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Karina E Guziewicz, Emily V Dutrow, Keiko Miyadera, Jackie Meyer, Ruchira Singh, Kathleen Boesze-Battaglia, David M Gamm, Gustavo D Aguirre; A newly recognized molecule in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):369.
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Genotype-phenotype correlations in human BEST1-associated disorders are considered complex. The pleiotropic effects of BEST1, together with incomplete penetrance and multifaceted clinical expression, strongly suggest that other factors modulate the rate of progression and severity of the disease. Still, no genetic components explaining this substantial clinical variability have been identified. Canine multifocal retinopathy (cmr) caused by mutations in the dog ortholog is a well-established model for human bestrophinopathies that captures the full clinical picture of the disease, including the variation in its clinical presentation. To expand the spectrum of potential disease modifiers, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to explore the pool of molecules in a healthy and cmr-affected retinal pigment epithelium (RPE).
LC-MS/MS technology (Thermo LTQ-Orbitrap XL) performed on total canine and human RPE lysates was utilized in these discovery studies. The findings were verified by western blot and immunohistochemistry (mouse monoclonal EM-09) in vivo and in vitro, and visualized using confocal microscopy (Leica TCS SP5). Exon-intron boundaries of the canine gene were defined by direct sequencing and verified against cDNA library.
Mass spectrometry-based RPE proteome profiling revealed a molecule previously uncharacterized in the eye, desmoyokin. Its RPE-specific expression in the retina was confirmed by western blot analysis and immunohistochemistry in both retinal cryosections of dog and human and in the RPE-derived in vitro model systems such as canine primary RPE, hfRPE, hiPSC-RPE and ARPE19 cells. Confocal microscopy defined desmoyokin as a plasma membrane-associated protein localized at the intracellular face of the plasmalemma, however, its subcellular localization depends on the formation of cell-cell contacts. The canine gene contains two non-coding and three protein-coding exons (5549aa) and exhibits high level of sequence conservation with its human ortholog (5890aa).
Here, we report a novel protein that is RPE-specific in the retina and has not been previously characterized in the eye. Our preliminary findings suggest its role in cell-cell adhesion and cytoarchitecture of the plasma membrane; however, its exact function in a healthy and BEST1-affected RPE is still under investigation.
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