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Jeremy Cook, Roxana Radu, Hui Sun, Gabriel Travis; Functional Characterization of Peropsin in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3764.
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Peropsin (RRH) is a non-visual opsin of unknown function located in apical processes of the retinal pigment epithelium (RPE). The current study is an attempt to determine the function of peropsin. We began with the hypothesis that peropsin is involved in the processing of visual retinoids. To this end, we compared levels of visual retinoids in wild-type and peropsin-knockout (rrh-/-) mice under dark-adapted conditions, and at different times following a deep photobleach. We are employing a cell-culture system to study the role of peropsin on retinoid processing by enzymes of the visual cycle in RPE cells.
Mice homozygous for a null mutation in the rrh gene were generated using conventional gene-targeting methods. Both the rrh-/- and strain-129 control mice (WT) were homozygous for the L450 (normal) allele of rpe65. Mice were dark-adapted overnight. A subset of WT and rrh-/- mice were euthanized and their eyes collected, while the remainder were exposed to fluorescent light at 1,000 lux for 5 minutes. Subsets of these mice were euthanized immediately following light exposure, or returned to darkness for different time periods before euthanasia. Retinas and RPE-containing eyecups minus retinas were dissected, homogenized, and extracted with hexane for HPLC analysis of retinoids. To assay peropsin expression and function in cultured HEK-293T cells, a construct containing the gene for mouse peropsin was subcloned into pcDNA and used for transient transfection. Expression was confirmed by immunoblot analysis and confocal microscopy, retinoid uptake was examined by HPLC.
Levels of all-trans-retinyl esters were several-fold lower in RPE from rrh-/- versus WT mice under all light conditions. Five minutes following the photobleach, levels of 11-cis-retinaldehyde and 11-cis-retinyl esters were higher in the RPE of rrh-/- versus WT mice. Levels of LRAT, Rpe65, RGR-opsin and LRAT were similar in RPE from the two strains. Peropsin immunoreactivity was observed in the plasma membranes of 293T cells following transient transfection with the peropsin expression plasmid.
The altered retinoid dynamics in rrh-/- mice suggest delayed removal of 11-cis-retinaldehyde from RPE and delayed uptake of all-trans-retinol by RPE from bleached photoreceptors. Peropsin-transfected HEK-293T cells represent a suitable system to study the role of peropsin on the uptake and processing of visual retinoids.
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