Synthesis of the All-trans-retinal Chromophore of Retinal G Protein-coupled Receptor Opsin in Cultured Pigment Epithelial Cells
2002; Elsevier BV; Volume: 277; Issue: 5 Linguagem: Inglês
10.1074/jbc.m108946200
ISSN1083-351X
Autores Tópico(s)Advanced Fluorescence Microscopy Techniques
ResumoLight-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cisisomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[3H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[3H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[3H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis. Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cisisomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[3H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[3H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[3H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis. retinal pigment epithelium RPE retinal G protein-coupled receptor high performance liquid chromatography bovine serum albumin phosphate-buffered saline The retinal pigment epithelial (RPE)1 cells are highly active in the metabolism of retinoids and are essential for the synthesis of the 11-cis-retinal chromophore of visual pigments (1Saari J.C. Invest. Ophthalmol. Visual Sci. 2000; 41: 337-348PubMed Google Scholar, 2McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Osborne N.N. Chader G.J. Progress in Retinal and Eye Research. 20. Elsevier Science Ltd., U. K.2001: 469-529Google Scholar). Many specialized enzymes and retinoid-binding proteins are involved in the production of 11-cis-retinal from all-trans-retinol. Lecithin-retinol acyltransferase is among the most active enzymes in retinoid processing and acts early in the retinoid cycle by catalyzing the esterification of all-trans-retinol soon after uptake into the RPE cells (3Saari J.C. Bredberg D.L. J. Biol. Chem. 1988; 263: 8084-8090Abstract Full Text PDF PubMed Google Scholar, 4Saari J.C. Bredberg D.L. J. Biol. Chem. 1989; 264: 8636-8640Abstract Full Text PDF PubMed Google Scholar, 5Shi Y.Q. Hubacek I. Rando R.R. Biochemistry. 1993; 32: 1257-1263Crossref PubMed Scopus (37) Google Scholar). The retinyl esters are normally the predominant retinoids, even while the content and distribution of retinoids in the RPE may vary under different light and dark conditions (6Zimmerman W.F. Vision Res. 1974; 14: 795-802Crossref PubMed Scopus (100) Google Scholar, 7Bridges D.D. Exp. Eye Res. 1976; 22: 435-455Crossref PubMed Scopus (129) Google Scholar, 8Saari J.C. Garwin G.G. Van Hooser J.P. Palczewski K. 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Biochemistry. 2000; 39: 11370-11380Crossref PubMed Scopus (87) Google Scholar). The retinoid-binding proteins in the RPE include cellular retinol-binding protein (22Bok D. Ong D.E. Chytil F. Invest. Ophthalmol. Visual Sci. 1984; 25: 877-883PubMed Google Scholar, 23Eisenfeld A.J. Bunt-Milam A.H. Saari J.C. Exp. Eye Res. 1985; 41: 299-304Crossref PubMed Scopus (47) Google Scholar), cellular retinaldehyde-binding protein (24Saari J.C. Bredberg L. Garwin G.G. J. Biol. Chem. 1982; 257: 13329-13333Abstract Full Text PDF PubMed Google Scholar, 25Bunt-Milam A.H. Saari J.C. J. Cell Biol. 1983; 97: 703-712Crossref PubMed Scopus (364) Google Scholar), and a unique opsin, RPE retinal G protein-coupled receptor (RGR) (26Shen D. Jiang M. Hao W. Tao L. Salazar M. Fong H.K.W. Biochemistry. 1994; 33: 13117-13125Crossref PubMed Scopus (78) Google Scholar, 27Tao L. Shen D. Pandey S. Hao W. Rich K.A. Fong H.K.W. Mol. Vis. 1998; 4: 25(http://www.molvis.org/molvis/v4/p25)PubMed Google Scholar). RGR is a membrane-bound opsin with seven transmembrane domains and is expressed in Müller cells as well as in the RPE (28Jiang M. Pandey S. Fong H.K.W. Invest. Ophthalmol. Visual Sci. 1993; 34: 3669-3678PubMed Google Scholar, 29Pandey S. Blanks J.C. Spee C. Jiang M. Fong H.K.W. Exp. Eye Res. 1994; 58: 605-614Crossref PubMed Scopus (69) Google Scholar). It is closely related in amino acid sequence to invertebrate visual pigments and retinochrome, a photoisomerase that catalyzes the conversion of all-trans- to 11-cis-retinal in squid photoreceptors (30Ozaki K. Hara R. Hara T. Kakitani T. Biophys. J. 1983; 44: 127-137Abstract Full Text PDF PubMed Scopus (26) Google Scholar). The RGR opsin is bound in the dark to all-trans-retinal and has absorption maxima at ∼469 and ∼370 nm (31Hao W. Fong H.K.W. Biochemistry. 1996; 35: 6251-6256Crossref PubMed Scopus (71) Google Scholar, 32Hao W. Fong H.K.W. J. Biol. Chem. 1999; 274: 6085-6090Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Illumination in vitro results in the stereospecific conversion of the bound all-trans-retinal to the 11-cis isomer. RGR is involved in the formation of 11-cis-retinal in mice and is necessary for maintaining normal steady-state levels of both 11-cis-retinal and rhodopsin in a light-adapted eye (33Chen P. Hao W. Rife L. Wang X.P. Shen D. Chen J. Ogden T. Van Boemel G.B. Wu L. Yang M. Fong H.K.W. Nat. Genet. 2001; 28: 256-260Crossref PubMed Scopus (141) Google Scholar). These results indicate that RGR functions to generate 11-cis-retinal in vivo and participates in a light-dependent visual cycle. Mutations in humanRGR, which is located on chromosome 10q23 (34Chen X.-N. Korenberg J.R. Jiang M. Shen D. Fong H.K.W. Hum. Genet. 1996; 97: 720-722Crossref PubMed Scopus (16) Google Scholar), are associated with cases of recessive and dominant retinitis pigmentosa (35Morimura H. Saindelle-Ribeaudeau F. Berson E.L. Dryja T.P. Nat. Genet. 1999; 23: 393-394Crossref PubMed Scopus (116) Google Scholar). Although RGR is a major all-trans-retinal-binding protein, it is unclear how the all-trans-retinal chromophore is generated in RPE and Müller cells. Only low amounts of all-trans-retinal, if any, have been reported in the RPE (6Zimmerman W.F. Vision Res. 1974; 14: 795-802Crossref PubMed Scopus (100) Google Scholar, 7Bridges D.D. Exp. Eye Res. 1976; 22: 435-455Crossref PubMed Scopus (129) Google Scholar, 8Saari J.C. Garwin G.G. Van Hooser J.P. Palczewski K. Vision Res. 1998; 38: 1325-1333Crossref PubMed Scopus (112) Google Scholar, 9Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (136) Google Scholar, 10Qtaishat N.M. Okajima T.-I.L. Li S. Naash M.I. Pepperberg K.R. Invest. Ophthalmol. Visual Sci. 1999; 40: 1040-1049PubMed Google Scholar), yet the RPE must be able to synthesize the chromophore of RGR. Indeed, the role of RGR in a photic visual cycle may require synthesis of the all-trans-retinal chromophore directly from all-trans-retinol. These considerations suggest that a novel all-trans-retinol dehydrogenase exists in the RPE to provide the chromophore of RGR. Recently, we demonstrated the uptake of exogenous all-trans-retinol into lentivirus-transduced ARPE-hRGR cells and subsequent incorporation of the retinoid into a bound ligand of RGR (36Yang M. Wang X.-G. Stout J.T. Chen P. Hjelmeland L.M. Appukuttan B. Fong H.K.W. Mol. Vis. 2000; 6: 237-242(http://www.molvis.org/molvis/v6/a32)PubMed Google Scholar). In this paper, we confirm that all-trans-retinal is synthesized in RPE cells and becomes bound to RGR physiologically. We also present evidence for a novel all-trans-retinol dehydrogenase in both cultured ARPE-hRGR and isolated bovine RPE cells. All-trans-[11,12-3H]Retinol (50 Ci/mmol) was obtained from PerkinElmer Life Sciences. All-trans-retinol, all-trans-retinal, and all-trans-retinyl palmitate standards were purchased from Sigma. 11-Cis-retinal was provided by Dr. Rosalie Crouch (Medical University of South Carolina, Charleston). 11-Cis-retinol was prepared by the reduction of 11-cis-retinal in the presence of NaBH4, as described previously (37Landers G.M. Methods Enzymol. 1990; 189: 70-80Crossref PubMed Scopus (21) Google Scholar). NADP and NAD were from Sigma. Organic solvents were HPLC grade. Dichloromethane and hexane were obtained from Fisher. Diethyl ether and methanol were from J. T. Baker Inc. Ethanol was from Gold Shield Chemical Company. ARPE-19 cells, a human RPE cell line, maintain many characteristics of normal RPE cells but do not express detectable levels of RGR opsin (38Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1027) Google Scholar). ARPE-hRGR cells, which stably express human RGR, were obtained by transduction of ARPE-19 cells with a recombinant lentivirus-human RGR vector (36Yang M. Wang X.-G. Stout J.T. Chen P. Hjelmeland L.M. Appukuttan B. Fong H.K.W. Mol. Vis. 2000; 6: 237-242(http://www.molvis.org/molvis/v6/a32)PubMed Google Scholar). The ARPE-19 and ARPE-hRGR cell lines were cultured in Dulbecco's modified Eagle's medium/F-12 (1:1) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and 1% glutamine-penicillin-streptomycin (Irvine Scientific) at 37 °C in a 5% CO2 incubator. Cells at passages 15–25 were grown and maintained at confluence for 1–2 weeks before use in each experiment. ARPE-hRGR and ARPE-19 cells were preincubated overnight with serum-free (retinol-free) RPMI 1640 medium (Invitrogen) at 37 °C in 5% CO2. The cells were then washed with RPMI 1640 medium and incubated in the dark with a mixture of all-trans-[3H]retinol (0.25 μCi/ml, 50 Ci/mmol), 500 μg/ml fatty acid-free bovine serum albumin (BSA), and 0.5% sucrose in RPMI 1640 medium. After incubation for various lengths of time at 37 °C in 5% CO2, the cells were washed with phosphate-buffered saline (PBS), collected by scraping in 2 ml of 67 mm sodium phosphate buffer, pH 6.7, and homogenized with a Dounce glass homogenizer. After the pH was adjusted to 8.0 with 1n NaOH, 38 mg/ml NaBH4 was added to the suspension. The membranes were centrifuged at 150,000 ×g for 1 h at 4 °C. 3H-Labeled proteins were analyzed by gel electrophoresis and fluorography. After separation by 12% SDS-PAGE, the proteins were fixed, and the gel was soaked in ENLIGHTNING Rapid Autoradiography Enhancer (PerkinElmer Life Sciences). The gel was dried and exposed to Kodak X-Omat AR 5 autoradiographic film (Eastman Kodak Co.). The autoradiographic films were scanned, and relative band densities were determined using Scion Image 1.62c software (Scion Corp., Frederick, MD). Bovine eyes were obtained from a local abattoir. The RPE cells were isolated under ambient illumination 2–3 h after enucleation. The anterior segments, lens, vitreous, and neural retina were removed, and the eyecups were washed twice with ice-cold PBS to remove retina debris. The RPE cells were scraped off gently with a metal spatula and collected in ice-cold PBS. The cells were then centrifuged at 800 × g for 5 min at 4 °C. The pellet was washed once with ice-cold PBS and centrifuged again. The following procedures were performed in the dark. The RPE cells were resuspended in 10 ml of Dulbecco's modified Eagle's medium/F-12 medium (1:1) supplemented with all-trans-[3H]retinol (1.0 μCi/ml, 50 Ci/mmol), 500 μg/ml fatty acid-free BSA, and 0.5% sucrose and incubated in a culture flask for 3 h at 37 °C in 5% CO2. After incubation, the cells were collected with a cell scraper, centrifuged, and washed once with PBS. The cells were resuspended in 600 μl of PBS and homogenized with a Dounce glass homogenizer. The sample was treated with NaBH4, as described above. The membranes were then centrifuged at 150,000 ×g for 1 h at 4 °C and analyzed by gel electrophoresis and fluorography to detect 3H-labeled proteins. Alternatively, part of the cell homogenate was saved for Western blot assay, or the retinoids were extracted in the presence of hydroxylamine, as described below. All procedures were performed under dim red light. Retinal isomers were extracted by the method of hydroxylamine derivatization, as described previously (39De Groenendijk G.W.T. Grip W.J. Daemen F.J.M. Anal. Biochem. 1979; 99: 304-310Crossref PubMed Scopus (57) Google Scholar, 40De Groenendijk G.W.T. Grip W.J. Daemen F.J.M. Biochim. Biophys. Acta. 1980; 617: 430-438Crossref PubMed Scopus (141) Google Scholar). Cultured ARPE-hRGR, ARPE-19, or bovine RPE cells were washed with PBS and homogenized in 300 μl of PBS using a Potter-Elvehjem microtissue grinder. The whole homogenate or membranes were mixed with 300 μl of methanol and then 30 μl of 2m NH2OH. After the mixture was incubated at room temperature for 5 min, 300 μl of CH2Cl2was added and mixed by vortexing for 30 s. The organic and aqueous phases were separated by centrifuge at 14,000 × g for 1 min. The aqueous phase was extracted twice more with CH2Cl2. The pooled CH2Cl2 solution was dried under nitrogen gas flow, dissolved in 600 μl of hexane, filtered through glass wool, dried again, and then stored at −80 °C for later analysis. The isomers of retinaloximes were analyzed by HPLC, as described previously (41Ozaki K. Terakita A. Hara R. Hara T. Vision Res. 1986; 26: 691-705Crossref PubMed Scopus (32) Google Scholar, 42Hao W. Chen P. Fong H.K.W. Methods Enzymol. 2000; 316: 413-422Crossref PubMed Google Scholar). The extracted retinaloximes were dissolved in hexane and separated on a Resolve Silica column (3.9 × 150 mm, 5 μm) (Waters Corp.) using a Waters 2690 HPLC module. The running buffer consisted of hexane supplemented with 8% diethyl ether and 0.33% ethanol and was pumped at a flow rate of 0.3 ml/min. Absorbance was measured at 360 nm and 320 nm with a Waters 2487 Dual Wavelength Absorbance Detector. The absorbance peaks were analyzed with the Millennium 32 Chromatography Manager software, version 3.20 (Waters Corp.). The HPLC column was calibrated before each run using all-trans- and 11-cis-retinaloxime and all-trans-retinol standards. Identification of the retinaloxime isomers was based on the retention times of the known retinaloxime products. The proportion of each isomer in the loading sample was determined from the total peak areas of both its syn- and anti-retinaloxime and was based on the following extinction coefficients (ε360, in hexane): all-trans syn, 54,900; all-trans anti, 51,600; 11-cis syn, 35,000; 11-cis anti, 29,600; 13-cis syn, 49,000; and 13-cis anti, 52,100 (40De Groenendijk G.W.T. Grip W.J. Daemen F.J.M. Biochim. Biophys. Acta. 1980; 617: 430-438Crossref PubMed Scopus (141) Google Scholar, 41Ozaki K. Terakita A. Hara R. Hara T. Vision Res. 1986; 26: 691-705Crossref PubMed Scopus (32) Google Scholar). The HPLC system was calibrated with 9.1–0.91 pmol of syn-all-trans-retinaloxime standard. The all-trans-retinyl palmitate standard was eluted in running buffer composed of hexane supplemented with 8% diethyl ether and 0.33% ethanol, or hexane supplemented with 0.3% ethyl acetate. The 3H-labeled retinoids were separated as described above. Four drops of the HPLC eluate were collected manually per fraction and were mixed with 10 ml of ScintiVerse BD scintillation mixture (Fisher Scientific). The amount of radioactivity was measured with a Beckman LS 6000IC counter. Identification of 3H-labeled retinoids was based on the retention times of known standards. ARPE-hRGR and ARPE-19 cells were preincubated overnight in serum-free RPMI 1640 medium at 37 °C in 5% CO2. Total cell membranes were prepared from bovine RPE, cultured ARPE-hRGR, and ARPE-19 cells. The cells were homogenized in buffer containing 67 mm sodium phosphate, pH 6.6, and 250 mm sucrose. The homogenate was centrifuged at 300 × g. Thereafter, the supernatant was centrifuged at 150,000 × g for 1.5 h at 4 °C. The membrane pellets were saved and stored at −80 °C. Microsomal membranes were prepared as described previously (26Shen D. Jiang M. Hao W. Tao L. Salazar M. Fong H.K.W. Biochemistry. 1994; 33: 13117-13125Crossref PubMed Scopus (78) Google Scholar, 36Yang M. Wang X.-G. Stout J.T. Chen P. Hjelmeland L.M. Appukuttan B. Fong H.K.W. Mol. Vis. 2000; 6: 237-242(http://www.molvis.org/molvis/v6/a32)PubMed Google Scholar). The following procedures were performed in darkness or under dim red light. Microsomal membranes were washed and resuspended in buffer containing 50 mm Tris-HCl, pH 7.5, and 0.1% BSA. The reactions were initiated by the addition of the membranes to 50 mm Tris-HCl, pH 7.5, 0.1% BSA, NADP or NAD, and 1 μmall-trans-[3H]retinol (reaction volume, 300 μl). The specific activity of all-trans-[3H]retinol was made 5 Ci/mmol by dilution with unlabeled all-trans-retinol. Solutions of 10 mm NADP or NAD were made fresh and added to give the indicated final concentrations. After incubation at 37 °C for the specified amount of time, the reactions were terminated by the addition of 300 μl of methanol and then 30 μl of 2 mNH2OH. The retinaloximes were extracted and analyzed by HPLC, as described previously. The HPLC fractions were collected, and the amount of radioactivity was determined. When nonradioactive substrates were used, retinol dehydrogenase activity was assayed with 5 μm exogenous all-trans-retinol or 11-cis-retinol in the presence of 200 μm NADP. Retinals were extracted by hydroxylamine derivatization and analyzed by HPLC. The extraction efficiency was monitored by the addition of all-trans-[3H]retinol to the methanol-denatured samples. Under the experimental conditions, the reaction rate for production of all-trans-retinal was linear within the initial 10 min. Microsomal membranes from bovine RPE, ARPE-hRGR, and ARPE-19 cells were washed and resuspended in buffer containing 50 mm Tris-HCl, pH 7.5, and 0.1% BSA. The reactions were initiated in the dark by addition of the membranes to 50 mmTris-HCl, pH 7.5, 0.1% BSA, 200 μm NADP or none, and 0.2 μm all-trans-[3H]retinol (10 μCi/ml, 50 Ci/mmol). After incubation at 37 °C for 30 min, the membranes were sedimented by centrifugation at 150,000 ×g for 1 h at 4 °C. The pellet was washed, resuspended in 1 ml of PBS, and mixed with 38 mg of NaBH4. The membranes were then centrifuged, resuspended in PBS containing 0.1% SDS, and analyzed by gel electrophoresis and fluorography, as described previously. The membranes were resuspended in PBS, and protein concentration was measured by the Bio-Rad protein assay. The samples were separated by 12% SDS-PAGE and then electrotransferred to an Immobilon-P membrane (Millipore). An affinity-purified antipeptide antibody (28Jiang M. Pandey S. Fong H.K.W. Invest. Ophthalmol. Visual Sci. 1993; 34: 3669-3678PubMed Google Scholar, 29Pandey S. Blanks J.C. Spee C. Jiang M. Fong H.K.W. Exp. Eye Res. 1994; 58: 605-614Crossref PubMed Scopus (69) Google Scholar), which is directed against the carboxyl terminus of bovine RGR, and the ECL detection reagents (Amersham Biosciences, Inc.) were used to detect RGR. Prestained protein standards (Invitrogen) were used for molecular weight markers. ARPE-hRGR cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium (1:1) containing 10% fetal bovine serum. The cells were maintained overnight in culture flasks wrapped in aluminum foil and were then processed under dim red light. The cells were washed with PBS, removed with a cell scraper, centrifuged, and resuspended in 300 μl of PBS. The suspended cells were transferred to a quartz cuvette and illuminated at room temperature for 5 min with 470-nm monochromatic light from an Oriel light source, model 66057, equipped with a 150-watt xenon arc lamp. An equivalent aliquot of ARPE-hRGR cells was kept in the dark as a control. Retinal isomers were extracted in the presence of hydroxylamine and and analyzed by HPLC, as described previously (39De Groenendijk G.W.T. Grip W.J. Daemen F.J.M. Anal. Biochem. 1979; 99: 304-310Crossref PubMed Scopus (57) Google Scholar, 40De Groenendijk G.W.T. Grip W.J. Daemen F.J.M. Biochim. Biophys. Acta. 1980; 617: 430-438Crossref PubMed Scopus (141) Google Scholar, 41Ozaki K. Terakita A. Hara R. Hara T. Vision Res. 1986; 26: 691-705Crossref PubMed Scopus (32) Google Scholar, 42Hao W. Chen P. Fong H.K.W. Methods Enzymol. 2000; 316: 413-422Crossref PubMed Google Scholar). We have demonstrated previously that precursor all-trans-retinol can be incorporated into a bound ligand of RGR in cultured ARPE-hRGR cells (36Yang M. Wang X.-G. Stout J.T. Chen P. Hjelmeland L.M. Appukuttan B. Fong H.K.W. Mol. Vis. 2000; 6: 237-242(http://www.molvis.org/molvis/v6/a32)PubMed Google Scholar). ARPE-hRGR cells with high long term expression of human RGR were incubated with all-trans-[3H]retinol in serum-free RPMI 1640 medium. The results indicated that a specific ∼30-kDa protein bound [3H]retinoid (Fig.1A). The ∼30-kDa protein band was not detected in control ARPE-19 cells that lack RGR (36Yang M. Wang X.-G. Stout J.T. Chen P. Hjelmeland L.M. Appukuttan B. Fong H.K.W. Mol. Vis. 2000; 6: 237-242(http://www.molvis.org/molvis/v6/a32)PubMed Google Scholar). The binding of [3H]retinoid to RGR in vivo was dependent on the time of incubation with all-trans-[3H]retinol. Binding occurred within 20 min and reached the highest level by 2 h of incubation with all-trans-[3H]retinol (Fig. 1B). Subsequently, the amount of [3H]retinoid bound to RGR remained relatively steady for up to 6 h of incubation in the dark. Under the experimental conditions, no other membrane protein bound the radiolabeled retinoid. The detection of noncovalently bound [3H]retinoid would not be expected in this assay. The results are consistent with the formation of a Schiff base linkage between [3H]retinal and the ∼30-kDa protein and subsequent reduction of the bond to a stable secondary amine in the presence of sodium borohydride. The specific binding of [3H]retinoid to RGR suggests that all-trans-retinal is produced in ARPE-hRGR cells as a chromophore for the RGR opsin. To verify that all-trans-retinal is synthesized in ARPE-hRGR cells, we extracted 3H-labeled retinals after incubation of the cells in the presence of all-trans-[3H]retinol. The distribution of [3H]retinoids extracted from the ARPE-hRGR cells included a significant pool of retinyl esters (44%), all-trans-retinal (16%), and all-trans-retinol (40%) (Fig. 2, upper panel). The specific activity of all-trans-[3H]retinal from the cells was ∼51 Ci/mmol, as determined from the amount of radioactivity and the corresponding absorbance peak of all-trans-retinal in the HPLC chromatogram. This specific activity was virtually identical to that of the added exogenous all-trans-[3H]retinol. The control ARPE-19 cells had a distinct profile of [3H]retinoids and contained retinyl esters (66%), all-trans-retinal (3%), and all-trans-retinol (31%) (Fig. 2, lower panel). Neither type of cell contained significant amounts of 11-cis-retinal or 11-cis-retinol. The results indicate that the retinal isomer bound to RGR in the dark in ARPE-hRGR cells is solely all-trans-retinal. ARPE-hRGR cells cultured in growth medium also synthesize the all-trans-retinal chromophore from a precursor in serum (Fig. 3). The fresh culture medium supplemented with 10% fetal bovine serum contains at least 30 pmol/ml of all-trans-retinol. All-trans-retinal was found in ARPE-hRGR cells (Fig. 3A) but not in control ARPE-19 cells that were maintained in serum-containing medium (Fig.3B). When the ARPE-hRGR cells were preincubated in serum-free medium for 16 h, all-trans-retinal fell to an undetectable level (Fig. 3C). Irradiation of the ARPE-hRGR cells resulted in stereospecific isomerization of the endogenous all-trans-retinal to 11-cis-retinal (Fig. 4). The proportion of all-trans- to 11-cis-retinal in the ARPE-hRGR cells was 2.60:0 pmol in the dark and 1.12:0.55 pmol after illumination with 470-nm monochromatic light. Photoisomerization to 13-cis-retinal was not detected.Figure 4Photoisomerization of all-trans-retinal in ARPE-hRGR cells. ARPE-hRGR cells (∼1.5 × 107) were cultured in the dark in serum-containing (retinol-containing) medium. The cells were suspended in PBS and either kept in the dark (upper panel) or illuminated with 470-nm monochromatic light at 410 lux (lower panel). The retinal isomers were extracted by hydroxylamine derivatization and analyzed by HPLC. 11-cis, 11-cis-retinal; all, all-trans-retinal (in the form of syn-retinaloximes).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The production of the all-trans-retinal chromophore from all-trans-retinol requires an oxidation reaction. The proposed enzymatic reaction may be catalyzed by a previously uncharacterized all-trans-retinol dehydrogenase in the RPE. We tested microsomal membranes from ARPE-hRGR cells for all-trans-retinol dehydrogenase activity in the presence of nicotinamide dinucleotide cofactors. The membranes were capable of producing all-trans-retinal from all-trans-retinol in the presence of NADP but not NAD (Fig.5A). At concentrations of >8 μm NADP, the synthesis of all-trans-retinal increased 5-fold compared with activity without cofactor or with NAD. The putative NADPH-dependent retinol dehydrogenase strongly preferred the all-trans isomer of retinol and did not react with 11-cis-retinol (Fig. 5B). The microsomal membranes from control ARPE-19 cells also contained all-trans-retinol dehydrogenase activity in the presence of NADP (results not shown). Although the parental ARPE-19 cells maintain many characteristics of RPE cells, they are highly deficient in the expression of several RPE proteins, including RGR, and may not function normally. To demonstrate that the synthesis of all-trans-retinal and specific binding of the chromophore to RGR are physiological properties of normal RPE cells, we incubated freshly isolated bovine RPE cells with precursor all-trans-[3H]retinol (Fig.6). The uptake of all-trans-[3H]retinol resulted in radiolabeling of a specific protein band that was equal in size to the RGR opsin (Fig. 6A). RGR was detectable by Western blot assay after the short term incubation of the bovine RPE cells (Fig.6B) but not after 5 days of culture in serum-containing medium (results not shown). After 3 h of incubation, the cells synthesized retinyl esters (62%), all-trans-retinal (23%), 11-cis-retinal (2%), and 11-cis-retinol (2%) (Fig. 6C). All-trans-retinol (7%) and a small amount of 13-cis-retinal (3%) were also extracted from the cells. Because the accumulation of all-trans-retinal in ARPE-hRGR and ARPE-19 cells was dependent on the presence of RGR (Figs.2 and 3), the synthesis or stability of all-trans-retinal may be connected with its binding to the opsin. To investigate chromophore synthesis and binding to RGR in an in vitrosystem, we incubated isolated RPE membranes with all-trans-[3H]retinol in the presence of NADP and analyzed the binding of [3H]retinoid to RGR. The results indicated that all-trans-[3H]retinal was synthesized in membranes and bound directly to RGR with high specificity (Fig. 7). Nontransduced ARPE-19 cells did not contain the radiolabeled ∼30-kDa protein band (Fig. 7A). The binding of [3H]retinoid to RGR in bovine RPE microsomes was stimulated 3.1-fold by the addition of NADP (Fig. 7B). The results indicate that all-trans-retinal, which is synthesized by the membrane-associated all-trans-retinol dehydrogenase, can be channeled to the binding site of RGR in the cell-free system. Little is known about the synthesis of all-trans-retinal as a chromophore for the RGR opsin in RPE cells. The endogenous all-trans-retinal bound to RGR may be synthesized directly from all-trans-retinol that is generated upon phototransduction or from serum all-trans-retinol. This reaction would require a novel all-trans-retinol dehydrogenase in the RPE. In this paper, we demonstrate further evidence for the oxidation of precursor all-trans-retinol in RPE cells and its physiological incorporation into the chromophore of RGR in the dark. After its uptake into RPE cells, all-trans-retinol is converted rapidly into retinyl esters. The esterification of retinol is catalyzed by lecithin-retinol acyltransferase, the activity of which is higher than is known for most other visual cycle enzymes. In addition to esterification, our results indicate that retinol is oxidized efficiently and that significant amounts of all-trans-retinal accumulate in ARPE-hRGR and bovine RPE cells in the absence of light. Despite the high lecithin-retinol acyltransferase activity, the ratio of all-trans-retinal to retinyl esters formed after 3 h of incubation with precursor all-trans-retinol is 1:2.8 and 1:2.7 in ARPE-hRGR and bovine RPE cells, respectively. Because the specific activity of all-trans-[3H]retinal in the ARPE-hRGR cells was close to that of the precursor all-trans-[3H]retinol, all-trans-retinol can be converted directly into all-trans-retinal without entering the pool of endogenous retinyl esters. The results suggest that retinol esterification by lecithin-retinol acyltransferase and oxidation by an all-trans-retinol dehydrogenase represent an early and important bifurcation point in the processing of retinol after uptake into RPE cells. The esterification and oxidation are both critical reactions for continuance of the visual cycle in that they catalyze formation of the substrate for a putative isomerohydrolase (18Law W.C. Rando R.R. Biochemistry. 1988; 27: 4147-4152Crossref PubMed Scopus (30) Google Scholar, 19Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (155) Google Scholar, 20Canada F.J. Law W.C. Rando R.R. Yamamoto T. Derguini F. Nakanishi K. Biochemistry. 1990; 29: 9690-9697Crossref PubMed Scopus (48) Google Scholar) and the chromophore for RGR, respectively. The difference in steady-state levels of all-trans-retinal in ARPE-hRGR and ARPE-19 cells indicates that the accumulation of all-trans-retinal is highly dependent on the presence of RGR. Although the membranes from ARPE-19 and other cells contain constitutive NADPH-dependent all-trans-retinol dehydrogenase activity and are capable of synthesizing all-trans-retinal from all-trans-retinol, all-trans-retinal generally does not accumulate in these cells (43Williams J.B. Pramanik B.C. Napoli J.L. J. Lipid Res. 1984; 25: 638-645Abstract Full Text PDF PubMed Google Scholar, 44McCormick A.M. Napoli J.L. J. Biol. Chem. 1982; 257: 1730-1735Abstract Full Text PDF PubMed Google Scholar). Normal RPE and cultured ARPE-hRGR cells have uniquely elevated levels of all-trans-retinal, which may be stabilized by covalent binding to RGR. The sequestration of all-trans-retinal to RGR would block the retinoid from reversible reduction, oxidation to retinoic acid, formation of nonspecific Schiff bases, and possible generation of harmfulbis-retinoid,N-retinylidene-N-retinylethanolamine compounds within the RPE (45Parish C.A. Hashimoto M. Nakanishi K. Dillon J. Sparrow J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14609-14613Crossref PubMed Scopus (407) Google Scholar). Failure to detect all-trans-retinal in RPE may result from inefficient extraction methods or the loss of RGR expression in cultured cells. The irradiation of ARPE-hRGR cells resulted in stereospecific isomerization of all-trans-retinal to 11-cis-retinal, indicating a functional recombinant RGR and physiological activity of the endogenous all-trans-retinal. With the ARPE-hRGR cells, the 11-cis retinoid metabolic pathways that lie downstream of the RGR opsin can be analyzed under a variety of culture and lighting conditions. Our finding of all-trans-retinal synthesis and accumulation in RPE cells is not inconsistent with data from previous studies. Timmers et al. (46Timmers A.M. van Groningen-Luyben D.A. de Grip W.J. Exp. Eye Res. 1991; 52: 129-138Crossref PubMed Scopus (11) Google Scholar) have demonstrated a slow constant increase in the synthesis of all-trans-retinal in isolated bovine RPE cells. Others have reported NADPH-dependent all-trans-retinol dehydrogenase activity in RPE membranes. As early as 1975, Zimmerman et al. (14Zimmerman W.F. Lion F. Daemen F.J.M. Bonting S.L. Exp. Eye Res. 1975; 21: 325-332Crossref PubMed Scopus (50) Google Scholar, 15Zimmerman W.F. Exp. Eye Res. 1976; 23: 159-164Crossref PubMed Scopus (24) Google Scholar) noted that RPE microsomes contain a relatively high amount of all-trans-retinol dehydrogenase activity in membrane vesicles that fractionate separately from contaminant membranes with the photoreceptor all-trans-retinol dehydrogenase. These interesting findings further support the notion that the all-trans-retinal chromophore is synthesized by a novel all-trans-retinol dehydrogenase in the RPE. Nevertheless, the putative RPE all-trans-retinol dehydrogenase has not been identified unequivocally. Further characterization of the all-trans-retinol dehydrogenase in ARPE-hRGR and isolated RPE cells is required. Our results indicate that the retinol dehydrogenase in ARPE-hRGR cells is membrane-bound, prefers NADP as the cofactor in oxidation, and has high substrate stereospecificity for all-trans-retinol versus the 11-cisisomer. We hypothesize that this enzyme is responsible for chromophore synthesis in ARPE-hRGR cells. To investigate the coupling between chromophore synthesis and its binding to RGR, we examined the reactions in vitro. In a cell-free membrane system, RGR had apparent and specific access to the newly synthesized all-trans-retinal. The microsomal membranes of both the human ARPE-hRGR cell line and bovine RPE cells were sufficient for the synthesis of all-trans-retinal and its channeling to the binding site of RGR. The results indicate that both cells contain a membrane-bound all-trans-retinol dehydrogenase and may have a highly conserved system of providing the chromophore of RGR. The binding of all-trans-[3H]retinal to RGR was enhanced in the presence of NADP, although RGR was also radiolabeled in the absence of added cofactor. Like preparations of various dehydrogenases (47Kato T. Berger S.J. Carter J.A. Lowry O.H. Anal. Biochem. 1973; 53: 86-97Crossref PubMed Scopus (221) Google Scholar), it is likely that the washed membranes still contained a significant amount of endogenous enzyme-bound NADP that allowed background synthesis of all-trans-[3H]retinal. The physical relationship between the RPE all-trans-retinol dehydrogenase and RGR is unknown. The all-trans-retinal chromophore of RGR may be synthesized also from precursor β,β-carotene by the oxidative cleavage activity of an enzyme, β,β-carotene-15,15′-dioxygenase (Bcdo) that has been found in the RPE (48Redmond T.M. Gentleman S. Duncan T. Yu S. Wiggert B. Gantt E. Cunningham Jr., F.X. J. Biol. Chem. 2001; 276: 6560-6565Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 49Yan W. Jang G.F. Haeseleer F. Esumi N. Chang J. Kerrigan M. Campochiaro M. Campochiaro P. Palczewski K. Zack D.J. Genomics. 2001; 72: 193-202Crossref PubMed Scopus (137) Google Scholar). The oxidative cleavage of β,β-carotene by Bcdo would directly generate all-trans-retinal under dark or photic conditions. The all-trans-retinal synthesized from β,β-carotene may then bind to RGR physiologically. In humans, β,β-carotene and vitamin A are available to the RPE at plasma levels of 0.171–0.216 and 0.548–0.587 μg/ml, respectively (50Schünemann H.J. Grant B.J.B. Freudenheim J.L. Muti P. Browne R.W. Drake J.A. Klocke R.A. Trevisan M. Am. J. Respir. Crit. Care Med. 2001; 163: 1246-1255Crossref PubMed Scopus (141) Google Scholar). On the other hand, only all-trans-retinol is transported from the photoreceptors to the RPE as an intermediate in the visual cycle. Consequently, the RPE all-trans-retinol dehydrogenase would be required to process all-trans-retinol rapidly in a continuous photic visual cycle. Like rhodopsin, the RGR opsin relies on retinol dehydrogenases for the processing of its retinal chromophore in biochemical pathways that lie upstream and downstream of photoisomerization. In contrast to the two-cell rhodopsin system, the all-trans-retinal chromophore of RGR is synthesized by a proximal retinol dehydrogenase within membranes of the RPE itself. After irradiation of RGR, the bound 11-cis-retinal is dissociated and converted to the alcohol by 11-cis-retinol dehydrogenase (51Chen P. Lee T.D. Fong H.K.W. J. Biol. Chem. 2001; 276: 21098-21104Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). RGR and the 11-cis-retinol dehydrogenase copurify consistently and may be tightly associated in a protein complex. The evidence for functional interaction of all-trans-retinol dehydrogenase, RGR opsin, and 11-cis-retinol dehydrogenase suggests a model for the flow of retinoids in the photic visual cycle (Fig.8). In this model, all-trans-retinal is converted to 11-cis-retinal by rapid photoisomerization, and the overall rate of conversion of all-trans retinoids to the 11-cis isomer is limited by binding kinetics and the enzymatic reactions catalyzed by the retinol dehydrogenases. The ARPE-hRGR and isolated RPE cells provide a promising approach to compare and analyze the biochemistry and kinetics of retinoid processing in the RGR system. We thank Daiwei Shen and Pu Chen for technical assistance.
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