Molecular Characterization of a Novel Short-chain Dehydrogenase/Reductase That Reduces All-trans-retinal

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors. The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors. Vitamin A and its metabolites are active participants in a number of important physiological processes (see Fig. 1). All-trans-retinol is the precursor of other naturally occurring retinoids and appears to be indispensable in reproduction (1Davis J.T. Ong D.E. Biol. Reprod. 1995; 52: 356-364Crossref PubMed Scopus (39) Google Scholar). All-trans-retinal is an intermediate in the production of all-trans- and perhaps 9-cis-retinoic acids, which act as hormones, affecting important biological processes such as morphogenesis and differentiation through their interaction with ligand-gated transcription factors (2Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2610) Google Scholar). In most tissues, retinals do not accumulate in high concentrations, perhaps because of their chemical reactivity and potential toxicity. However, the visual system is unique in that its function is based on retinals and as a consequence, they accumulate in extraordinarily high concentrations, up to 3 mm in the outer segment of photoreceptor cells (3Dowling J.E. The Retina: An Approachable Part of the Brain. Harvard University Press, Cambridge1987Google Scholar). 11-cis-Retinal is the chromophore for all known visual pigments; however, this isomer is not found in other tissues in significant amounts. Thus, metabolism of 11-cis-retinal and its photoproduct all-trans-retinal, in particular, are of prime importance in the visual system. In the retina, light isomerizes 11-cis-retinal, activates rhodopsin, and initiates the process of phototransduction by which the visual sensation is produced. The product of photoisomerization, all-trans-retinal, enters into a series of reactions that regenerate the 11-cis-configuration and the native visual pigment. At any given level of illumination, a steady state is established in which the visual pigment photoisomerization rate is equal to and opposed by the rate of visual pigment regeneration (4Alpern M. J. Physiol. 1971; 217: 447-471Crossref PubMed Scopus (115) Google Scholar, 5Alpern M. Maaseidvaag F. Ohba N. Vision Res. 1971; 11: 539-549Crossref PubMed Scopus (54) Google Scholar). Thus, phototransduction and regeneration reactions are irrevocably linked in a cyclical process called the visual cycle (6Wald G. Science. 1968; 162: 230-239Crossref PubMed Scopus (805) Google Scholar). The regeneration reactions begin with the NADPH-dependent reduction of all-trans-retinal. The enzyme catalyzing this reaction, all-trans-retinol dehydrogenase (RDH), 1The abbreviations used are: RDHall-trans-retinol dehydrogenaseHPLChigh performance liquid chromatographyESTexpressed sequence tagRACErapid amplification of cDNA endsPCRpolymerase chain reactionkbkilobaseROSrod outer segmentHSDhydroxysteroid dehydrogenaseRPEretinal pigment epitheliumOSouter segmentSDRshort-chain dehydrogenase/reductaseMES(2-(N-morpholino) ethanesulfonic acid. plays an important role in photoreceptor physiology that is only beginning to be understood. RDH catalyzes the rate-limiting reaction of the visual cycle in rodent rod photoreceptors (7Saari J.C. Garwin G.G. Van Hooser J.P. Palczewski K. Vision Res. 1998; 44: 17-23Google Scholar) and plays an important role in phototransduction as the final step in the quenching of photoactivated rhodopsin (8Hofmann K.P. Pulvermüller A. Buczylko J. Van Hooser J.P. Palczewski K. J. Biol. Chem. 1992; 267: 15701-15706Abstract Full Text PDF PubMed Google Scholar, 9Palczewski K. Saari J.C. Curr. Opin. Neurobiol. 1997; 7: 500-504Crossref PubMed Scopus (69) Google Scholar). Furthermore, the activity of RDH controls the level of all-trans-retinal in the retina. This retinoid is responsible, in part, for setting the level of sensitivity of the visual system (10Palczewski K. Jager S. Buczylko J. Crouch R.K. Bredberg D.L. Hofmann K.P. Asson-Batres M.A. Saari J.C. Biochemistry. 1994; 33: 13741-13750Crossref PubMed Scopus (133) Google Scholar) and may play a role in retinal pathology. All-trans-retinal has been shown to be a constituent of the major fluorescent component of lipofuscin (Fig.1), a pigment that accumulates in retinal pigment epithelium (RPE) during aging and in pathological conditions (12Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). all-trans-retinol dehydrogenase high performance liquid chromatography expressed sequence tag rapid amplification of cDNA ends polymerase chain reaction kilobase rod outer segment hydroxysteroid dehydrogenase retinal pigment epithelium outer segment short-chain dehydrogenase/reductase (2-(N-morpholino) ethanesulfonic acid. The reactions and enzymes of phototransduction have been thoroughly characterized at a molecular level (12Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar); however, the molecular details of the visual cycle remain poorly characterized. The amino acid sequence is known for only one enzyme of the cycle, 11-cis-retinol dehydrogenase (13Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The remaining reactions have been characterized only as enzymatic activities in membrane preparations (14Saari J.C. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, New York1994: 351-385Google Scholar, 15Rando R.R. Chem. Biol. 1996; 3: 255-262Abstract Full Text PDF PubMed Scopus (74) Google Scholar). In the present study, we report the molecular cloning of a cDNA expressing a short-chain dehydrogenase/reductase (retSDR1), and demonstrate that this enzyme is highly abundant in cone outer segments. Expression of retSDR1 in other cells of the retina, and in other tissues, suggests that it may be involved more generally in retinoid metabolism. The molecular characterization of retSDR1 will open the way for further studies of this visual cycle enzyme, and of its potential role in retinal cone dystrophies. All-trans retinal was obtained from Sigma, [3H]NaBH4 was from NEN Life Science Products, and 11-cis-retinal was a gift from the National Eye Institute. Retinoids were purified by HPLC (16Saari J.C. Bredberg L. Garwin G.G. J. Biol. Chem. 1982; 257: 13329-13333Abstract Full Text PDF PubMed Google Scholar). A search of the EST data base with primer FH28 (5′-GGCCTGGTCAACAATGCTGG-3′) was performed in the GenBank data base with FASTA from the GCG package. Amino acid sequence alignments were generated with PILEUP. Total RNA was isolated from human retinal tissue, obtained from the Lions Eye Bank at the University of Washington, using the Ultraspec RNA Isolation system (Biotecx, Inc.) and reverse transcribed with oligo(dT) (Life Technologies, Inc.). Rapid amplification of cDNA ends (RACE) was performed to amplify the 3′-end of the selected EST cDNA using the Marathon cDNA amplification kit (CLONTECH) and the Expand high fidelity PCR system (Boehringer Mannheim). 3′-RACE was primed with an internal gene-specific primer (FH42: 5′-ATGGCGTGGAAACGGCTGGG-3′) and the Marathon adaptor primer (AP1) (CLONTECH). Samples were heated at 95 °C for 5 min and amplified for 40 cycles at 94 °C for 30 s, and 68 °C for 4 min. A secondary PCR reaction was carried out using the AP2 Marathon adapter primer and a nested gene-specific primer (FH41: 5′-CGGNGGCGGGAGAGGNATCGGG-3′) as described above. The 5′-end of the selected EST cDNA was amplified from a human retina cDNA library with primers FH43 (5′-TCCAAGAACTGGCCCAGGGTGTTG-3′, a gene-specific primer) and λgt10S. After heating at 95 °C for 5 min, the reactions were cycled 5 times through 94 °C for 30 s, 72 °C for 4 min; 5 times through 94 °C for 30 s, 70 °C for 4 min; and 30 times through 94 °C for 5 s, 68 °C for 4 min. Two amplification products for each PCR were cloned into pCRTM2.1 vector (TA cloning kit, Invitrogen) and sequenced by dyedeoxy-terminator sequencing (ABI-Prism, Perkin-Elmer). Total RNA was isolated from bovine retinas using the Ultraspec RNA isolation system (Biotecx, Inc.). cDNA used in the PCR was prepared by reverse transcription with oligo(dT) from 20 μg of total RNA in a 20-μl reaction (Life Technologies, Inc.). 5′-RACE PCR was carried out using 0.5 μl of cDNA and the Expand high fidelity PCR system (Boehringer Mannheim) with a gene-specific primer FH43 (5′-TCCAAGAACTGGCCCAGGGTGTTG-3′) and the Marathon adaptor primer (AP1) (CLONTECH). Samples were heated at 95 °C for 5 min and amplified for 5 cycles at 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 4 min and for 35 cycles at 94 °C for 30 s, and 68 °C for 4 min. A secondary PCR amplification was carried out using the AP2 Marathon adapter primer and primer FH43, for 5 cycles at 94 °C for 30 s, and 72 °C for 3 min; 5 cycles at 94 °C for 30 s, and 70 °C for 3 min; and 25 cycles at 94 °C for 30 s, and 68 °C for 3 min. 3′-RACE was performed with primers FH42 (5′-ATGGCGTGGAAACGGCTGGG-3′) and the Marathon adaptor primer (AP1) (CLONTECH) followed by a secondary PCR amplification carried out using the AP2 Marathon adapter primer and a nested gene-specific primer FH41 (5′-CGGNGGCGGGAGAGGNATCGGG-3′) using the same PCR conditions described for 5′-RACE. Two amplification products for each PCR were cloned in pCRTM2.1 vector (TA cloning kit, Invitrogen) and sequenced by dyedeoxy-terminator sequencing (ABI-Prism, Perkin-Elmer). The mouse cDNA sequence was amplified from a λZAPII both oligo(dT)- and random-primed mouse retinal cDNA library (obtained from Dr. W. Baehr, University of Utah) in two overlapping fragments. The 5′-end was amplified with primers FH59 (5′-TCATCGTCACTGTCCATCAAGCTT-3′, gene-specific primer) and FH100 (5′-TTGTAATACGACTCACTATAGGGCG-3′, covering T7 primer) for 5 cycles at 94 °C for 30 s, and 72 °C for 3 min; for 5 cycles at 94 °C for 30 s, and 70 °C for 3 min; and for 30 cycles at 94 °C for 30 s, and 68 °C for 3 min. A secondary nested PCR amplification was carried out with primers FH100 and FH40 (5′-CACGGCGGCATTGTTCACCAG-3′) for 5 cycles at 94 °C for 30 s, 64 °C for 30 s, and 68 °C for 1 min and for 25 cycles at 94 °C for 30 s, and 68 °C for 1 min. The 3′end was amplified with primers FH100 and FH54 (5′-TCGGGACTTGTCGCGGGAGTCA-3′, gene-specific primer) for 5 cycles at 94 °C for 30 s, and 72 °C for 3 min; for 5 cycles at 94 °C for 30 s, and 70 °C for 3 min; and for 30 cycles at 94 °C for 30 s, and 68 °C for 3 min. A secondary PCR amplification was performed with primers FH100 and FH58 (5′-ATCGGACGCCACCTCGCTCGGG-3′, nested gene-specific primer) for 25 cycles at 94 °C for 30 s, and 72 °C for 2 min. Two amplification products for each PCR were sequenced by dyedeoxy-terminator sequencing (ABI-Prism, Perkin-Elmer) after subcloning into pCRTM 2.1 vector (TA cloning kit, Invitrogen). Bovine retina poly(A)+RNA was purified from total RNA using the mRNA purification kit (Amersham Pharmacia Biotech), resolved by agarose gel electrophoresis in the presence of 0.66 m formaldehyde and transferred to nylon membranes. Hybridization with a 1.2-kb bovine dehydrogenase cDNA 32P-labeled with the Megaprime DNA labeling systems (Amersham Pharmacia Biotech) was performed in 40% formamide, 10% dextran sulfate, 1% SDS, 1 m NaCl, 50 mmTris, pH 7.4, 25 μg/ml herring sperm DNA and washed in 0.1× SSC at 58 °C. A human multiple tissue Northern blot containing 2 μg of poly(A)+ RNA from various human tissue (CLONTECH) was hybridized with the dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase 32P-labeled cDNA according to the manufacturer's instructions. The full-length 1.4-kb human retSDR1 cDNA was generated by cloning the 5′ and 3′ ends using the NarI restriction site present in the overlapping region. The coding sequence for retSDR1 was amplified from this plasmid by PCR with primers FH48 (5′-CATATGGCGTGGAAACGGCTGGGC-3′), which placed a NdeI restriction site on the ATG, and FH49 (5′-CTAGTGATGGTGATGGTGATGTGTCCGCCCTTTGAAAGTGTT-3′), which placed a 6-His tag at the 3′-end and using a denaturing temperature of 94 °C for 30 s, and an annealing and extension temperature of 68 °C for 2.5 min. The purified fragment was cloned into pCRTM 2.1 vector (TA cloning kit, Invitrogen). The insert was transferred as a NdeI-BamHI fragment into NdeI and BamHI sites of pET-3b (Novagen) and expressed in BL21(DE3) pLysS after induction with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. Proteins insoluble in bacteria were purified on Ni-NTA resin (Qiagen) under denaturing conditions following the manufacturer's instructions. The sequences of all constructs presented in this study were verified by DNA sequencing. The coding sequence for retSDR1 was amplified from the retSDR1 plasmid by PCR with primers FH48 (5′-CATATGGCGTGGAAACGGCTGGGC-3′), which placed a NdeI restriction site on the ATG, and FH50 (5′-CTATGTCCGCCCTTTGAAAGTGTT-3′) using a denaturing temperature of 94 °C for 30 s, and an annealing and extension temperature of 68 °C for 2.5 min. The purified fragment was cloned into pCRTM 2.1 vector (TA cloning kit, Invitrogen), designated pFR415. A fragmentXbaI-HindIII from pFR415 covering the human retSDR1 coding sequence was cloned between the XbaI and HindIII sites of pFastBac1 expression vector (Life Technologies, Inc.). The expression cassette was then transferred into the baculovirus shuttle vector (bacmid) by transposition. Sf9 insect cells were transfected with the recombinant bacmid using cationic liposome-mediated transfection (CellFECTIN reagent, Life Technologies, Inc.) according to the manufacturer's protocol. The coding sequence for 11-cis-retinol dehydrogenase was amplified from bovine retina cDNA by PCR with primers FH51 (5′-CATATGTGGCTGCCTCTGCTGCTG-3′), which placed a NdeI restriction site on the ATG, and FH52 (5′-TTAGTAGACTGTCTGGGCAGG-3′) for 32 cycles at 94 °C for 30 s, and 68 °C for 2.5 min and cloned into pCRTM 2.1 vector (TA cloning kit, Invitrogen) (designated pFR425) and then transferred into insect cells following the same procedure as for human retSDR1. For the expression of recombinant proteins, cells cultured at 27 °C in Sf-900 II SFM (Life Technologies, Inc.) were harvested by centrifugation at 1200 ×g, 72–96 h after infection. Variable expression and activity levels were observed between different preparations. The reasons for these differences were not investigated. An ATG and NdeI restriction site were introduced upstream of the amino acid 36 of retSDR1 by PCR on pFR415 plasmid with primers FH64 (5′-CATATGCTGTCGCGGGAGAACGTCC-3′) and FH50 (5′-CTATGTCCGCCCTTTGAAAGTGTT-3′) through 32 cycles using a denaturing temperature of 94 °C for 30 s and an annealing and extension temperature of 68 °C for 2.5 min. The purified fragment was cloned into pCRTM 2.1 vector (TA cloning kit, Invitrogen) and sequenced by dyedeoxy-terminator sequencing (ABI-Prism, Perkin-Elmer) and then transferred into insect cells following the same procedure as for the full-length retSDR1. RDH activity was measured by following the transfer of 3H from [3H]NADPH to all-trans-retinal (reduction) (17Saari J.C. Bredberg D.L. Garwin G.G. Buczylko J. Wheeler T. Palczewski K. Anal. Biochem. 1993; 213: 128-132Crossref PubMed Scopus (16) Google Scholar). [3H]NADPH was synthesized by chemical reduction of NADP (Sigma) with [3H]NaBH4 (18Chaykin S. Chakraverty K. King L. Watson J.G. Biochim. Biophys. Acta. 1966; 124: 1-12Crossref PubMed Scopus (19) Google Scholar), and purified on a Nucleosil NB 10 column (Macherey-Nagel) (17Saari J.C. Bredberg D.L. Garwin G.G. Buczylko J. Wheeler T. Palczewski K. Anal. Biochem. 1993; 213: 128-132Crossref PubMed Scopus (16) Google Scholar). Cells were homogenized in water containing 2 mm benzamidine, 0.1 mmNADP, 0.5 mm dithiothreitol. Cell membranes were washed in the same solution, pelleted by centrifugation at 40,000 rpm for 5 min, and resuspended in 5 times the volume of the pellet. The reaction mixture contained 10 mm MES, pH 5.5, 14.2 μm[3H]NADPH (83,000 dpm/nmol, both pro-4S and pro-4R isomers), and membranes from insect cells (2.5–3.0 mg/ml protein), in a final volume of 60 μl. All-trans-retinal or 11-cis-retinal in ethanol was added at a concentration of 10 μm (final concentration of ethanol <1%, v/v). Reactions were quenched with 400 μl of methanol, 50 μl of 10 mm NH2OH, pH 7.0, 50 μl of 0.1 m NaCl and extracted with 500 μl of petroleum ether. Radioactivity was determined in 250 μl of the organic phase. [3H]Pro-4S-NADPH was prepared from NADP and [2-3H,3-3H]glutamic acid (DuPont) in the presence of glutamate dehydrogenase (Sigma) as described previously (19Nakajima N. Nakamura K. Esaki N. Tanaka H. Soda K. J. Biochem. ( Tokyo ). 1989; 106: 515-517Crossref PubMed Scopus (6) Google Scholar). NADPH was purified on Mono Q column (Amersham Pharmacia Biotech) using a salt gradient from 0 to 500 mm NaCl in 10 mm(1,3-bis[tris(hydroxymethyl)methylamino]propane), pH 7.5, during 60 min at a flow rate of 1 ml/min. NADPH was eluted at ∼250 mm NaCl. Steroid dehydrogenase assays were performed using membranes from insect cells infected with a virus carrying the retSDR1 construct or a control helper virus (bacmid transposed with pFASTBACD). The following steroids were purchased from Sigma: estrone (1,3,5[10]-estratrien-3-ol-17-one), β-estradiol (1,3,5[10]-estratriene-3,17β-diol), 4-androstene-3,17-dione, testosterone (4-androsten-17β-ol-3-one), 11-dehydrocorticosterone (4-pregnen-21-ol-3,11,20-trione), corticosterone (4-pregnene-11β,21-diol-3,20-dione), 5α-androstane-3,17-dione, androsterone (5α-androstan-3α-ol-17-one), 5α-androstane-17β-ol-3-one, and 5α-androstane-3α,17β-diol (dihydroandrosterone). The steroids were dissolved in ethanol (15–50 mm stock solutions). The reduction of steroids was carried out in 25 mm MES, pH 5.5, containing 1 mg/ml membrane proteins, 20 μm pro-4S-[3H]NADPH and 0.5 mm steroids for 10 min at 30 °C. Steroids were extracted with 10-fold excess (volume) of dichloromethane. As a positive control, the reduction of 4-androstene-3,17-dione to testosterone by liver microsome was used. The product was identified by thin layer chromatography (40Chai X. Boerman M.H.E.M. Zhai Y. Napoli J.L. J. Biol. Chem. 1995; 270: 3900-3904Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Liver microsomes were prepared from fresh bovine liver by flotation in 45% sucrose, similar to the preparation of rod outer segments. BALB/c mice were immunized with his-tagged human retSDR1 expressed in E. coli retSDR1. A hybridoma cell line producing specific antibodies was prepared by fusion of BALB/c mouse myeloma cells with spleen cells from the immunized mouse according to the procedure described by Harlow and Lane (20Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 139-243Google Scholar). Supernatants from culture of the hydridomas were screened by immunocytochemistry and by immunoblotting. Subsequent cloning yielded mAb A11, which was an IgG1. The epitope recognized by mAb A11 has not been characterized in detail; however, it must be present in the C-terminal half of the protein because mAb A11 recognized a truncated protein comprising amino acid residues 169–303 (data not shown). An anti-11-cis-retinol dehydrogenase rabbit polyclonal antibody was generated using a peptide whose sequence (SKFLGLEAFSDSLRRDV) encompassed the catalytic region of the enzyme. The peptide was coupled to a carrier protein and used for immunization as described previously (21Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A, H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Crossref PubMed Scopus (82) Google Scholar). Anti-retSDR1 and anti-11-cis-retinol dehydrogenase reacted specifically with the dehydrogenase used for immunization. Samples of bovine, monkey and human retina were processed with sense and antisense digoxygenin-labeled riboprobes as described previously (21Otto-Bruc A. Fariss R.N. Haeseleer F. Huang J. Buczylko J. Surgucheva I. Baehr W. Milam A, H. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4727-4732Crossref PubMed Scopus (82) Google Scholar). The anterior segments of bovine, monkey, and human eyes were removed and the eye cups immersed in 4% paraformaldehyde, 0.13 m sodium phosphate, pH 7.4, at 4 °C for 6 h. For immunoperoxidase staining, the whole mount retinal sections were processed as described by Milam et al. (22Milam A.H. Possin D.E. Huang J. Fariss R.N. Flannery J.G. Saari J.C. Vis. Neurosci. 1997; 14: 601-608Crossref PubMed Scopus (14) Google Scholar). For confocal microscopy, agarose-embedded retinal sections (100 μm) were processed as described previously (22Milam A.H. Possin D.E. Huang J. Fariss R.N. Flannery J.G. Saari J.C. Vis. Neurosci. 1997; 14: 601-608Crossref PubMed Scopus (14) Google Scholar, 23Saari J.C. Huang J. Possin D.E. Fariss R.N. Leonard J. Garwin G.G. Crabb J.W. Milam A.H. Glia. 1997; 21: 259-268Crossref PubMed Scopus (38) Google Scholar). A 1.5-kb cDNA probe covering human retSDR1 was labeled with biotin-11-dUTP by nick translation (Life Technologies, Inc.). Metaphase chromosome preparations from lymphocytes of a human male were obtained using 75 mm KCl as a hypotonic buffer and methanol:acetic acid (3:1, v/v) as fixative. The hybridization was carried out as described previously by Edelhoff et al. (24Edelhoff S. Ayer D.E. Zervos A.S. Steingrimsson E. Jenkins N.A. Copeland N.G. Eiseman R.N. Brent R. Disteche C.M. Oncogene. 1994; 9: 665-668PubMed Google Scholar). Hybridization signals were detected using a detection system from Vector Laboratories. After incubation with goat anti-biotin antibody, the slides were rinsed in modified 2× SSC (2× SSC, 0.1% Tween 20, 0.15% bovine serum albumin). A second incubation with fluorescein-labeled anti-goat IgG and a rinse in modified 2× SSC followed. The chromosomes were banded using Hoechst 33258-actinomycin D staining and counterstained with propidium iodide. The chromosomes and hybridization signals were visualized by fluorescence microscopy, using a dual band pass filter (Omega). The model of retinol retSDR1 was constructed using the two crystal structures of ternary complexes of short-chain dehydrogenases with NADP and substrates. The structure of residues 35–286 is based on that of mouse carbonyl reductase, which has 26% sequence identity and 34% similarity (26Breton R. Housset D. Mazza C. Fontecilla-Camps J.C. Structure. 1996; 15: 905-915Abstract Full Text Full Text PDF Scopus (191) Google Scholar). Inserts (1, 4, 3, 1, 1, and 1 amino acids) and omissions (1 amino acid) were modeled using the Biopolymer (Tripos) and Chain (Baylor University) software. The conformation of the inserted loops was modeled to resemble that of the human 17β-hydroxysteroid dehydrogenase (17β-HSD), which has 26% sequence identity and 33% similarity (26Breton R. Housset D. Mazza C. Fontecilla-Camps J.C. Structure. 1996; 15: 905-915Abstract Full Text Full Text PDF Scopus (191) Google Scholar). The C-terminal part of retSDR1, residues 287–302, was modeled based on the fold of 17β-HSD. The model was allowed to relax from possible steric overlaps of the side chains and then was optimized by energy minimization using the Biopolymer software. The model of retinol was constructed using the coordinates of retinoic acid available in the Cambridge Structural Database. The retinol molecule was positioned in the active site of retSDR1 so that its oxygen atom corresponded to the oxygen atom of the propanol present in the structure of carbonyl reductase and the ligand position was optimized with the docking option of the Biopolymer software. The enzymatic properties of retinol dehydrogenase in ROS extracts, such as molecular weight, sensitivity to thiol-reactive reagents, and solubility, suggested that the enzyme was likely to be a member of the short-chain dehydrogenase/reductase (SDR) superfamily. A DNA sequence (∼40 base pairs) (see “Experimental Procedures”) corresponding to a conserved domain among retinol dehydrogenases was used to search for homology in an expressed sequence tag (EST) data base. One EST obtained from a human retinal cDNA library (W22782 generated by J. Macke, P. Smallwood, and J. Nathans) was similar to 11-cis-retinol dehydrogenase, and its translation product had amino acid sequence motifs conserved in the SDR superfamily (27Jörnvall H. Persson B. Krook M. Atrian S. Gonzalez-Duarte R. Jeffery J. Ghosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1167) Google Scholar). PCR with primers designed from this EST sequence amplified a product of 250 base pairs, which provided a probe to identify the putative retSDR1 cDNA among RACE products. Overlapping cDNA 5′ and 3′ ends obtained by RACE were combined to construct a 1.4-kb human cDNA. This full-length cDNA contains an open reading frame encoding a basic protein (pI = 9.07) of 302 amino acids with a predicted molecular mass of 33,520 Da (Fig. 2A). The first ATG (55–57 nucleotides) matched the Kozak consensus sequence for translation initiation (28Kozak M. Mamm. Genome. 1996; 7: 563-574Crossref PubMed Scopus (759) Google Scholar). Bovine and mouse cDNAs were cloned following the same strategy, and sequences are shown in Fig. 2A. The amino acid sequences of human, bovine and mouse retSDR1s are 94–98% identical (Table I). This high homology is absent in the 5′- and 3′- untranslated regions of bovine, human, and mouse retSDR1 (data not shown).Table ISequence comparison of retSDR1 with selected SDRsSDRAmino acid similarityAmino acid identity%%hretSDR1100100bretSDR198.798.3mretSDR19694.4RODHI39.129RODHII34.325.7RODHIII34.325CRAD33.926.411cisRDH37.627.69cisRDH39.429BDH3221.811βHSD131.724.1MLCR3425.817βHSD130.825PGDH33.720.3The comparison was made using amino acid sequences obtained in this study and deposited in the GenBank (see Fig 2 B for definitions of abbreviations and accession numbers) using the GAP program from the GCG package. The gap creation and gap extension penalties were set on 6 and 4, respectively. Open table in a new tab The comparison was made using amino acid sequences obtained in this study and deposited in the GenBank (see Fig 2 B for definitions of abbreviations and accession numbers) using the GAP program from the GCG package. The gap creation and gap extension penalties were set on 6 and 4, respectively. The retSDR1 sequence contains all the motifs present in the SDR superfamily of enzymes including the invariant YXXXK motif (amino acids 188–192), the catalytic Ser-175, the highly conserved Ser before Ly-192 residue, the highly conserved nucleotide binding motif TGXXXGXG (44–51), and the conserved Gly, Asp, Gly, Leu, Asn, Ala, Asn, Gly, Ile, Leu, Val, and Pro at sequence positions 62, 96, 115, 120, 123, 124, 147, 168, 170, 202, 212, and 219, respectively. The presence of RTEK at positions 71–74 is consistent with the specificity of this enzyme for NADPH (29Tsigelny I. Baker M.E. Biochem. Biophys. Res. Commun. 1996; 226: 118-127Crossref PubMed Scopus (12) Google Scholar). retSDR1s are related to other SDRs, including those with retinol dehydrogenase activity, as shown in a dendrogram (Fig. 2 B). Tissue distribution of retSDR1 mRNA was assessed by Northern blot analysis of several human tissues and bovine retina. A transcript of ∼1.8 kb is present in placenta, lung, liver, kidney, pancreas, and retina but was not detected in brain. The size of the human retSDR1 detected by blotting is similar to that of the partial (perhaps lacking fragments of untranslated regions) isolated cDNAs (1.401 for human, 1.478 for mouse, and 1.460 for bovine retSDR1). A transcript of smaller size (∼1.4 kb) was observed in heart and skeletal muscle (Fig.3). This transcript could arise from tissue-specific polyadenylation of the retSDR1 mRNA or splicing of the retSDR1 pre-mRNA, because a similar hybridization pattern was observed with the probe derived from the 3′-untranslated region of retSDR1 (data not shown). The presence of the retSDR1 mRNA, and not a cross-reacting transcript, in other tissues was also supported by a homology search of EST data bases, which revealed sequences identical to retSDR1 in several m