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Originally published In Press as doi:10.1074/jbc.M706372200 on October 9, 2007

J. Biol. Chem., Vol. 282, Issue 49, 35621-35628, December 7, 2007
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Overproduction of Bioactive Retinoic Acid in Cells Expressing Disease-associated Mutants of Retinol Dehydrogenase 12*

Seung-Ah Lee, Olga V. Belyaeva, Ivan K. Popov, and Natalia Y. Kedishvili1

From the Department of Biochemistry and Molecular Genetics, School of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, August 2, 2007 , and in revised form, October 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin A derivatives serve two different physiological functions: detection of light in vision and regulation of gene transcription during differentiation and development of cells and tissues. The first function is carried out by 11-cis-retinaldehyde, which serves as a visual chromophore (1). Upon absorption of light, 11-cis-retinaldehyde covalently bound to rod and cone opsins is isomerized to all-trans-retinaldehyde (1). All-trans-retinaldehyde dissociates from the opsin and is reduced in photoreceptors to all-trans-retinol, which is translocated to retinal pigment epithelium (RPE) and esterified to all-trans-retinyl esters. The resulting retinyl esters are isomerized and hydrolyzed to produce 11-cis-retinol. 11-cis-retinol is oxidized in RPE to 11-cis-retinaldehyde, which is then returned to photoreceptor cells. This sequence of events is known as the retinoid visual cycle and allows for regeneration of 11-cis-retinaldehyde for the next round of visual signal transduction.

The function of vitamin A in gene transcription is mediated by retinoic acid, the activating ligand of nuclear transcription factors, retinoic acid receptors (2). Retinoic acid is required for differentiation and development of many tissues and is also of key importance for eye and photoreceptor development in vertebrates (3–11). Deficiency in retinoic acid causes microphthalmia and other defects (6, 12). However, excess of retinoic acid is equally harmful and was shown to accelerate photoreceptor cell death by apoptosis (13–16).

Retinoic acid is synthesized from retinol in two consecutive steps: the oxidation of retinol followed by the oxidation of retinaldehyde (17). The first step, the oxidation of retinol to retinaldehyde is rate-limiting and determines the overall rate of retinoic acid biosynthesis. The levels of retinoic acid in tissues are tightly regulated, but the mechanisms underlying this regulation are not yet fully understood.

Recently, mutations in one of the retinoid-active enzymes, human retinol dehydrogenase 12 (RDH12),2 have been genetically linked to the severe early-onset autosomal recessive retinal dystrophy (arRD) (18, 19) and Leber's congenital amaurosis (LCA) (20, 21). The biochemical causes of photoreceptor cell death in patients with arRD and LCA are not clear, because the physiological functions of RDH12 in photoreceptors have not yet been established.

In vitro, RDH12 catalyzes the oxidoreductive interconversions of all-trans- and cis-retinoids and is also active toward medium-chain peroxidic aldehydes, such as nonanal, trans-2-nonenal, and 4-hydroxynonenal (4-HNE) (22). This substrate specificity prompted speculations that RDH12 may contribute to the reduction of all-trans-retinaldehyde to all-trans-retinol in the visual cycle (18–21) and/or to detoxification of lipid peroxidation products (22). However, RDH12-null mice exhibit normal visual cycle function and have normal levels of peroxidic aldehydes (23, 24), indicating that RDH12 fulfills a different physiological need.

To better understand the contribution of RDH12 to photoreceptor physiology, we investigated the role of RDH12 in retinoid metabolism of living cells and examined the effect of disease-associated mutations on RDH12 retinoid activity. The results of this study provide a new insight into the general mechanisms that regulate retinoic acid biosynthesis in the cells and suggest an underlying biochemical cause for retinal degeneration in patients carrying loss-of-function mutations in RDH12.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Anti-RDH12 Polyclonal Antiserum—Polyclonal antiserum was raised in rabbits against bacterially expressed RDH12 that lacked the N-terminal hydrophobic segment (amino acids 1–23). The vector for expression of truncated RDH12 was prepared as follows. The RDH12 cDNA in pET28a vector (22) was PCR-amplified using primers 5'-CTCATATGATCAGGAAGTTCTTTGCTGGTG-3' (NdeI restriction site underlined) and 5'-GCGGATCCCTACTCCCACCGGATCCTAGAAGCT-3' (BamHI restriction site underlined). The PCR product was gel-purified, cleaved with restriction endonucleases NdeI and BamHI, and cloned into the corresponding sites of pET19b vector (Novagen, Madison, WI). The resulting construct encoded truncated RDH12 (amino acids 24–116) fused to the N-terminal His10 tag. Truncated RDH12 was expressed in Escherichia coli and purified using Ni2+ affinity chromatography. Rabbit polyclonal antiserum against purified RDH12 was raised by Cocalico Biologicals, Inc. (Reamstown, PA). The antiserum was affinity purified using RDH12 immobilized on polyvinylidene difluoride membrane. At a 1:1,000 dilution, the purified antiserum detected less than 10 ng of RDH12 using ECL Western blotting detection reagents (Amersham Biosciences).

Western Blot Analysis—Freshly isolated Macaca mulatta retina was homogenized on ice in 50 mM Tris-HCl, pH 7.4, containing aprotinin (2 µg/ml), leupeptin (2 µg/ml), pepstatin-A (1 µg/ml), phenylmethylsulfonyl fluoride (5 mM), EDTA (0.1 mM), benzamidine (5 mM), and dithiothreitol (1 mM). Microsomes were isolated from the post-12,000 x g supernatant by centrifugation at 105,000 x g for 1 h and resuspended in 90 mM KH2PO4, 40 mM KCl, pH 7.4, and 20% glycerol. Protein concentrations were determined using Bio-Rad DC Protein Assay.

Protein samples were separated by electrophoresis in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE) and transferred to Hybond-P membrane (Amersham Biosciences). The membrane was blocked with a solution of 5% bovine serum albumin in 20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween 20, and incubated with affinity purified anti-RDH12 polyclonal antiserum at a 1:1,000 dilution. RDH12 was detected by chemiluminescence using goat anti-rabbit antibodies conjugated to horseradish peroxidase (at a 1:10,000 dilution). Western blot analysis of RDH12 expressed in HEK293 cells was carried out as described above except whole cell lysates were used for analysis after centrifugation at 5,000 x g for 15 min at 4 °C. The blots were re-stained for β-actin using anti-β-actin monoclonal antibody (Sigma-Aldrich) at a 1:5,000 dilution to control for protein loading.

Endoglycosidase H (Endo H) Treatment—Fifty micrograms of monkey retina microsomal protein were resuspended in 50 mM sodium phosphate buffer, pH 5.5, containing 0.5% SDS and 0.1 M β-mercaptoethanol. Protease inhibitor phenylmethylsulfonyl fluoride was added to the final concentration of 1 mM. One-half of the mixture was treated with 3 µl (15 units) of Endo H (Roche Applied Sciences, Mannheim, Germany), and the other half received 3 µl of the buffer. Both samples were incubated overnight at room temperature, then denatured with SDS-PAGE loading buffer for 5 min at 94 °C and analyzed by Western blotting as described above. Microsomal preparation of FLAG-tagged 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expressed in Sf9 cells served as a positive control for deglycosylation by Endo H as described previously (25).

Construction of Eukaryotic Expression Vectors—The vector for expression of RDH12 in HEK293 cells was prepared by PCR-amplifying the RDH12 cDNA in pET28a vector (22) using primers 5'-TTGGAATTCATGCTGGTCACCTTGGGACTGC-3' and 5'-GCTGGATCCGCAGCCGGATCTCAGTGGTG-3' (EcoRI and BamHI restriction sites underlined), and cloning the PCR product after digestion with EcoRI and BamHI endonucleases into the corresponding sites of pIRESneo plasmid (Clontech, Mountain View, CA). The resulting construct encoded human RDH12 protein as a fusion to the C-terminal His6 tag.

To prepare the expression constructs for RDH12 mutants, site-directed mutagenesis was carried out on wild-type RDH12 cDNA in pET28a vector (22) using an ExSite PCR-based kit (Stratagene, La Jolla, CA). Primers used for mutagenesis are summarized in Table 1. The mutagenized cDNAs were released from pET28a by digestion with XbaI and NotI endonucleases and cloned into the respective sites of modified pVL1393 vector (22) in-frame with the C-terminal His6 tag. Recombinant baculoviruses were produced by cotransfection of Sf9 cells with the transfer vectors and the linearized Sapphire Baculovirus DNA (Orbigen Inc., San Diego, CA) according to the manufacturer's instructions.


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  		TABLE 1
Primers for mutagenesis

 
For expression in HEK293 cells, the mutagenized RDH12 cDNAs in pET28a were PCR-amplified using primers 5'-TTGGAATTCATGCTGGTCACCTTGGGACTGC-3' (forward primer, EcoRI site underlined) and 5'-GCTGGATCCGCAGCCGGATCTCAGTGGTG-3' (reverse primer, BamHI site underlined) to create the restriction sites matching those in pIRESneo vector. PCR products were digested with the corresponding restriction endonucleases, purified, and cloned into the EcoRI/BamHI sites in pIRESneo.

Expression of RDH12 in HEK293 Cells and Enzyme Assays—HEK293 cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO2. Cells were plated in 35-mm dishes and transiently transfected with expression constructs for wild-type or mutant RDH12 in pIRESneo vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transfection efficiency was monitored by cotransfecting the cells with an expression construct for β-galactosidase in pCMV vector and assaying β-galactosidase activity using o-nitrophenyl-D -galactopyranoside.

Twenty-four hours after transfection, the cells were incubated with either 5 µM all-trans-retinaldehyde or 10 µM all-trans-retinol (Sigma-Aldrich) for 3 h or 24 h, respectively. Media and cells were collected separately under reduced light. Retinoids were extracted into hexane and separated by normal phase high performance liquid chromatography (HPLC) using Waters Alliance Separation Module and 2996 Photodiode Array Detector. Peaks were identified by comparison to retention times of retinoid standards and evaluation of wavelength maxima and quantified as described previously (22).

Purification and Characterization of Wild-type and Mutant RDH12—Expression and purification of His6-tagged RDH12 variants was carried out following the previously established protocol (22). Specific activities of purified enzymes were determined using 6.5 µM all-trans-retinaldehyde and 1 mM NADPH. The apparent Km values for the reduction of all-trans-retinaldehyde were determined at 1 mM NADPH and six concentrations of all-trans-retinaldehyde (0.1–5 µM). The apparent Km values for cofactors were determined at a fixed saturating concentration of all-trans-retinaldehyde or all-trans-retinol and six concentrations of NADPH or NADP+ (1.25–1,000 µM). The amount of protein was varied between 0.035 µg and 0.175 µgso that the amount of the product did not exceed 10% of the substrate. Reaction rates were determined based on the percent substrate conversion as described previously (25). Initial velocities (nmol/min of product formed per mg of protein) were obtained by non-linear regression analysis. Kinetic constants were calculated using GraFit (Erithacus Software Ltd., UK) and expressed as the means ± S.D. The results shown are representative of three to four experiments.

MTT Assay—Cell viability was assessed using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) assay (26). Briefly, cells seeded in a 24-well plate were transfected with an empty pIRESneo vector or with RDH12/pIRESneo expression vector. Twenty-four hours after transfection, fresh media (0.5 ml) containing various concentrations (0, 5, 10, and 30 µM) of all-trans-retinaldehyde (four-replicate wells for each concentration) was added. Cells were exposed to all-trans-retinaldehyde for 24 h. Medium containing 0.5 mg/ml MTT solution was added to each well, and plates were incubated at 37 °C for 2 h. The medium was decanted, and the reaction was stopped by the addition of 0.5 ml dimethyl sulfoxide. Plates were incubated at 37 °C for several minutes, and 0.2 ml of the solubilized formazan solution was transferred to a 96-well plate. The absorbance was read at 595 nm using an ELISA plate reader.

RNA Isolation and Northern Blot Analysis—Total RNA was isolated from the wild-type and mutant RDH12-transfected HEK293 cells using RNeasy Mini kit (Qiagen Inc. Valencia, CA). RNA (8 µg of each sample) was subjected to 1.2% agarose gel containing 2.2 M formaldehyde and transferred to Hybond-XL nylon membrane (Amersham Biosciences). The membrane was hybridized with 32P-labeled RDH12 cDNA probe in ExpressHyb hybridization solution (Clontech), according to the manufacturer's instructions. The mRNA bands were visualized by exposure to x-ray film at –80 °C with two intensifying screens for several hours. A cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Statistical Analysis—Statistical significance was determined by a two-tailed unpaired Student's t test using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA). p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Model for the Analysis of RDH12 Function in Retinoid Metabolism—In humans, RDH12 is expressed almost exclusively in the inner segments of photoreceptors (23, 25). To our knowledge, there are no available immortalized photoreceptor cell lines. Thus, to investigate the role of RDH12 in retinoid metabolism, we created a cell model based on HEK293 cell line, which was shown to be an excellent choice for characterization of retinoid-active enzymes (27).

HEK293 cells were transiently transfected with the vector encoding RDH12 and the expression of RDH12 protein was analyzed by Western blotting using anti-RDH12 antiserum. Consistent with the previous reports (18, 19), RDH12 was localized in the microsomal fraction. However, anti-RDH12 antibodies detected a single immunoreactive protein band of ~35 kDa (data not shown). This was surprising, because RDH12 previously expressed in COS-7 cells exhibited three immunoreactive bands and appeared to be glycosylated on two endogenous N-glycosylation consensus motifs (172NVS, 297NKT) (18, 19), implying that RDH12 localizes on the luminal side of the endoplasmic reticulum (ER) membrane.

To test whether RDH12 had the correct transmembrane orientation in our expression system, we examined the glycosylation state of the native RDH12. M. mulatta retina microsomes containing RDH12 protein were treated with Endo H and the electrophoretic mobility of RDH12 before and after the treatment was examined by Western blotting. As a positive control, we used recombinant 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which has three glycosylation consensus motifs and is known to be glycosylated (28). As shown in Fig. 1, the electrophoretic mobility of retina RDH12 did not change upon treatment with Endo H, consistent with the lack of glycosylation. The same result was obtained for Endo H treatment of recombinant human RDH12 expressed in HEK293 cells (data not shown). At the same time, 11β-HSD1 was efficiently deglycosylated (Fig. 1), as evidenced by the disappearance of the slower moving glycosylated forms of the protein. These results demonstrated that the lack of glycosylation of RDH12 expressed in HEK293 cells was not due to a deficiency in glycosylation capacity of the cells but, instead, accurately represented the state of the native protein. The lack of glycosylation suggested that RDH12 faces the cytosolic side of the ER membrane, like the closely related RalR1 (72% identity), which has two endogenous N-glycosylation consensus motifs (174NVS and 298NET) in positions similar to RDH12 and was shown not to be glycosylated (25). Together, these results indicated that HEK293 cell model was appropriate for characterization of RDH12 activity.


Figure 1
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  		FIGURE 1.
Analysis of glycosylation state of RDH12. Fifty micrograms of microsomes isolated from monkey retina were incubated with (+) or without (–) Endo H, and RDH12 protein was visualized by Western blot analysis using affinity-purified rabbit anti-RDH12 polyclonal antiserum as described under "Experimental Procedures." Untagged human recombinant RDH12 (RDH12) in Sf9 microsomes served as a reference standard for electrophoretic mobility. FLAG-tagged 11β-HSD1 (11β-HSD1) in Sf9 microsomes was used as a positive control for Endo H deglycosylation activity. 11β-HSD1 was visualized using anti-FLAG polyclonal antibody at a 1:2,000 dilution. The three protein bands corresponding to 11β-HSD1 N-glycosylated on three, two, and one residue, respectively, are indicated by asterisks (*).

 
Role of RDH12 in Retinoid Metabolism—In vitro, RDH12 catalyzes the reduction of retinaldehyde to retinol or the oxidation of retinol to retinaldehyde depending on the substrate and cofactor availability. To determine whether the RDH12-catalyzed reaction is reversible in the cells, we incubated RDH12-transfected and mock-transfected cells with all-trans-retinaldehyde or all-trans-retinol. From all-trans-retinaldehyde, RDH12-transfected cells generated 2.2-fold less retinoic acid (p = 0.002) and 1.9-fold more retinol (p = 0.015) than mock-transfected cells (Fig. 2A). The amount of retinyl esters detected in RDH12-transfected cells was also greater than that in mock-transfected cells (720 ± 120 versus 500 ± 16 nmol/mg) (p < 0.05), consistent with the greater availability of retinol. These results indicated that RDH12 metabolized retinaldehyde to retinol, thereby decreasing the amount of retinaldehyde available for retinoic acid biosynthesis.


Figure 2
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  		FIGURE 2.
Enzymatic activity of RDH12 in transiently transfected HEK293 cells. HEK293 cells transfected with an expression construct for RDH12 (RDH12) or empty vector (mock) were incubated with 5 µM all-trans-retinaldehyde for 3 h (A) or 10 µM all-trans-retinol for 24 h (B). The products were extracted and analyzed by HPLC. ROL, retinol; RA, retinoic acid; RE, retinyl ester. Data are mean ± S.D., n = 4. Statistical analysis was done using two-tailed unpaired Student's t test comparing RDH12-transfected cells to mock-transfected cells.

 
The amount of retinoic acid produced from retinol was severalfold lower than that produced from retinaldehyde (Fig. 2B), consistent with the oxidation of retinol being the rate-limiting step in retinoic acid biosynthesis (17). However, the amount of retinoic acid was further decreased in the presence of RDH12 (2.1-fold, p = 0.0002), indicating that despite the excess of retinol, RDH12 again acted in the reductive direction and converted the retinaldehyde produced in the cells by endogenous retinol dehydrogenases back to retinol. The levels of retinyl esters in RDH12-transfected cells were 1.2-fold greater than in mock-transfected cells (p = 0.03) (Fig. 2B). Because retinol was provided in excess, this smaller difference in the levels of retinyl esters could be due to the limiting catalytic capacity of the cellular acyl transferases.

Together, these results demonstrated for the first time that, in intact cells, RDH12 acts unidirectionally as a retinaldehyde reductase and shifts the flow of the cellular retinoid metabolism toward the increased production of the storage forms, retinol and retinyl esters, and a decreased production of bioactive retinoic acid. This implied that RDH12 can regulate retinoic acid biosynthesis from retinol or retinaldehyde and protect the cells against potentially toxic levels of retinoic acid.

RDH12-mediated Protection against Retinaldehyde-induced Cell Death—To determine the efficiency of RDH12 at different retinaldehyde concentrations, RDH12-transfected and mocktransfected cells were incubated with 5, 10, and 30 µM retinaldehyde. Analysis of retinoid metabolites showed that the contribution of RDH12 as a retinaldehyde reductase increased dramatically at higher doses of retinaldehyde. The efficiency of retinaldehyde conversion to retinol and retinyl esters in RDH12-transfected cells was much greater at 30 µM than at 5 µM retinaldehyde (Fig. 3, A and B). At the same time, the presence of RDH12 effectively minimized the increase in retinoic acid levels caused by the increasing doses of retinaldehyde (Fig. 3C). This retinoic acid-lowering effect of RDH12 resulted in a progressively larger difference between the amounts of retinoic acid generated in RDH12-expressing versus RDH12-lacking cells.

Interestingly, the yields of the total cellular protein after treatments with retinaldehyde were consistently greater from the cells expressing RDH12, suggesting that these cells exhibited greater cell survival. To test whether RDH12 exhibited a protective effect against retinaldehyde-induced cell death, we examined cell viability by MTT assay (26). As shown in Fig. 3D, cells that expressed RDH12 were essentially insensitive to high doses of retinaldehyde whereas mock-transfected cells exhibited significantly reduced cell survival. At 30 µM retinaldehyde, 91% of RDH12-expressing cells were alive compared with only 48% of mock-transfected cells (p = 0.0003). Importantly, the greater survival of RDH12-expressing cells correlated with the lower levels of retinoic acid generated from retinaldehyde.

These results demonstrated that: 1) RDH12 was highly efficient at metabolizing a wide range of retinaldehyde concentrations; 2) high concentrations of retinaldehyde caused cell death; and 3) RDH12 rescued the cells by efficiently converting retinaldehyde to retinol and minimizing the increase in bioactive retinoic acid at higher concentrations of retinaldehyde. These results also suggested that mutations inactivating RDH12 could lead to increased toxicity due to accumulation of retinaldehyde and overproduction of retinoic acid.

Catalytic Properties of RDH12 Mutants—To examine the ability of disease-associated RDH12 mutants to control retinoid conversions in the cells, we selected six naturally occurring RDH12 variants that were confirmed by at least two independent groups and had substitutions in the cofactor-binding site 46GANTGIG (T49M (19, 21), I51N (20)), in the central core of the polypeptide (L99I (19, 20), H151D (19, 20)), or near the substrate-binding site 200YCHSK (S175P (19, 20), Y226C (19, 20)), and determined the effects of these mutations on RDH12 function in retinoid metabolism. The six RDH12 variants were expressed in Sf9 cells as fusions to the C-terminal His6 tag and purified using Ni2+ affinity chromatography. The first noticeable difference between the wild-type and mutant RDH12 was in the yields of purified recombinant proteins. Although the expression and purification of all seven proteins was carried out under identical conditions, the yields of mutant RDH12 proteins were noticeably lower than that of wild-type RDH12 (Fig. 4). Proteins with H151D and Y226C mutations were undetectable by silver staining after separation in denaturing polyacrylamide gel (Fig. 4), whereas the yields of other mutant proteins constituted less than 30% of wild-type RDH12.


Figure 3
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  		FIGURE 3.
Effect of RDH12 for retinaldehyde-induced cell death and analysis of retinoid metabolites. HEK293 cells were transfected with an expression construct for RDH12 (dark gray) or empty vector (light gray) and incubated for 24 h with different concentrations of all-trans-retinaldehyde as indicated. A–C, retinoids were extracted from the cells and media and separated by normal phase HPLC. Data shown represent the sum of retinoids from the cells and medium produced at the indicated concentrations of all-trans-retinaldehyde. RE, retinyl ester; ROL, retinol; RAL, retinal; RA, retinoic acid. Data are mean ± S.D., n = 3. D, cell viability was determined by MTT assay, and the optical density (OD) was monitored at 595 nm using an ELISA plate reader. Data are mean ± S.D., n = 4. Statistical analysis was done using two-tailed unpaired Student's t test comparing RDH12-transfected cells to mock-transfected cells. *, p < 0.05; **, p < 0.002.

 


Figure 4
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  		FIGURE 4.
Purification of wild-type and mutant RDH12-His6. Wild-type (RDH12) and mutant RDH12 variants T49M, I51N, L99I, H151D, S175P, and Y226C were expressed in Sf9 cells and purified as described under "Experimental Procedures." Equal fractions of purified proteins were analyzed by SDS-PAGE followed by silver staining. All proteins appeared as single bands of expected size (~36.6 kDa). Positions of molecular weight standards are indicated on the right.

 
Activity assays using 6.5 µM retinaldehyde as substrate revealed that purified T49M had a ~2-fold greater specific activity (950 nmol/min·mg–1) than wild-type RDH12 (430 nmol/min·mg–1), whereas both I51N and L99I had somewhat lower but still measurable specific activities (300 and 140 nmol/min·mg–1, respectively). L99I variant was quite unstable when stored frozen at –80 °C and lost a significant fraction of its activity over time. Interestingly, no activity was detected for the abundantly expressed and apparently stable S175P mutant. Thus, mutations in different segments of RDH12 polypeptide appeared to have a very different impact on its properties, relatively stable proteins were completely devoid of activity whereas some of the catalytically active mutants were rather unstable. However, two of the disease-associated mutants, T49M and I51N retained significant catalytic activity, suggesting that they could exert some level of control over retinoid metabolism in the cells. To determine whether these mutants had the same catalytic efficiency as wild-type RDH12, we carried out their kinetic characterization.

Remarkably, the kcat values of T49M and I51N variants were greater than the kcat value of wild-type RDH12. However, their apparent Km values for retinaldehyde were increased 5–7-fold, resulting in somewhat lower catalytic efficiencies (kcat/Km) compared with wild-type RDH12 (Table 2). The most dramatic change was observed in the Km values of T49M and I51N for nucleotide cofactors (Fig. 5), consistent with the close proximity of the mutations to the cofactor binding site. The apparent Km values of T49M and I51N for NADPH and NADP+ were at least 30-fold and over 100-fold greater, respectively, than those of wild-type RDH12 (Table 3). This reduced affinity could potentially impair T49M and I51N activities in the cells, because the cellular concentrations of NADP+/NADPH are generally low (~35 µM (29)). For retina, the amounts of NADP+ and NADPH were reported to be 33–324 µmol/kg dry tissue compared with 630–2170 µmol/kg dry tissue for NAD+ and NADH (30).


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  		TABLE 2
Kinetic constants for the reduction of all-trans-retinaldehyde

Kinetic constants were determined in the presence of saturating concentration of NADPH (1 mM).

 


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  		TABLE 3
The apparent Km values for cofactors

The apparent Km values for NADPH and NADP+ were determined in the presence of 5 µM all-trans-retinaldehyde or 5 µM all-trans-retinol, respectively.

 


Figure 5
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  		FIGURE 5.
Determination of Km values for NADPH. Shown is Lineweaver-Burk plot of wild-type RDH12 ({square}), T49M ({circ}), and I51N (•) activity as a function of NADPH concentration. Data were fitted using GraFit 3.0 and are representative of at least three independent experiments.

 
Together, these results suggested that out of six RDH12 mutants characterized in this study only two, T49M and I51N, had significant retinaldehyde reductase activity. To determine whether these mutants could contribute to the regulation of retinoic acid biosynthesis, we examined their performance in retinoid metabolism of living cells.

Analysis of the Retinoid Activities of T49M and I51N Mutants in Intact Cells—The protein levels of both T49M and I51N variants expressed in HEK293 cells were significantly lower compared with the wild-type protein (Fig. 6, A and B). This was not caused by lower stability of the mutant mRNAs, as established by Northern blot analysis (Fig. 6C). Rather, the reduced expression of T49M and I51N variants appeared to occur at the protein level and could be due to reduced biosynthesis and/or increased degradation.

Analysis of retinoid metabolites produced from retinaldehyde in I51N-transfected cells revealed that I51N variant was essentially inactive, since the levels of retinoic acid or retinol produced by these cells were almost identical to those in mock-transfected cells (Fig. 6D). T49M variant appeared to exhibit some retinaldehyde reductase activity, because T49M-transfected cells produced somewhat lower amounts of retinoic acid than mock-transfected cells, but the difference did not reach statistical significance (Fig. 6D). The lack of activity for the mutant proteins was in sharp contrast to the robust activity of wild-type RDH12, which effectively decreased the amount of cellular retinoic acid (2.4-fold, p = 0.009) and increased cellular retinol (2.6-fold, p = 0.004). These results demonstrated that despite considerable residual activity retained by purified T49M and I51N mutants in vitro, neither mutant enzyme was able to influence the direction of retinoid metabolism in the cells. Thus, photoreceptors expressing the partially active T49M or I51N variants of RDH12 would likely be at risk of overproducing retinoic acid.


Figure 6
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  		FIGURE 6.
Analysis of mutant RDH12 expression and activity in living cells. HEK293 cells were transfected with empty vector (mock) or expression constructs for wild-type (wt) or mutant RDH12 (T49M and I51N). A, expression levels of corresponding proteins were analyzed in cell lysates (100 µg) using anti-RDH12 antiserum. β-Actin immunostaining was used for control of protein loading. The experiment was repeated three times with similar results. B, quantification of the data shown in panel A. Band intensities were normalized to the loading control β-actin. Data are mean ± S.D., n = 3. C, mRNA expression levels were analyzed by Northern blot hybridization with a human RDH12-specific 32P-labeled probe. mRNA loading was analyzed using 32P-labeled probe directed against human GAPDH. Shown is the representative result of three independent experiments. D, transfected cells were treated with 5 µM all-trans-retinaldehyde for 3 h. The retinoids extracted from cells and media were separated by normal phase HPLC. Data shown represent combined retinoids extracted from medium and cells transfected with the indicated expression vectors. ROL, retinol; RA, retinoic acid. Data are mean ± S.D., n = 3. Statistical analysis was done using two-tailed unpaired Student's t test comparing cells transfected with wild-type or mutant RDH12 to mock-transfected cells. **, p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study demonstrate that, in living cells, RDH12 acts exclusively in the reductive direction and shifts the retinoid metabolism toward the increased production of retinol and retinyl esters. Importantly, the retinaldehyde reductase activity of RDH12 down-regulates the biosynthesis of retinoic acid, and not only from retinaldehyde as the starting substrate, but also from retinol, indicating that RDH12 is able to utilize the retinaldehyde produced in the cells by endogenous retinol dehydrogenases. Because the direction of RDH12-catalyzed reaction in the cells cannot be reversed by the excess of retinol, it must be determined by the RDH12 preference for NADP+/ NADPH as cofactors. As suggested by the lack of glycosylation, RDH12 faces the cytosolic side of the ER membrane, and in the cytosol of most cells, the concentration of NADPH is believed to be significantly greater than that of NADP+ (31). In retina, the ratio of NADPH to NADP+ varies throughout different layers but the concentration of NADPH exceeds that of NADP+ in the inner segments where RDH12 is located, suggesting that RDH12 also functions in the reductive direction in photoreceptors (30).

Genetic linkage studies indicate that mutations in RDH12 cause early-onset retinal degeneration (18–21), however, the etiology of photoreceptor cell death associated with mutations in RDH12 remains unknown. The results of this study suggest that a functional RDH12 is essential to photoreceptor physiology because of its role in the regulation of retinoic acid biosynthesis. Retinoic acid is produced in photoreceptors from retinaldehyde diffusing from illuminated rhodopsin into the inner segments of photoreceptors (32), where retinaldehyde is oxidized irreversibly to retinoic acid by cytosolic aldehyde dehydrogenases (33). The amount of all-trans-retinaldehyde generated in the eye is variable, because it depends on ambient light intensity, and can theoretically reach concentrations as high as 3 mM (13). Uncontrollable conversion of such high concentrations of retinaldehyde to retinoic acid may result in cytotoxicity because excessive levels of retinoic acid are known to induce apoptosis in many different cell types including photoreceptors (14–16). Our data suggest that RDH12 is ideally suited for protection of photoreceptors against overproduction of retinoic acid because it can metabolize a wide-range of retinaldehyde concentrations and is highly efficient at minimizing the fluctuations in retinoic acid levels at varied concentrations of retinaldehyde. Furthermore, the presence of RDH12 in the cells clearly protects these cells against death caused by high concentrations of retinaldehyde and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. This finding agrees remarkably well with the recent report that mice lacking RDH12 are highly susceptible to light-induced retinal apoptosis in both cone and rod photoreceptors (24).

At least 32 disease-associated mutations of RDH12 have been identified to date (18–21). Most of RDH12 mutants have a reduced ability to catalyze the reduction of retinaldehyde to retinol in vitro. However, some of the mutant forms of RDH12 appear to be quite active. Analysis of RDH12 mutants carried out in this study sheds a new light onto the discrepancy between the high in vitro catalytic activity of one such mutant, T49M, and the severity of retinal degeneration associated with this mutation. T49M was initially reported to have a twice greater activity than wild-type RDH12 (18, 19); however, a more recent study challenged this finding by showing that T49M has little or no activity when assayed in cell lysates (21). Here, we demonstrate that purified T49M as well as the previously uncharacterized variant, I51N, do, in fact, have kcat values greater than wild-type RDH12. However, when examined in living cells, these mutants are unable to control retinoic acid biosynthesis because of their low protein levels and reduced affinity for NADPH. Thus, the severity of retinal degeneration observed in human patients carrying T49M and I51N mutations is consistent with the lack of significant retinaldehyde reductase activity for these mutants in living cells.

Knock-out mouse models have been created to study the pathogenesis of retinal degeneration associated with the lack of RDH12 (23, 24). Surprisingly, the phenotype of RDH12-null mice is much less severe than that associated with mutations in human RDH12. It is possible that the mouse RDH12 has catalytic properties different from human RDH12. This appears to be the case for some of the human and rodent RDH orthologs (22). Alternatively, the retinaldehyde reductase activity of RDH12 may not be as critical for the control of retinoic acid biosynthesis in nocturnal animals as it is in humans. In this respect, it is interesting that the retinal structure observed in one of the patients with the loss of RDH12 function was found to have especially poor organization in the foveal region which is exposed to the highest intensities of light (21), consistent with the light-induced overproduction of retinoic acid.


   FOOTNOTES
 
* This work was supported by NIAAA, National Institutes of Health Grant AA12153 (to N. Y. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama-Birmingham, 720 20th St. South, 440B Kaul Genetics Bldg., Birmingham, AL 35294. Tel.: 205-996-4023; Fax: 205-934-0758; E-mail: nkedishvili{at}uab.edu.

2 The abbreviations used are: RDH12, retinol dehydrogenase 12; RalR1, retinaldehyde reductase 1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Endo H, endoglycosidase H. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Judith M. Thomas in the Dept. of Surgery at the University of Alabama, Birmingham, for sharing samples of M. mulatta tissues for Western blot analysis of RDH12 expression. We would also like to thank Dr. Christine Curcio in the Dept. of Ophthalmology, University of Alabama, Birmingham, for help with dissection of retina.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Saari, J. C. (2000) Invest. Ophthalmol. Vis. Sci. 41, 337–348[Free Full Text]
  2. Mangelsdorf, D., Umesono, K., and Evans, R. M. (1994) in The Retinoids: Biology, Chemistry and Medicine (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds), pp. 319–350, Raven Press, New York
  3. Hyatt, G. A., and Dowling, J. E. (1997) Invest. Ophthalmol. Vis. Sci. 38, 1471–1475[Free Full Text]
  4. Wagner, E., McCaffery, P., and Dräger, U. C. (2000) Dev. Biol. 222, 460–470[CrossRef][Medline] [Order article via Infotrieve]
  5. Levine, E. M., Fuhrmann, S., and Reh, T. A. (2000) Cell. Mol. Life Sci. 57, 224–234[CrossRef][Medline] [Order article via Infotrieve]
  6. Hyatt, G. A., Schmitt, E. A., Fadool, J. M., and Dowling, J. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13298–13303[Abstract/Free Full Text]
  7. De Leeuw, A. M., Gaur, V. P., Saari, J. C., and Milam, A. H. (1990) J. Neurocytol. 19, 253–264[CrossRef][Medline] [Order article via Infotrieve]
  8. Kastner, P., Mark, M., Ghyselinck, N., Krezel, W., Dupe, V., Grondona, J. M., and Chambon, P. (1997) Development 124, 313–326[Abstract]
  9. Li, A., Zhu, X., and Craft, C. M. (2002) Investig. Ophthalmol. Vis. Sci. 43, 1375–1383[Abstract/Free Full Text]
  10. Li, A., Zhu, X., Brown, B., and Craft, C. M. (2003) Investig. Ophthalmol. Vis. Sci. 44, 996–1007[Abstract/Free Full Text]
  11. Boatright, J. H., Stodulkova, E., Do, V. T., Padove, S. A., Nguyen, H. T., Borst, D. E., and Nickerson, J. M. (2002) Vision Res. 42, 933–938[CrossRef][Medline] [Order article via Infotrieve]
  12. Kalter, H., and Warkany, J. (1959) Physiol. Rev. 39, 69–115[Free Full Text]
  13. Saari, J. C. (1994) in The Retinoids: Biology, Chemistry, and Medicine (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds), pp. 351–385, Raven Press, New York, NY
  14. Birnbach, C. D., and Reh, T. A. (1997) Investig. Ophthalmol. Vis. Sci. 38, S311
  15. Smith, S. B., Bora, N., McCool, D., Kutty, G., Wong, P., Kutty, R. K., and Wiggert, B. (1995) Investig. Ophthalmol. Vis. Sci. 36, 2193–2201[Abstract/Free Full Text]
  16. Soderpalm, A. K., Fox, D. A., Karlsson, J. O., and van Veen, T. (2000) Investig. Ophthalmol. Vis. Sci. 41, 937–947[Abstract/Free Full Text]
  17. Napoli, J. L. (1999) Biochim. Biophys. Acta 1440, 139–162[Medline] [Order article via Infotrieve]
  18. Janecke, A. R., Thompson, D. A., Utermann, G., Becker, C., Hubner, C. A., Schmid, E., McHenry, C. L., Nair, A. R., Ruschendorf, F., Heckenlively, J., Wissinger, B., Nurnberg, P., and Gal, A. (2004) Nat. Genet. 36, 850–854[CrossRef][Medline] [Order article via Infotrieve]
  19. Thompson, D. A., Janecke, A. R., Lange, J., Feathers, K. L., Hubner, C. A., McHenry, C. L., Stockton, D. W., Rammesmayer, G., Lupski, J. R., Antinolo, G., Ayuso, C., Baiget, M., Gouras, P., Heckenlively, J. R., den Hollander, A., Jacobson, S. G., Lewis, R. A., Sieving, P. A., Wissinger, B., Yzer, S., Zrenner, E., Utermann, G., and Gal, A. (2005) Human Mol. Genet. 14, 3865–3875[Abstract/Free Full Text]
  20. Perrault, I., Hanein, S., Gerber, S., Barbet, F., Ducroq, D., Dollfus, H., Hamel, C., Dufier, J. L., Munnich, A., Kaplan, J., and Rozet, J. M. (2004) Am. J. Hum. Genet. 75, 639–646[CrossRef][Medline] [Order article via Infotrieve]
  21. Sun, W., Gerth, C., Maeda, A., Lodowski, D. T., Van Der Kraak, L., Saperstein, D. A., Héon, E., and Palczewski, K. (2007) Vision Res. 47, 2055–2066[CrossRef][Medline] [Order article via Infotrieve]
  22. Belyaeva, O. V., Korkina, O. V., Stetsenko, A. V., Kim, T., Nelson, P. S., and Kedishvili, N. Y. (2005) Biochemistry 44, 7035–7047[CrossRef][Medline] [Order article via Infotrieve]
  23. Kurth, I., Thompson, D. A., Ruther, K., Feathers, K. L., Chrispell, J. D., Schroth, J., McHenry, C. L., Schweizer, M., Skosyrski, S., Gal, A., and Hubner, C. A. (2007) Mol. Cell. Biol. 27, 1370–1379[Abstract/Free Full Text]
  24. Maeda, A., Maeda, T., Imanishi, Y., Sun, W., Jastrzebska, B., Hatala, D. A., Winkens, H. J., Hofmann, K. P., Janssen, J. J., Baehr, W., Driessen, C. A., and Palczewski, K. (2006) J. Biol. Chem. 281, 37697–37704[Abstract/Free Full Text]
  25. Belyaeva, O. V., Stetsenko, A. V., Nelson, P., and Kedishvili, N. Y. (2003) Biochemistry 42, 14838–14845[CrossRef][Medline] [Order article via Infotrieve]
  26. Gerlier, D., and Thomasset, N. (1986) J. Immunol. Methods 94, 57–63[CrossRef][Medline] [Order article via Infotrieve]
  27. Chen, Y., Ma, J. X., and Crouch, R. K. (2003) Mol. Vis. 9, 345–354[Medline] [Order article via Infotrieve]
  28. Odermatt, A., Arnold, P., Stauffer, A., Frey, B. M., and Frey, F. J. (1999) J. Biol. Chem. 274, 28762–28770[Abstract/Free Full Text]
  29. Bauldry, S. A., Nasrallah, V. N., and Bass, D. A. (1992) J. Biol. Chem. 267, 323–330[Abstract/Free Full Text]
  30. Matschinsky, F. M. (1968) J. Neurochem. 15, 643–657[CrossRef][Medline] [Order article via Infotrieve]
  31. Veech, R. L., Eggleston, L. V., and Krebs, H. A. (1969) Biochem. J. 115, 609–619[Medline] [Order article via Infotrieve]
  32. Dräger, U. C., Wagner, E., and McCaffery, P. (1998) J. Nutr. 128, 463S–466S[Medline] [Order article via Infotrieve]
  33. Pinazo-Duran, M. D., Verdejo, C., Azorin, I., Renau-Piqueras, J., and Iborra, F. J. (2000) Ophthalmic Res. 32, 61–68[CrossRef][Medline] [Order article via Infotrieve]

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O. V. Belyaeva, M. P. Johnson, and N. Y. Kedishvili
Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase
J. Biol. Chem., July 18, 2008; 283(29): 20299 - 20308.
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