Effect of lipid peroxidation products on the activity of human retinol dehydrogenase 12 (RDH12) and retinoid metabolism

Mutations in human Retinol Dehydrogenase 12 (RDH12) are known to cause photoreceptor cell death but the physiological function of RDH12 in photoreceptors remains poorly understood. In vitro, RDH12 recognizes both retinoids and medium-chain aldehydes as substrates. Our previous study suggested that RDH12 protects cells against toxic levels of retinaldehyde and retinoic acid [S.A. Lee, O.V. Belyaeva, I.K. Popov, N.Y. Kedishvili, Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12, J. Biol. Chem. 282 (2007) 35621-35628]. Here, we investigated whether RDH12 can also protect cells against highly reactive medium-chain aldehydes. Analysis of cell survival demonstrated that RDH12 was protective against nonanal but not against 4-hydroxynonenal. At high concentrations, nonanal inhibited the activity of RDH12 towards retinaldehyde, suggesting that nonanal was metabolized by RDH12. 4-Hydroxynonenal did not inhibit the RDH12 retinaldehyde reductase activity, but it strongly inhibited the activities of lecithin:retinol acyl transferase and aldehyde dehydrogenase, resulting in decreased levels of retinyl esters and retinoic acid and accumulation of unesterified retinol. Thus, the results of this study showed that RDH12 is more effective in protection against retinaldehyde than against medium-chain aldehydes, and that medium-chain aldehydes, especially 4-hydroxynonenal, severely disrupt cellular retinoid homeostasis. Together, these findings provide a new insight into the effects of lipid peroxidation products and the impact of oxidative stress on retinoid metabolism.     

Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12

Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12(-/-) mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage(1).     

cis-Retinol/androgen dehydrogenase, isozyme 3 (CRAD3): a short-chain dehydrogenase active in a reconstituted path of 9-cis-retinoic acid biosynthesis in intact cells

9-cis-Retinoic acid activates retinoid X receptors, which serve as heterodimeric partners with other nuclear hormone receptors, yet the enzymology of its physiological generation remains unclear. Here, we report the identification and molecular/enzymatic characterization of a previously unknown member of the short-chain dehydrogenase/reductase family, CRAD3 (cis-retinoid/androgen dehydrogenase, type 3), which catalyzes the first step in 9-cis-retinoic acid biosynthesis, the conversion of 9-cis-retinol into 9-cis-retinal. CRAD3 shares amino acid similarity with other retinoid/steroid short-chain dehydrogenases/reductases: CRAD1, CRAD2, and RDH4. Relative to CRAD1, CRAD3 has greater 9-cis-retinol/all-trans-retinol discrimination and lower efficiency as an androgen dehydrogenase. CRAD3 has apparent efficiency (V/K(m)) for 9-cis-retinol about equivalent to that for CRAD1 and 3 orders of magnitude greater than that for RDH4. (CRAD2 does not recognize 9-cis-retinol as a substrate). CRAD3 contributes to 9-cis-retinoic acid production in intact cells, in conjunction with each of three retinal dehydrogenases that recognize 9-cis-retinal (RALDH1/AHD2, RALDH2, and ALDH12). Liver and kidney, two tissues reportedly with the highest concentrations of 9-cis-retinoids, show the most intense mRNA expression of CRAD3, but expression also occurs in testis, lung, small intestine, heart, and brain. These data are consistent with the participation of CRAD3 in the biogeneration of 9-cis-retinoic acid.     

Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.     

A vertebrate aldo-keto reductase active with retinoids and ethanol

Enzymes of the short chain and medium chain dehydrogenase/reductase families have been demonstrated to participate in the oxidoreduction of ethanol and retinoids. Mammals and amphibians contain, in the upper digestive tract mucosa, alcohol dehydrogenases of the medium chain dehydrogenase/reductase family, active with ethanol and retinol. In the present work, we searched for a similar enzyme in an avian species (Gallus domesticus). We found that chicken does not contain the homologous enzyme from the medium chain dehydrogenase/reductase family but an oxidoreductase from the aldo-keto reductase family, with retinal reductase and alcohol dehydrogenase activities. The amino acid sequence shows 66-69% residue identity with the aldose reductase and aldose reductase-like enzymes. Chicken aldo-keto reductase is a monomer of M(r) 36,000 expressed in eye, tongue, and esophagus. The enzyme can oxidize aliphatic alcohols, such as ethanol, and it is very efficient in all-trans- and 9-cis-retinal reduction (k(cat)/K(m) = 5,300 and 32,000 mm(-1).min(-1), respectively). This finding represents the inclusion of the aldo-keto reductase family, with the (alpha/beta)(8) barrel structure, into the scenario of retinoid metabolism and, therefore, of the regulation of vertebrate development and tissue differentiation.     

Reduction of all-trans-retinal in vertebrate rod photoreceptors requires the combined action of RDH8 and RDH12

In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde all-trans-retinal that is released by photoactivated rhodopsin, to all-trans-retinol (vitamin A). Previous studies have shown that RDH8 localizes to photoreceptor outer segments and is a strong candidate for performing this role. However, RDH12 function in the photoreceptor inner segments is also key, because loss of function mutations cause retinal degeneration in some forms of Leber congenital amaurosis. To investigate the in vivo roles of RDH8 and RDH12, we used fluorescence imaging to examine all-trans-retinol production in single isolated rod cells from wild-type mice and knock-out mice lacking either one or both RDHs. Outer segments of rods deficient in Rdh8 failed to reduce all-trans-retinal, but those deficient in Rdh12 were unaffected. Following exposure to light, a leak of retinoids from outer to inner segments was detected in rods from both wild-type and knock-out mice. In cells lacking Rdh8 or Rdh12, this leak was mainly all-trans-retinal. Wild-type rods incubated with all-trans-retinal reduced moderate loads of retinal within the cell interior, but this ability was lost by cells deficient in Rdh8 or Rdh12. Our findings are consistent with localization of RDH8 to the outer segment where it provides most of the activity needed to reduce all-trans-retinal generated by the light response. In contrast, RDH12 in inner segments can protect vital cell organelles against aldehyde toxicity caused by an intracellular leak of all-trans-retinal, as well as other aldehydes originating both inside and outside the cell.     

Evidence that proteosome inhibitors and chemical chaperones can rescue the activity of retinol dehydrogenase 12 mutant T49M

Retinol dehydrogenase 12 (RDH12) is a microsomal enzyme that catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol when expressed in cells. Mutations in RDH12 cause severe retinal degeneration; however, some of the disease-associated RDH12 mutants retain significant catalytic activity. Our previous study (Lee et al., 2010 [9]) demonstrated that the catalytically active T49M and I51N variants of RDH12 undergo accelerated degradation through the ubiquitin-proteosome system, which results in reduced levels of these proteins in the cells. Here, we investigated whether the stabilization of T49M or I51N RDH12 protein levels through the inhibition of proteosome activity or improved folding could rescue their retinaldehyde reductase activity. For the T49M variant, the inhibition of proteosome activity resulted in an increased level of T49M protein in the microsomal fraction. The higher level of the T49M variant in microsomes correlated with the higher microsomal retinaldehyde reductase activity. T49M-expressing living cells treated with the inhibitors of proteosome activity or with dimethyl sulfoxide exhibited an increase in the conversion of retinaldehyde to retinol, consistent with the recovery of functional RDH12 protein. On the other hand, accumulation of the I51N variant in the microsomes did not result in higher retinaldehyde reductase activity of the microsomes or cells. These results provide a proof of concept that, at least in the case of the T49M variant, the prevention of accelerated degradation could lead to restoration of its function in the cells. This finding justifies further search for more efficient and clinically relevant compounds for stabilizing the T49M variant activity.     

Characterization of a dehydrogenase activity responsible for oxidation of 11-cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene. A model for the human hereditary disease fundus albipunctatus.

In the vertebrate retina, the final step of visual chromophore production is the oxidation of 11-cis-retinol to 11-cis-retinal. This reaction is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs), prior to the chromophore rejoining with the visual pigment apo-proteins. The RDH5 gene encodes a dehydrogenase that is responsible for the majority of RDH activity. In humans, mutations in this gene are associated with fundus albipunctatus, a disease expressed by delayed dark adaptation of both cones and rods. In this report, an animal model for this disease, 11-cis-rdh-/- mice, was used to investigate the flow of retinoids after a bleach, and microsomal membranes from the retinal pigment epithelium of these mice were employed to characterize remaining enzymatic activities oxidizing 11-cis-retinol. Lack of 11-cis-RDH leads to an accumulation of cis-retinoids, particularly 13-cis-isomers. The analysis of 11-cis-rdh-/- mice showed that the RDH(s) responsible for the production of 11-cis-retinal displays NADP-dependent specificity toward 9-cis- and 11-cis-retinal but not 13-cis-retinal. The lack of 13-cis-RDH activity could be a reason why 13-cis-isomers accumulate in the retinal pigment epithelium of 11-cis-rdh-/- mice. Furthermore, our results provide detailed characterization of a mouse model for the human disease fundus albipunctatus and emphasize the importance of 11-cis-RDH in keeping the balance between different components of the retinoid cycle.     

Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.     

Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol

Retinol dehydrogenase (RDH), the enzyme that catalyzes the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment, was the first visual cycle enzymatic activity to be identified. Previous work has shown that this enzyme utilizes NADPH, shows a marked preference for all-trans-retinal over 11-cis-retinal, and is tightly associated with the outer segment membrane. This paper reports the identification of a novel member of the short chain dehydrogenase/reductase family, photoreceptor RDH (prRDH), using subtraction and normalization of retina cDNA, high throughput sequencing, and data base homology searches to detect retina-specific genes. Bovine and human prRDH are highly homologous and are most closely related to 17-beta-hydroxysteroid dehydrogenase 1. The enzymatic properties of recombinant bovine prRDH closely match those previously reported for RDH activity in crude bovine rod outer segment preparations. In situ hybridization and RNA blotting show that the PRRDH gene is expressed specifically in photoreceptor cells, and protein blotting and immunocytochemistry show that prRDH localizes exclusively to both rod and cone outer segments and that prRDH is tightly associated with outer segment membranes. Taken together, these data indicate that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment.     

Identification of a mouse short-chain dehydrogenase/reductase gene,retinol dehydrogenase-similar. Function of non-catalytic amino acid residues in enzyme activity

We report a mouse short-chain dehydrogenase/reductase (SDR), retinol dehydrogenase-similar (RDH-S), with intense mRNA expression in liver and kidney. The RDH-S gene localizes to chromosome 10D3 with the SDR subfamily that catalyzes metabolism of retinoids and 3 alpha-hydroxysteroids. RDH-S has no activity with prototypical retinoid/steroid substrates, despite 92% amino acid similarity to mouse RDH1. This afforded the opportunity to analyze for functions of non-catalytic SDR residues. We produced RDH-S Delta 3 by mutating RDH-S to remove an "additional" Asn residue relative to RDH1 in its center, to convert three residues into RDH1 residues (L121P, S122N, and Q123E), and to substitute RDH1 sequence G208FKTCVTSSD for RDH-S sequence F208-FLTGMASSA. RDH-S Delta 3 catalyzed all-trans-retinol and 5 alpha-androstane-3 alpha,17 alpha-diol (3 alpha-adiol) metabolism 60-70% as efficiently (Vm/Km) as RDH1. Conversely, substituting RDH-S sequence F208FLTGMASSA into RDH1 produced a chimera (viz. C3) that was inactive with all-trans-retinol, but was 4-fold more efficient with 3 alpha-adiol. A single RDH1 mutation in the C3 region (K210L) reduced efficiency for all-trans-retinol by >1250-fold. In contrast, the C3 area mutation C212G enhanced efficiency with all-trans-retinol by approximately 2.4-fold. This represents a >6000-fold difference in catalytic efficiency for two enzymes that differ by a single non-catalytic amino acid residue. Another chimera (viz. C5) retained efficiency with all-trans-retinol, but was not saturated and was weakly active with 3 alpha-adiol, stemming from three residue differences (K224Q, K229Q, and A230T). The residues studied contribute to the substrate-binding pocket: molecular modeling indicated that they would affect orientation of substrates with the catalytic residues. These data report a new member of the SDR gene family, provide insight into the function of non-catalytic SDR residues, and illustrate that limited changes in the multifunctional SDR yield major alterations in substrate specificity and/or catalytic efficiency.     

Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic acid, however, initial assays suggested that RDH10 prefers NADP(+) as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD(+)-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) among all NAD(+)-dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD(+), RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNA-mediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.     

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Evaluation of the role of the retinal G protein-coupled receptor (RGR) in the vertebrate retina in vivo

The retinal G protein-coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein-coupled receptors. RGR may play a role as a photoisomerase in the production of 11-cis-retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11-cis-retinol dehydrogenase (RDH5), is to regenerate 11-cis-retinal under light conditions and RDH5 is expected to function in the light-independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr-/- single and rdh5-/-rgr-/- double knockout mice under a number of light conditions. The most striking phenotype of rgr-/- mice after a single flash of light includes light-dependent formation of 9-cis- and 13-cis-retinoid isomers. These isomers are not formed in wild-type mice because either all-trans-retinal is bound to RGR and protected from isomerization to 9-cis- or 13-cis-retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all-trans-retinyl esters and an attenuated recovery of 11-cis-retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic-like photoisomerization-driven production of 11-cis-retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod-dominated mice.     

Gene structure, expression analysis, and membrane topology of RDH4

The murine retinol dehydrogenase RDH4 oxidizes several cis-isomers of retinol into their corresponding aldehydes. We have determined the structure of the murine gene, investigated the temporal and spatial expression of the enzyme, and analyzed the membrane topology of the enzyme. The gene has four translated exons, and several alternatively spliced exons in the 5'-untranslated region were identified. Immunohistochemical analysis showed expression of RDH4 in developing and adult mouse eye, particularly in the retinal pigment epithelium. In nonocular adult tissues, including liver, kidney, lung, and skin, RDH4 expression was widespread. The results suggest that RDH4 may have a dual and tissue-specific role in oxidation of 9-cis- and 11-cis-isomers of retinol into 9-cis-retinal and 11-cis-retinal, respectively. Furthermore, the lumenal orientation of the enzyme domain in the ER suggests that oxidation of both cis-isomers of retinol occurs in the ER.     

Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo

The regeneration of 11-cis-retinal, the universal chromophore of the vertebrate retina, is a complex process involving photoreceptors and adjacent retinal pigment epithelial cells (RPE). 11-cis-Retinal is coupled to opsins in both rod and cone photoreceptor cells and is photoisomerized to all-trans-retinal by light. Here, we show that RPE microsomes can catalyze the reverse isomerization of 11-cis-retinol to all-trans-retinol (and 13-cis-retinol), and membrane exposure to UV light further enhances the rate of this reaction. This conversion is inhibited when 11-cis-retinol is in a complex with cellular retinaldehyde-binding protein (CRALBP), providing a clear demonstration of the protective effect of retinoid-binding proteins in retinoid processes in the eye, a function that has been long suspected but never proven. The reverse isomerization is nonenzymatic and specific to alcohol forms of retinoids, and it displays stereospecific preference for 11-cis-retinol and 13-cis-retinol but is much less efficient for 9-cis-retinol. The mechanism of reverse isomerization was investigated using stable isotope-labeled retinoids and radioactive tracers to show that this reaction occurs with the retention of configuration of the C-15 carbon of retinol through a mechanism that does not eliminate the hydroxyl group, in contrast to the enzymatic all-trans-retinol to 11-cis-retinol reaction. The activation energy for the conversion of 11-cis-retinol to all-trans-retinol is 19.5 kcal/mol, and 20.1 kcal/mol for isomerization of 13-cis-retinol to all-trans-retinol. We also demonstrate that the reverse isomerization occurs in vivo using exogenous 11-cis-retinol injected into the intravitreal space of wild type and Rpe65-/- mice, which have defective forward isomerization. This study demonstrates an uncharacterized activity of RPE microsomes that could be important in the normal flow of retinoids in the eye in vivo during dark adaptation.     

Targeted disruption of the murine retinal dehydrogenase gene Rdh12 does not limit visual cycle function

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.