Quantitative analysis of hydroxyapatite-binding plasma proteins in genotyped individuals with late-stage age-related macular degeneration. (July 2018)

• Record Type: Journal Article
• Authors: Arya, Swati; Emri, Eszter; Synowsky, Silvia A.; Shirran, Sally L.; Barzegar-Befroei, Neda; Peto, Tunde; Botting, Catherine H.; Lengyel, Imre; Stewart, Alan J.
• Is Part Of: Experimental eye research. Volume 172(2018)
• Page Start: 21
• Page End: 29
• Publication Date: 2018-07
• Subjects: Drusen -- Sub-retinal pigment epithelial deposits -- Mineral-protein interactions -- Retinal disease -- Quantitative proteomics; AMD age-related macular degeneration -- APOC4 apolipoprotein C4 -- BCA bicinchoninic acid -- C4A complement factor C4A -- C4B complement factor C4B -- C8A complement factor C8A -- C8B complement factor C8B -- CFH complement factor H -- ELISA enzyme-linked immunosorbent assay -- FDR false discovery rate -- FHR1 factor H-related protein 1 -- FHR3 factor H-related protein 3 -- HAP hydroxyapatite -- IC1 plasma protease C1 inhibitor -- iRT indexed retention time -- PZP pregnancy zone protein -- RPE retinal pigment epithelium -- SNP single nucleotide polymorphism -- SWATH-MS sequential window acquisition of all theoretical fragment-ion spectra by mass spectrometry; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2018.03.023
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (p < 0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation. Highlights: Hydroxyapatite (HAP) structures are thought to initiate sub-RPE deposit formation. HAP-binding plasma proteins from individuals with AMD were analysed using SWATH-MS. Certain proteins exhibited differing abundance between CFH-based genotypic groups. Six proteins were present in the RPE/choroid and four were associated with HAP. Genetic differences can influence proteins involved in sub-RPE deposit formation.