Purchase this article with an account.
Yusuke Takahashi, Gennadiy P Moiseyev, Jian-Xing Ma; Identification of Key Residues Enhancing Isomerohydrolase Activity of Human RPE65 for More Efficient Gene Therapy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3476.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RPE65, retinoid isomerohydrolase, is a membrane-associated protein, which is predominantly expressed in the retinal pigment epithelium (RPE) and converts all-trans retinyl ester to 11-cis retinol, a key reaction in the retinoid visual cycle. We have previously reported that RPE65 in cone-dominant chicken (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits approximately 7 fold higher isomerohydrolase activity than that of bovine RPE65 and hRPE65. We expected that some key residues are responsible for the higher enzymatic activity of cRPE65. The purpose of this study was to identify the key residues responsible for the higher enzymatic activity of cRPE65.
Based on the amino acid sequence comparison of mammalian and chicken RPE65, 8 residues of hRPE65 were replaced by their counterparts of cRPE65 using site-directed mutagenesis. The wt hRPE65, cRPE65 and the site-directed mutants were expressed in 293A-LRAT, a cell line stably expressing human lecithin retinol acyltransferase (LRAT). The expression levels of generated mutants were examined by Western blot analysis and semi-quantified by densitometry. The enzymatic activities of the mutants were measured by in vitro isomerohydrolase activity assay, and the generated retinoids were analyzed by HPLC.
Among the mutants analyzed, we identified that two single mutations of N170K and K297G in hRPE65 and the double mutant, N170K/K297G, exhibited significantly higher catalytic activity than that of wt hRPE65. Further, when the amino terminal end (1Met-33Arg) fragment of the N170K/K297G double mutant of hRPE65 was replaced by the corresponding cRPE65 fragment, the isomerohydrolase activity and protein level were further increased to a level similar to that of cRPE65.
We successfully engineered more efficient human isomerohydrolase. This finding contributes to the understanding of structural basis for isomerohydrolase activity and may improve the efficacy of RPE65 gene therapy for retina degeneration caused by RPE65 mutations.
This PDF is available to Subscribers Only