Retinoid Binding Properties of RPE65 and its Role in the Visual Cycle

Abstract

Abstract: : Purpose: RPE65 is an abundant protein of the retinal pigment epithelium (RPE) whose function remains undetermined. The biochemical phenotype in rpe65 null mutant mice is massive accumulation of all-trans retinyl esters (atRE) and undetectable 11-cis retinoids. Studies were conducted to resolve the function of RPE65 and its role in the visual cycle. Methods: RPE65 was expressed in SF9 cells and purified by anion-exchange and size-exclusion chromatography. The purified protein was used in retinoid binding studies, which included UV-vis analyses of binding in solution and fluorescence microscopy of binding in RPE65-proteoliposomes. Rpe65-/- knock-out mice and age/strain-matched controls were used to assess the function of RPE65 in vivo. In these studies, retinoids were extracted from ocular tissues and analyzed by HPLC. Time-course profiles were obtained for the endogenous retinoids during periods of light- and dark-adaptation. In addition, the metabolism of basally and apically derived all-trans-retinol (atROL) was measured following administration of [³H]-atROL by intra-peritoneal and intra-ocular injections, respectively. Results: Spectroscopic analysis of mixtures containing RPE65 and all-trans-retinyl palmitate (atRP) revealed the presence of a single molecular species defined by an isosbestic point at 296 nm. Binding of RPE65 to atRP was concentration- and time-dependent, and not observed when heat-denatured RPE65 protein was used. We corroborated these findings by incubating atRP with either RPE65-proteoliposomes or "empty" proteoliposomes. Following removal of excess atRP, the liposomes were analyzed by fluorescence microscopy. Only RPE65-proteoliposomes demonstrated fluorescence properties typical of atRP. Studies of vitamin A metabolism in RPE65 knock-out mice revealed a light-dependent flux of atRE within membranes and cytosol of the RPE. Light-adaptation was associated with decreased atRE pools. The atRE pools increased during dark adaptation. Delivery of [³H]-atROL to the basal RPE resulted in cytosolic accumulation of newly formed [³H]-atRE, while delivery of [³H]-atROL to the apical RPE resulted transient [³H]-atROL accumulation in both cytosol and membrane compartments and was slowly processed into membranes as [³H]-atRE. Conclusions: These observations support the hypothesis that RPE65 is an atRE binding-protein, which may function to maintain retinyl ester homoestasis by mediating retinyl ester flux between membranous to cytosolic compartments of the RPE cell.

Keywords: retinal pigment epithelium • retinal degenerations: cell biology • brightness and lightness