Initial screening of prostate short chain dehydrogenase/reductase I (PSDR1) expression, an enzyme cloned by Nelson and co-workers (20
) from prostate epithelium, reveals that this enzyme is also expressed in the eye (data not shown). Therefore, the name PSDR1 was changed into RDH11 to reflect its broader expression.
–14 Sequence Analyses and Gene Structures
—Nucleic acid and protein sequence databases were searched with the RDH11 cDNA sequence (identical to the sequence of the PSDR1
gene product (20
)) using Blast. This search identified full-length cDNA clones that show homology to RDH11 and encode RDH12 (first deposited by T. Isogai and under accession number AK054835), RDH13 (expressed sequence tag (EST) deposited by R. Strausberg and under accession number BE736147), and RDH14 (also named PAN2 and deposited by Z. Krozowski under accession number AF237952). The analysis of these cDNAs shows open reading frames of 316, 331, and 336 amino acids for RDH12, RDH13, and RDH14, respectively, encoding proteins of ~35, ~36, and ~37 kDa. RDH11 shares 79% similarity with RDH12 and ~60% similarity with RDH13 and RDH14. RDH12, RDH13, and RDH14 share ~60% similarity among themselves (, A
Fig. 1 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), (more ...)
These proteins contain two motifs highly conserved among SDRs, the cofactor-binding site (GXXX
G) and catalytic residues (YXXX
K). SDRs contain a motif at the amino terminus consisting of β-strand A, α-helix B, β-strand B, and α-helix C (part of the βA-αB-βB-αC-βC-αD-βD that forms the Rossman fold), which interacts with the adenosine monophosphate moiety of the cofactor. The residues present at the junctions βA-αB and βB-αC are thought to be important in selectivity for NAD(H) versus
NADP(H). For favorable interaction with NADP(H), positively charged residues are present at the βA-αB junction (glycine-rich motif) and/or at the beginning of α-helix C (7
). For all RDH11–14, no charged amino acids are present in the Gly-rich motif (GANTGIG for RDH11–13 or GANSGLG for RDH14), and there are positively charged residues present at the βB-αC junction (R
A for RDH11, -12, -13, and -14, respectively), suggesting a preference for NADP/NADPH ().
The tissue distribution of RDH11–14 was deduced from the array of ESTs displayed in databases corresponding to these RDHs. RDH11 was reported to be expressed abundantly in prostate tissue but also in eye, kidney, pancreas, liver, testis, heart, and brain (20
). In addition, ESTs corresponding to RDH11 were also found in libraries from eye, skin, and muscle. ESTs matching RDH12 were identified in multiple tissues, most of them from eye, but also some from kidney, brain, skeletal muscle, and stomach. RDH13 ESTs were obtained mostly from eye, pancreas, placenta, and lung. Many ESTs from brain, kidney, pancreas, and placenta correspond to RDH14.
Genomic clones were identified by GenBank™ data base searches with the coding sequences of the RDH
s. Clone Hs14_10185 contains both entire human RDH11
genes. The RDH11
gene is located ~30 kb from the RDH12
gene in the 3′-RDH11
-3′ orientation. This genomic clone originates from chromosome 14 at q23.3. These genes are located at the locus for a recessive blinding disease, Leber's congenital amaurosis 3 (LCA3) (www.sph.uth.tmc.edu/Retnet
). Clone AC011476.7, obtained from chromosome 19 at q13.42, contains the complete RDH13
gene. Clone Hs2_16082 contains the RDH14
gene and originates from chromosome 2 at p24.1. Comparison of the RDH
cDNAs with these genomic clones solved the gene structures. The gene structures of RDH11
, and RDH13
are almost identical and are interrupted by six introns. The intron/exon junctions of RDH11
are at the same positions, whereas intron 6 of RDH13
is positioned 35 amino acids upstream compared with intron 6 of RDH11
has only one intron, which is located at the same position as intron 3 of RDH11
, and RDH13
, and is a relatively small gene (~6 kb compared with 13–18 kb for the RDH11-13
) (). This gene structure is different from other SDR superfamily RDH
s expressed in the eye (30
Localization of RDH11–14 in the Eye—A monoclonal antibody specific for RDH11 did not cross-react with RDH5, a prominent enzyme present in the RPE as shown by immuno-blotting (). The lack of cross-reactivity is apparent because RDH5 and RDH11 have different molecular masses. Strong immunoreactivity was detected in bovine and monkey RPE (, C and F) and was blocked by RDH11 peptides (, D and G). A lower level of RDH11 expression was also detected in the Müller cells, as demonstrated by a double immunolabeling study with the Müller cell marker glial fibrillary acidic protein ().
Fig. 2 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 (more ...)
A digoxigenin-conjugated RDH12 antisense RNA probe hybridized to the base of monkey photoreceptor inner segments (, A and B, left). To visualize the chromogenic signal in RPE cells, an albino mouse retina was examined in this study (). In albino mouse (BALB/c) retina (, left), signals were not observed in the RPE cell layer. As a negative control, the sense RDH12 RNA probe did not produce significant hybridization signals in monkey or mouse retina (, A and B, right). No specific anti-RDH12 antibodies have been generated so far.
Fig. 3 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. (more ...)
Antibodies recognizing RDH13 () labeled inner segments of the photoreceptor cells of human and monkey retina (, C and F). Weak signals were observed in a small population of inner nuclear neurons and the inner plexiform layer. Higher magnification images localized RDH13 expression to inner segments of rod and cone photoreceptors (, inset). This immunoreactivity is specific as it was blocked by purified RDH13 protein. RDH14 yielded hybridization signals in the photoreceptor nuclear layer, and this enzyme appears to be expressed at low levels in the eye, although RDH14 immunolabeling was clearly observed in the bovine cone and ROS with a weaker signal in Müller cells (Supplemental Fig. 1).
Fig. 4 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 (more ...)
RDH Activity of RDH11, RDH12, and RDH14—RDH11 catalyzed the reduction of all-trans-retinal and its 9-cis-, 11-cis-, and 13-cis-retinal isomers. The activity was observed in the presence of NADPH and with Sf9 insect cell membranes only when Sf9 cells were transfected with the cDNA encoding RDH11 (). The products were clearly identified by the characteristic spectrum for each retinol isomer and a retention time that was similar to authentic standards (). This analysis avoids problems associated with the isomerization among retinols during incubation or sample handling. The activity toward 13-cis- was the lowest of the retinoid substrates tested and was only detected using high sensitivity HPLC analysis. The summary of the product conversion is illustrated in . RDH12 and RDH14 have very similar properties to those of RDH11 (, B and C). However, RDH13, expressed in insect cells (), displayed no RDH activity. The double specificity exhibited by RDH11, RDH12, and RDH14 toward cis- and all-trans-retinoids makes these enzymes unique among short chain RDHs.
Fig. 5 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, (more ...)
Fig. 6 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 (more ...)
RDH11, RDH12, and RDH14 demonstrate a clear specificity for the pro-S
hydrogen on C4 of NADPH (shown only for one enzyme, ) and the pro-R
hydrogen on C15
of all highly active retinols (). These properties resemble those of the photoreceptor dehydrogenase, prRDH (24
), and not those of the RPE enzyme RDH5 (16
), which is active toward the pro-S
position of both substrates. The results also suggest that these enzymes catalyze the reaction in both directions, NADPH/retinals ↔ NADP/retinols. RDH11, RDH12, and RDH14 show equal utilization of 11-cis
-retinal and all-trans
-retinal when these substrates are present at equal concentrations in the same mixture (data not shown), a property that suggests similar efficiency toward both substrates. No steroid dehydrogenase activity was detected for RDH11, RDH12, and RDH14. The activity of the RDH11–14, photoreceptor prRDH, and RDH5 was potently inhibited by retinoic acids (for example, 9-cis
-retinoic acid, KI
for RDH11), recombinant CRBP1 (Supplemental Table 1, prRDH), and CRALBP (Supplemental Table 2, RDH5).
Co-purification of RDH5 and RDH11 and Expression of RDHs in Immortal ARPE19 Cells—When RDH5 was purified from RPE membranes using anti-RDH5 monoclonal antibody affinity chromatography, NAD(H)-dependent (RDH5) and NADP(H)-dependent (RDH11) enzymes were also isolated based on immunoblotting and retinol activity profiles (). The activity was suppressed by diluting [4-3H]NADH with NADH and NADPH. Because the anti-RDH5 antibody did not cross-react with RDH11 (), these results suggest that both enzymes may form a larger oligomeric structure and/or interact with each other. However, when RDH5 was expressed and purified from insect cells using anti-RDH5 monoclonal antibody affinity chromatography, only NADH- and cis-retinoid preferable properties were observed (). Qualitatively, the stereospecificity of the mixture of RDHs isolated from RPE membranes () matched the sum of stereo-preferences toward retinals of RDH5 () and RDH11 (Figs. and ) or the sum of stereo-preferences of RDH5 () and the remaining activity in RPE membranes derived from rdh5−/− mice (). This suggests that the enzyme responsible for oxidation of 11-cis-retinol in these membranes is RDH11.
Fig. 7 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 (more ...)
As with many enzymes involved in retinoid metabolism, the expression of RDH11 and RDH5 are lost in ARPE19, an immortalized RPE cell line (Supplemental Fig. 2A), although other functions of retinal epithelium are preserved. These cells also lack RDH activity toward retinals (Supplemental Fig. 2B). These findings are not due to a secondary effect caused by a lack of other retinoid-processing enzymes because transfecting these cells with the RDH11 or 12 cDNAs restores RDH activity. This observation supports the hypothesis that RDH11 is involved in 11-cis-retinol oxidation in the RPE (Supplemental Fig. 2C).