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G. P. Moiseyev, O. Nikolaeva, K. Farjo, Y. Takahashi, J.-X. Ma; A2E Inhibits Isomerohydrolase Activity Through Direct Interaction With RPE65. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1304.
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Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in RPE cells with age and in age-related macular degeneration (AMD). The purpose of this study is to determine the effect of A2E on the retinoid isomerase reaction, the key step of the visual cycle, catalyzed by RPE65.
RPE65 was expressed in 293-LRAT cells using adenovirus vector. Isomerohydrolase activity was measured using either retinyl palmitate incorporated in liposomes as a substrate for in vitro assays or all-trans retinol in cell culture assays. The product of the reaction, 11-cis retinol, was extracted and analyzed by normal phase HPLC. A2E binding to purified RPE65 was measured using fluorescence spectra of A2E.
A2E potently inhibited generation of 11-cis retinol in in vitro isomerohydrolase assays in a concentration-dependent manner. LRAT activity was not inhibited by A2E at the same concentrations. The inhibition type is competitive with Ki = 13.6 µM. Fluorimetric titration of purified RPE65 by A2E resulted in an increase of A2E fluorescence, suggesting a direct, specific binding of A2E to RPE65. To measure cellular RPE65 activity, 293-LRAT cells were transfected with a CRALBP expression vector and infected with adenovirus expressing RPE65, and then treated with all-trans retinol. Addition of A2E to the cell culture media significantly decreased the amount of 11-cis retinol generated.
A2E potently and selectively inhibited conversion of all-trans retinyl ester to 11-cis retinol through direct binding to RPE65 isomerohydrolase. This interaction may disturb the visual cycle and impair vision in patients with AMD.
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