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Swati Arya, Silvia Synowsky, Sally Shirran, Catherine Botting, Imre Lengyel, Alan Stewart; Identifying the hydroxyapatite interactome: Involvement of serum proteins in the formation of sub-RPE deposits. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5257.
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© ARVO (1962-2015); The Authors (2016-present)
A feature of the ageing retina is the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. Although deposit formation is central to the initial steps leading to age-related macular degeneration (AMD), the factors that control this are not well understood. Recently, we identified the presence of hydroxyapatite (HAP) spherules within sub-RPE deposits in human eyes to which several AMD-associated proteins were bound including complement proteins that could have originated from plasma. Here we tested the hypothesis that circulating plasma proteins may contribute to sub-RPE deposits formation by binding to HAP spherules in the sub-RPE space.
We performed Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) proteomics on plasma samples from 30 AMD patients. Samples with CFH genotypes homozygous to T1277 or C1277 (“at-risk” SNP (T1277C)) were depleted of albumin and IgG and incubated with HAP-coated ceramic beads (Bio-Rad). Bound proteins were eluted and analysed by SWATH-MS using ProteinPilot, PeakView and MarkerView software packages. Proteins levels for some of the relevant proteins were verified in each sample using commercially available ELISA kits. The presence of the relevant proteins in deposits in human cadaver eyes was verified using immunofluorescence on samples obtained from the Pathology Archive at the UCL Institute of Ophthalmology. The study adhered to the Helsinki convention and had full ethical permission.
A library of 242 plasma proteins (1% false discovery rate) was generated. SWATH-MS revealed significant differences in HAP binding of 23 proteins (p≥0.05) between the two homozygous groups. These included apolipoproteins and complement factors. Those displaying most significantly altered levels of binding between groups were verified using ELISA. The presence of new proteins like histidine rich glycoprotein in sub-retinal epithelial deposits were confirmed by immunolabelling of sectioned archived cadaveric tissues.
This study provides qualitative and quantitative information relating to the degree by which certain plasma proteins may contribute to sub-RPE deposit formation in individuals with different genetic backgrounds through binding to HAP. The data also strongly supports a role for plasma proteins in drusen formation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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