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M. Jin, N.L. Mata, M.C. Koag, S.N. M. Reid, J.S. Lee, J. Garcia, D.B. Farber, G.H. Travis; Cloning and Characterization of Two New Retinol Dehydrogenases (RDH15 and RDH16) from Cone–dominant Retinas and RPE . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1263.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies suggest that 11–cis–retinaldehyde (11cRAL) chromophore may be regenerated in daylight by pathways involving (i) an unidentified 11–cis–retinol dehydrogenase (11cRDH) in cones, and (ii) RGR–opsin in RPE cells. To analyze the molecular basis of these pathways, we have characterized two new retinol dehydrogenases (RDH’s) in cones and RPE cells. Neither enzyme has been previously described. Methods: We screened cDNA libraries from wild–type mouse and cone–dominant ground–squirrel eyecups, and RNA from cone–dominant nrl–/– mouse eyecups by degenerate PCR. Primers were based on the conserved regions of short–chain dehydrogenases (SDR’s). To clone full–length cDNAs, we used the same templates for 5'– and 3'–RACE. Polyclonal Ab’s to the cloned proteins were raised in rabbits. Profiles of cellular expression were established by Northern blotting, Western blotting, and in situ hybridization. Kinetic properties of the enzymes were determined using Sf9–expressed proteins. Results: A novel RDH (RDH15) was cloned from cone–dominant nrl–knockout mouse eyecup . This protein contained 313 residues. RDH15 mRNA was present at higher levels in cone– versus rod–dominant retinas, suggesting that it is cone specific. Preliminary in situ hybridization analysis of the RDH15 mRNA in mouse retinas agreed with this expression pattern. RDH15 demonstrated oxidoreductase activity against 11–cis–retinol (11cROL) and all–trans–retinol (atROL) (Vmax = 125 pmole/min/mg; KM = 4 µM), but not 13–cis– or 9–cis–retinol. RDH15 exclusively used pro–S NADPH/NADP+ as a cofactor. A second RDH (RDH16) was cloned from the cDNA of wild–type mouse eyecups. The RDH16 mRNA was expressed in RPE cells. The protein contained 323 residues and showed unusual substrate specificity. As an oxidase, RDH16 was similarly active on atROL and 11cROL substrates (Vmax = 50–70 pmole/min/mg; KM = 10–15 µM). However, as a reductase, RDH16 showed specificity for 11cRAL, with no activity towards all–trans–retinaldehyde (atRAL). Similar to RDH15, RDH16 used pro–S NADPH/NADP+ as a cofactor and was not active against 13–cis– or 9–cis–retinoids. Conclusions: RDH15 may represent the cone 11cRDH in the recently proposed cone visual cycle. RDH16 may function cooperatively with RGR–opsin by providing atRAL for photo–isomerization. If so, the retinoid specificity of RDH16 would tend to drive all–trans to 11–cis photo–isomerization by providing substrate and specifically removing the 11cRAL product.
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