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Y. Takahashi, G. Moiseyev, J.-X. Ma; Identification of Key Residues Enhancing the Isomerohydrolase Activity of RPE65. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2997.
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RPE65, the retinoid isomerohydrolase, is a membrane-associated protein which abundantly 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. Recently we reported that cone-dominant chicken RPE65 (cRPE65) sharing 90% identity with human RPE65 (hRPE65) exhibited 7-10 times higher isomerohydrolase activity than that of native bovine RPE65 and recombinant hRPE65. The purpose of this study was to identify the key residues responsible for the higher enzymatic activity of cRPE65.
We chose specific residues in RPE65 conserved in cone-dominant species and different from the mammalian counterparts as the candidate residues. The hRPE65 cDNA was used as a template and the candidate residues of hRPE65 were replaced by the residues of cRPE65 using site-directed mutagenesis. The hRPE65 and cRPE65 cDNAs were digested into six fragments and the chimeric RPE65 of hRPE65 and cRPE65 were constructed. The wt hRPE65 and cRPE65, the site-directed and chimeric mutants were expressed in 293A-LRAT, a cell line stably expressing human lecithin retinol acyltransferase (LRAT). Their expression levels were examined by Western blot analysis and semi-quantified by densitometry. Their enzymatic activities were measured by in vitro isomerohydrolase activity assay, and the retinoid profiles were analyzed by HPLC.
Under the same culture conditions, most of the single, double and triple mutants of hRPE65 showed protein levels and isomerohydrolase activities similar to that of wt hRPE65. By combining the single mutations, we have identified a triple mutant of hRPE65 in which 3 residues were replaced by the counterparts of cRPE65 with 2-fold higher catalytic activity than that of wt hRPE65. Furthermore, a hybrid hRPE65 which replaced another fragment of the triple mutant by the cRPE65 fragment displayed a further increase in isomerohydrolase activity.
The hybrid hRPE65 is a more efficient isomerohydrolase than wt hRPE65. This mutant might be useful to improve the efficacy of gene therapy for RPE65 deficient patients.
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