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Minghao Jin, Songhua Li, Minsup Lee; Interphotoreceptor retinoid-binding protein (IRBP) promotes RPE uptake and esterification of all-trans-retinol in the interphotoreceptor matrix (IPM). Invest. Ophthalmol. Vis. Sci. 2017;58(8):3580.
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© ARVO (1962-2015); The Authors (2016-present)
IRBP, the most abundant soluble protein in the IPM, is known to play an important role in the visual cycle. However, the molecular mechanism by which IRBP facilitates the visual cycle in the IPM remains largely unknown. In this study, we test whether IRBP plays a role in RPE uptake and esterification of all-trans-retinol (atROL) released into the IPM after photobleaching the visual pigments.
Dark-adapted 3-week-old wild-type (WT) and Irbp-/- mice were exposed to light for 30 or 60 min. Ocular retinoids in these mice were measured by high performance liquid chromatography. Expression levels of lecithin-retinol acyltransferase (LRAT) and diacylglycerol O-acyltransferase type 1 (DGAT1) in the eyes were determined by immunoblot analysis. Subcellular localization patterns of LRAT and RPE65 in RPE were analyzed by immunocytochemistry. The effects of IRBP on RPE uptake and esterification of atROL were evaluated by measuring all-trans retinyl esters (atRE) in RPE of Irbp-/- eyecups incubated with atROL in serum-free media containing 4 μM IRBP or bovine serum albumin (BSA). IRBP was prepared by transient transfection of 293T-LC cells followed by 100-kD cutoff filtration. Its purity and identity was confirmed by Coomassie Brilliant blue-staining and immunoblot analysis using an antibody against IRBP.
The total amounts of retinoid in dark-adapted Irbp-/- mice were similar to those in WT mice. The amounts of atROL in the eyes of Irbp-/- mice exposed to light for 1 h were significantly greater than those in WT eyes whereas the amounts of atRE in the same Irbp-/- eyes were approximately half of atRE amount in the same WT eyes. However, the expression levels of LRAT and DGAT1 in dark-adapted and light-exposed Irbp-/- mice were similar to those in WT mice under the same light conditions. Immunocytochemistry showed that subcellular distribution patterns of LRAT and PE65 in RPE of light-exposed Irbp-/- mice were similar to those in WT mice. The amounts of atRE in RPE explants incubated with atROL in the presence of IRBP were at least 1.6-fold greater than those in RPE incubated with atROL in the presence of BSA.
RPE may express a membrane receptor/s of IRBP and/or an intracellular regulator/s that promotes RPE to uptake and/or esterify all-trans-retinol bound with IRBP in the IPM.
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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