late-onset retinal macular degeneration........................................................ 40 1.7.1 Diseased retinal structure................................................................... 40 1.7.2 Sub-RPE deposits in L-ORMD.......................................................... 41 1.7.3 Identification of a disease-causing mutation in C1QTNF5 ................ 44 1.7.4 C1QTNF5 shares homology with short chain collagens.................... 44 1.7.5 C1QTNF5 protein properties.............................................................. 47 1.7.6 C1QTNF5 tissue expression............................................................... 48 1.7.7 The effects of the Ser163Arg mutation on C1QTNF5 in a cellular system................................................................................................. 48 v 1.7.7.1 Wild-type C1QTNF5 is secreted but the mutant protein is not ....... 49 1.7.7.2 Different cellular trafficking of the mutant protein ......................... 49 1.7.7.3 C1QTNF5 and RPE cell adhesion to Bruch’s membrane................ 50 1.7.8 C1QTNF5 interacts with CFH ........................................................... 51 1.7.9 C1QTNF5 and obesity........................................................................ 51 1.7.10 Membrane-type Frizzled-Related Protein (MFRP)............................ 52 1.7.10.1 MFRP mutation and expression in the mouse eye........................... 52 1.7.10.2 MFRP mutation in the human eye ................................................... 55 1.7.10.3 MFRP interacts with C1QTNF5...................................................... 55 1.8 Aims of this study.......................................................................................... 56 2 CHAPTER 2 : MATERIALS & METHODS................................... 57 2.1 Cell culture procedures .................................................................................. 58 2.1.1 Mammalian cell culture...................................................................... 58 2.1.2 Bacterial cell culture........................................................................... 59 2.1.3 Yeast cell culture................................................................................ 59 2.2 Stably transfected cell line creation ..............................................................
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