RPE65 Mutations From Patients With Retinal Dystrophies Decrease RPE65 Expression

Abstract

Abstract: : Purpose: RPE65 is a membrane–associated protein predominantly expressed in the retinal pigment epithelium (RPE). It plays a key role in the visual cycle. It has been reported that some of RPE65 mutations are associated with retinal dystrophies such as autosomal recessive retinitis pigmentosa (ADRP) and Leber’s congenital amaurosis (LCA). The purpose of this study is to reveal how RPE65 mutants identified in the patients affect RPE65 structure and function. Methods: RPE65 mutations, Arg91Trp and Tyr368His, were generated by site–directed mutagenesis. Wild–type (wt) RPE65 and the mutants were expressed in HEK293 cells by the adenovirus system. Their expression levels were examined by Western blot analysis. The subcellular localizations were determined by immunocytochemistry. The activities of RPE65 and the mutants were measured by in vitro isomerase activity assay, and the products analyzed by HPLC. Results: Wt RPE65 was expressed at high levels in HEK293 cells. In contrast, the Arg91Trp and Tyr368His mutants showed drastically decreased protein levels under the same conditions. However, both of the mutants showed a subcellular localization similar to that of wt RPE65. When co–expressed with lecithin retinol acyltransferase (LRAT), wt RPE65 converted all–trans retinol to 11–cis retinol, while both the mutants have no detectable isomerase activities. Conclusions: RPE65 mutantions, Arg91Trp and Tyr368His, as identified in patients with retinal dystrophies may affect the stability of RPE65 and thus, interrupt the visual cycle.

Keywords: retinal pigment epithelium • adenovirus • retinoids/retinoid binding proteins