Primary sequences and the gene structures of RDHs expressed in the retina. A, alignment of the deduced amino acid sequences of human RDH11 (AF167438), human RDH12 (sequence identical to unnamed XM_085058), human RDH13 (sequence identical to unknown AAH09881), human PAN2 (RDH14) (NM_020905), human RDH5 (HSU43559), human prRDH (NM_015725), and human retSDR1 (AF061741). The identical residues in all sequences are shown in white letters on a black background. The identical residues in RDH11–14 sequences are shown in dark gray. The conserved residues in RDH11–14 sequences are shown in light gray. The amino acids used to develop anti-RDH11 monoclonal antibodies are indicated by white letters on the black background. B, phylogenetic tree. The tree was built with a bootstrap analysis of neighbor-joining distance using PAUPSearch in GCG (Genetics Computer Group). The numbers represent the percentage of identities with RDH12. The percentage of similarities is indicated in parentheses. C, gene structure of RDH11–14. The coding regions are shown as black boxes, and the noncoding regions are shown as white boxes. The thick lines and the numbers represent the introns and their sizes, respectively.

Immunolocalization of RDH11 in bovine and monkey retina. A, specificity of anti-RDH5 (lane 1) and anti-RDH11 (lanes 2–5) antibodies. Lanes 1 and 2, bovine RPE; lane 3, bovine ROS; lane 4, Sf9 cell lysate expressing recombinant RDH11; lane 5, Sf9 cell lysate; lane 6, purified RDH5-His₆. B–D, immunofluorescence localization of RDH11 in monkey retina. B, control bright field image of monkey retina. C, RDH11 immunolabeling is predominant in the RPE cell layer of monkey retina. D, addition of purified peptide (0.5 μg/ml) abolishes RDH11 immunoreactivity. E–G, immunofluorescence localization of RDH11 in bovine retina. E, control bright field image of bovine retina. F, RDH11 immunolabeling is predominant in the RPE cell layer of bovine retina. G, addition of purified peptide (0.5 μg/ml) abolishes RDH11 immunoreactivity. Bar, 50 μm. H, co-localization of RDH11 and glial fibrillary acidic protein in the inner retina. The bovine retina section was double-labeled with antibodies to glial fibrillary acidic protein and RDH11. Arrows show the colocalization of glial fibrillary acidic protein (green) and RDH11 (red) in Müller cells. Bar,50 μm. OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve-fiber layer.

In situ hybridization of monkey and mouse RDH12. A, in situ hybridization of RDH12 transcripts in monkey retina using antisense (left) and sense (right) RNA. The strongest signal is detected in photoreceptor (cones and rods) inner segments and cell bodies. B, in situ hybridization of RDH12 transcripts in mouse retina using antisense (left) and sense (right) RNA. The strongest signal is detected in photo-receptor inner segments. Bar, 50 μm. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve-fiber layer.

Immunolocalization of RDH13 in human and monkey retina. A, specificity of anti-RDH13 antibodies. Lane 1, SF9 cell lysate expressing recombinant RDH11; lane 2, SF9 cell lysate expressing recombinant RDH12; lane 3, SF9 cell lysate expressing recombinant RDH13; lane 4, SF9 cell lysate expressing recombinant RDH14; lane 5, monkey retinal homogenate; lane 6, human retinal homogenate. B–D, immunofluorescence localization of RDH13 in human retina. B, bright field image of human retina. C, RDH13 immunolabeling is predominant in the photoreceptor inner segments of human retina. Weak signals were observed in the inner plexiform layer and inner nuclear neurons proximal to the outer plexiform layer. D, addition of purified RDH13 (2 μg/ml) abolishes RDH13 immunoreactivity. E–G, immunofluorescence localization of RDH13 in monkey retina. E, bright field image of monkey retina. F, RDH13 immunolabeling is predominant in the photoreceptor inner segments of monkey retina. Weak signals were observed in inner plexiform layer and inner nuclear neurons proximal to the outer plexiform layer. Bar, 50 μm. G, addition of purified RDH13 (2 μg/ml) abolishes RDH13 immunoreactivity. H, localization of RDH13 (red) in monkey retina. The cone sheath was simultaneously visualized by fluorescein-conjugated peanut agglutinin (green). Inset, higher magnification image. Arrows show the localization of RDH13 (red) in cone inner segments surrounded by the cone sheath (green). Asterisks indicate the localization of RDH13 in rod inner segments. Bar, 50 μm. RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve-fiber layer.

RDH activity with various isomers of retinals and NADH and NADPH dinucleotides. In each panel, the first (from the top) and third chromatograms were from bacmid in Sf9 cells with NADH (solid lines) and NADPH (dashed lines) as dinucleotide substrates, respectively. The second and fourth chromatograms were from recombinant RDH11 expressed in Sf9 cells using NADH (dotted lines) and NADPH (dashed-dotted lines) as dinucleotide substrates, respectively. Retinols (indicated by arrows identifying specific elution times and spectra (insets)) were only produced when RDH11 expressed in Sf9 cells and NADPH were used. The assays (reduction of retinals) were carried out using non-radiolabeled retinals (90 μm) and dinucleotides (50 μm), as described under “Materials and Methods.” The retinoids were separated using a normal phase HPLC column (Beckman, Ultrasphere-Si, 2.1 mm × 250 mm) developed with 10% ethyl acetate, 90% hexane at a flow rate of 0.5 ml/min. Note that the spontaneous isomerization of retinal generated a mixture of aldehydes, for example all-trans-retinal (1) is contaminated by 13-cis-retinal (4), and the production of 13-cis-retinol is at the level of background (fourth panel). mAU, milliabsorbance units.

Stereoisomeric specificities of RDH11–14. A–C, different geometric isomers of retinals (1, all-trans-retinal; 2, 9-cis-retinal; 3, 11-cis-retinal; 4, 13-cis-retinal, 90 μm each) were tested with various RDHs in the presence of stereospecifically radiolabeled pro-S [4-³H]NADPH (18 μm). The assay (reduction of retinals) and HPLC conditions were the same as in Fig. 5. The corresponding retinol fraction was collected and subjected to scintillation counting. D, stereospecifically radiolabeled pro-R [4-³H]NADPH (20 μm) or pro-S [4-³H]NADPH (18 μm) was tested with RDH11 using various retinals (1-3). The partition assay for retinal reduction was used to measure the activity, as described under “Materials and Methods.” E, stereospecifically labeled pro-R [15-³H]retinols (1′, all-trans-retinol; 2′, 9-cis-retinol; 3′, 11-cis-retinol, 40–60 μm) or pro-S [15-³H]retinols (40–60 μm) were examined with RDH12 in the presence of NADP (700 μm) at pH 8.0. The partition assay for retinol oxidation was used to measure the activity as described under “Materials and Methods.”

Enzymatic activities and RDH11 immunoreactivity of affinity column-purified of RDH5. A, RDH5 from bovine RPE microsomes was purified as described under “Materials and Methods.” The assay with pro-S [4-³H]NAD(P)H and geometric isomers of retinals in the presence or absence of NAD(P)H was carried out for 50 min at 37 °C using the phase partition assay as described under “Materials and Methods,” and the amount of [15-³H]retinol isomer was quantified. B, RDH5-His₆ from transfected Sf9 cells was purified as described under “Materials and Methods.” The assay was carried out for 50 min at 37 °C using phase partition assay as described under “Materials and Methods.” Indicated is the amount of the corresponding [15-³H]retinol isomer formed in pmol/min. Inset, immunoblot of the purified fraction on anti-RDH5 antibody column probed with specific anti-RDH5 (lane 1) and RDH11 antibodies (lane 3). Lane 2 is a 33-kDa molecular marker. C, stereospecificity toward retinal in RPE membranes derived from rdh5−/− mice (16).

Isomeric specificity of the retinoid cycle reactions in the vertebrate retina. Light causes the isomerization of rhodopsin chromophore, 11-cis-retinal, to all-trans-retinal, which is next reduced in the reaction catalyzed by all-trans-retinal-specific RDH(s). This dehydrogenase activity utilizes pro-S [4-H_a] of NADPH and does not bind NADH to generate pro-R [15-H_a]all-trans-retinol. Next, pro-R [15-H_a]all-trans-retinol or its derivative is isomerized with the inversion of the C₁₅ prochiral methylene hydroxyl group configuration, specifically generating pro-S [15-H_a]11-cis-retinol isomer. Pro-S [15-H_a]11-cis-retinol isomer is then oxidized by RDH5 activity (resulting in the loss of pro-S [15-H_a]) or by RDH11 (resulting in the loss of pro-R [15-H_b]) to 11-cis-retinal with concomitant generation of pro-S [4-H_a]NADH or pro-S [4-H_b]NADPH to complete the cycle (modified version from (24)).