From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
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ABSTRACT |
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We report the cDNA sequence and catalytic
properties of a new member of the short chain dehydrogenase/reductase
superfamily. The 1134-base pair cDNA isolated from the human liver
cDNA library encodes a 317-amino acid protein, retinol
dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with
rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and
RoDH-3, and mouse cis-retinol/androgen dehydrogenase
(73% identity). The mRNA for RoDH-4 is abundant in adult liver,
where it is translated into RoDH-4 protein, which is associated with
microsomal membranes, as evidenced by Western blot analysis.
Significant amounts of RoDH-4 message are detected in fetal liver and
lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect
cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to
corresponding aldehydes and oxidizes the 3
-hydroxysteroids
androstane-diol and androsterone to dihydrotestosterone and
androstanedione, respectively. NAD+ and NADH are the
preferred cofactors, with apparent Km values
250-1500 times lower than those for NADP+ and NADPH.
All-trans-retinol and 13-cis-retinol inhibit
RoDH-4 catalyzed oxidation of androsterone with apparent
Ki values of 5.8 and 3.5 µM,
respectively. All-trans-retinol bound to cellular
retinol-binding protein (type I) exhibits a similar Ki value of 3.6 µM. Unliganded
cellular retinol-binding protein has no effect on RoDH activity. Citral
and acyclic isoprenoids also act as inhibitors of RoDH-4 activity.
Ethanol is not inhibitory. Thus, we have identified and characterized a
sterol/retinol-oxidizing short chain dehydrogenase/reductase that
prefers NAD+ and recognizes all-trans-retinol
as substrate. RoDH-4 can potentially contribute to the biosynthesis of
two powerful modulators of gene expression: retinoic acid from retinol
and dihydrotestosterone from 3
-androstane-diol.
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INTRODUCTION |
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Short chain alcohol dehydrogenases/reductases are either cytosolic or membrane-bound enzymes with a subunit molecular mass of 25-35 kDa that utilize a vast variety of substrates, including steroids and prostaglandins (1). Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2-6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7). All-trans-retinoic acid is produced from all-trans-retinol in two oxidative steps: all-trans-retinol is oxidized to all-trans-retinal and then further to all-trans-retinoic acid. Retinol dehydrogenases catalyze the rate-limiting step: the oxidation of retinol to retinaldehyde (8). Although the effects of retinoic acid on gene transcription and regulation have been intensively studied during the last decade, the exact enzymes that synthesize this morphogen and the mechanisms that regulate its production in tissues are not fully understood. Enzymatic activity capable of oxidizing retinol to retinaldehyde is readily detected in the cytosolic and microsomal fractions of total cell homogenates (9). The cytosolic activity has been linked to the NAD+-dependent medium-chain alcohol dehydrogenases (ADHs),1 which, in addition to trans and cis forms of retinol, oxidize a variety of aliphatic and a number of cyclic alcohols (10). In the cells, most retinol is bound to the cellular retinol-binding protein (CRBP) (11). ADHs cannot oxidize the bound form of retinol.2
The first enzyme purified by following its ability to oxidize CRBP-bound retinol, RoDH-1, turned out to be a microsomal NADP+-dependent short chain dehydrogenase/reductase (3). The cDNA for RoDH-1 was initially isolated from a rat liver cDNA library, and later on, its mouse homolog was cloned and found to share 98% amino acid sequence identity with the rat enzyme (6). Two more closely related enzymes were subsequently cloned, RoDH-2 (4), with 82% sequence identity to RoDH-1, and RoDH-3 (12), with 95% sequence identity, establishing a multigene family of all-trans-retinol dehydrogenases.
The physiological role of these NADP+-dependent enzymes in retinoic acid biosynthesis in vivo has been questioned because of the cellular ratios of the reduced and oxidized forms of NADP+ (13). In the liver cytosol, and presumably other tissues, the NADP+/NADPH ratio is about 0.01, whereas the NAD+/NADH ratio is about 1000 (14), suggesting that enzymes that prefer NADP+ will function in the reductive rather than oxidative direction.
We became interested in finding an isoenzyme of RoDH that would function efficiently in the oxidative direction (i.e. prefer NAD+ as cofactor) and would recognize all-trans-retinol as substrate, because it was clear that RoDH exists in multiple isoenzymic forms. Here, we report a cDNA sequence and catalytic properties of a new human short chain dehydrogenase/reductase that shares more than 70% sequence identity with rat RoDHs, recognizes all-trans-retinol, and prefers NAD+ over NADP+.
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MATERIALS AND METHODS |
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cDNA Cloning--
A human liver gt10 cDNA library
(CLONTECH Inc., Palo Alto, CA) was screened with
the [
-32P]dATP-labeled coding region of rat RoDH-1
prepared by PCR amplification of the rat liver mRNA with
gene-specific primers designed according to a published sequence (3).
The hybridization conditions were as follows: 25% formamide, 5×
Denhardt's solution (0.1% bovine serum albumin, 0.1%
polyvinylpyrrolidone, 0.1% Ficoll 400), 5× saline-sodium-phosphate-EDTA, 0.1 mg/ml salmon sperm DNA, and 0.1% SDS
at 42 °C overnight. After hybridization, the Nytran filters were
washed several times in 6× SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.5), 0.1% SDS at room temperature,
and the final wash was performed in 0.2× SSC, 0.1% SDS. Positive
recombinant phage plaques were purified, and phage DNA was isolated
using a Qiagen Lambda kit (Qiagen, Chatsworth, CA). The DNA insert was obtained by digestion with EcoRI and subcloned into a
M13mp19RF digested with EcoRI. Sense and antisense
single-stranded M13 DNAs were each sequenced at least twice.
Northern Blot Analysis--
The cDNA probe was prepared by
EcoRI digestion of the cDNA clone in M13 vector and
purified by electrophoresis in 1% agarose gel. The human adult and
fetal multiple tissue Northern blots (CLONTECH)
were hybridized with 32P-labeled cDNA probe in
ExpressHyb hybridization solution according to the manufacturer's
instructions (CLONTECH). Briefly, the blots were
prehybridized in ExpressHyb solution for 30 min at 68 °C and
transferred to a fresh solution containing 2 ×106 cpm/ml
of the denatured radiolabeled cDNA. The hybridization was performed
at 68 °C for 1 h. The blots were rinsed in 2× SSC, 0.05% SDS
several times at room temperature and washed in 0.1× SSC, 0.1% SDS
for 40 min at 50 °C. The blots were exposed to x-ray film at
70 °C with two intensifying screens for 1 week.
Expression of RoDH-4 in Escherichia coli--
The N-terminal
fragment of RoDH-4 cDNA in M13 vector was amplified by PCR using
primers Eco5' (sense, CGG GAA TTC CAG GTG CTG AGC CAC CTG;
nucleotides at position 200-217) and Hin3' (antisense, AGA AAG
CTT TGT CTC TCA CGC ACT CC; nucleotides at position 430-446). The primers carried recognition sites for restriction endonucleases EcoRI and HindIII, respectively (underlined). The
C-terminal fragment of RoDH-4 cDNA was amplified using primers
Hin5' (sense, CCA AAG CTT GTG TGG TCA ACG TCT CCA GT;
nucleotides at position 609-628) and Xho3' (antisense, AGA CTC
GAG TGG CAT CCC AGC CAG CTG; nucleotides at position 981-998),
which carried recognition sites for restriction endonucleases
HindIII and XhoI, respectively (underlined). Both PCRs were heated for 5 min at 94 °C and cooled to 72 °C, 2 µl of Pfu polymerase (Stratagene) were added, and 30 cycles
were run as follows: denaturing at 94 °C for 45 s, annealing at
52 °C for 45 s, and extension at 72 °C for 6 min. The PCR
fragments were purified by electrophoresis in 1% agarose gel and
subcloned into pET32a vector (Novagen) digested with
EcoRI/HindIII and
HindIII/XhoI, respectively, using Rapid Ligation
kit (Boehringer Mannheim, Indianapolis, IN). Competent BL21 E. coli cells were transfected with ligation mixtures and spread onto
TY hard agar plates containing 200 µg/ml ampicillin. The pET32a
vectors that contained inserts were sequenced to verify the sequence of
the inserts and were transfected into BL21(DE3) cells. The expression
of recombinant proteins in BL21(DE3) cells was induced by 0.4 mM isopropyl-1-thio--D-galactopyranoside at
A600 of 0.8-1.0. The cultures were incubated
for 24 h at 30 °C. Cell pellets were resuspended in ice-cold
1× binding buffer (5 mM imidazole, 0.5 M NaCl,
20 mM Tris-HCl, pH 7.9) and homogenized twice using a
French press. The homogenate was centrifuged at 20,000 × g for 15 min, the supernatant was discarded, and the pellet
was resuspended in 1× binding buffer, sonicated, and centrifuged to
remove all soluble proteins. The pellet was resuspended in the 1×
binding buffer supplemented with 6 M urea and incubated on
ice for 1 h shaking. The solubilized proteins were loaded onto a
His Bind Resin charged with NiSO4 at room temperature and
equilibrated with 1× binding buffer plus 6 M urea. Protein
was eluted with 1× elute buffer plus 6 M urea. From a
1-liter culture, 5.5 mg of the N-terminal fragment and 3.5 mg of the
C-terminal fragment were obtained. The purified proteins were dialyzed
against 10 mM Tris-HCl, pH 7.4, to remove urea. Rabbits
were injected subcutaneously with 500-µg portions of each protein
mixed 1:1 with adjuvant five times at 3-week intervals. A 1:2000
dilution of each anti RoDH-4 antiserum detected ~1 ng of the
corresponding recombinant protein.
Expression of RoDH-4 in Sf9 Cells-- Sf9 cells were purchased from Invitrogen and were grown in a monolayer at 27 °C in Grace's insect cell culture medium (Life Technologies, Inc.) supplemented with yeastolate and lactalbumin hydrolysate (each to 3330 mg/liter), fetal bovine serum (10%), gentamycin (10 µg/ml), and amphotericin (2.5 µg/ml). At confluency, cells were sloughed off and split 1:3.
The RoDH-4 cDNA was subcloned into the XbaI/BglII restriction sites of pVL1392 vector (Pharmingen, San Diego, CA). The expression construct was sequenced to verify the sequence of RoDH-4. The cotransfection of Sf9 cells with RoDH-4-pVL1392 and BaculoGold DNA was performed according to manufacturer's protocol (Pharmingen). Briefly, 2.5 million Sf9 cells were cotransfected with a mixture of 2 µg of sterile RoDH-4-pVL1392 and 0.5 µg of BaculoGold DNA. The infected cells were incubated for 5 days at 27 °C, and the medium was collected and centrifuged for 10 min at 5000 × g to remove detached cells. The supernatant was amplified two more times, and the titer of the amplified supernatant was determined by plaque assay. To produce recombinant RoDH-4, attached Sf9 cells were infected at virus:cell ratio of 10:1. Cells were collected after 3 days of incubation at 27 °C; resuspended in 0.01 M potassium phosphate, pH 7.4, 0.25 M sucrose, 0.1 mM EDTA, 0.1 mM DTT; and homogenized using a Dounce homogenizer. The unbroken cells, cellular debris, and mitochondria were removed by centrifugation at 20,000 × g for 15 min. Microsomes were pelleted by centrifugation at 105,000 × g for 2 h and resuspended in 0.1 M potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.1 mM DTT, 20% glycerol. Microsomal suspension was aliquoted into small portions and stored frozen atWestern Blot Analysis-- A sample of frozen human liver was homogenized in 50 mM Hepes, pH 6.8, 0.5% Triton X-100, 2 mM DTT, 1 mM benzamidine, and 1 mM EDTA. The homogenate was centrifuged at 20,000 × g for 30 min, and the supernatant was recentrifuged at 105,000 × g for 2 h to isolate the membranes. Twenty-one micrograms of protein from each fraction, 105,000 × g supernatant, and 105,000 × g pellet were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 3% bovine serum albumin in PBS, washed several times with PBST, and incubated with a 1:2000 dilution of either anti-N-terminal or anti-C-terminal antiserum overnight. After washing with PBST, the membrane was incubated with a 1:2,000 dilution of 125I-labeled protein A. The bands were visualized by overnight exposure to x-ray film (Kodak X-OMAT AR).
Preparation of CRBP-bound Retinol--
The coding region of CRBP
(type I) cDNA (16) was amplified from rat liver total RNA by
reverse transcription-PCR using Pfu polymerase. The
gene-specific nucleotide primers carried restriction sites for
BamHI and EcoRI endonucleases. The PCR product
was subcloned into the corresponding sites in pGEX-2T expression vector
and sequenced. The recombinant protein was produced at 30 °C
overnight in TG-1 E. coli cells in the presence of 0.2 mM isopropyl-1-thio--D-galactopyranoside and
200 µg/ml ampicillin as a fusion with glutathione
S-transferase. The cell pellet was resuspended in ice-cold
PBS, 2 mM EDTA, 0.1%
-mercaptoethanol and lysed using a
French press. The fusion protein was purified to homogeneity using
affinity chromatography on a glutathione-agarose column. The purified
fusion protein was cleaved with thrombin in 50 mM Tris, pH
8.0, 0.1%
-mercaptoethanol, 150 mM NaCl, 2.5 mM CaCl2. CRBP was separated from glutathione
S-transferase by elution with 0-500 mM NaCl
gradient in 10 mM Tris, pH 7.4, 1 mM DTT from a
Q Sepharose column. The yield of CRBP was about 14 mg per liter of
culture. The final preparation exhibited a single protein band of
approximately 16 kDa by SDS-polyacrylamide gel electrophoresis. The
amount of functional protein was determined from the fluorescence
titration curve of apo-CRBP with retinol (17). Excitation was at 350 nm; emission was measured at 480 nm. The fluorescence values were
corrected for contribution of free retinol.
Determination of Kinetic Constants--
Steady-state kinetics
were performed in 90 mM potassium phosphate, pH 7.3, and 40 mM KCl at 37 °C in siliconized glass tubes. The
radiolabeled steroids (NEN Life Science Products)
5-androstan-3
,17
-diol (3
-adiol) (41 Ci/mmol),
5
-androstan-3
-ol-17one (androsterone) (45 Ci/mmol), and
5
-androstan-17
-ol-3-one (dihydrotestosterone) (43.5 Ci/mmol) were
diluted with cold steroids (Sigma) to achieve the required specific
radioactivity of each steroid. Aqueous solutions of the substrates were
prepared by adding 100× stock of the radiolabeled substrate in
dimethyl sulfoxide (Me2SO), so that the final concentration of Me2SO in the reaction mixture did not exceed 1%.
Equimolar amounts of bovine serum albumin were added to improve the
solubility of steroids. The suspensions were sonicated for 10 min, and
the concentration of the radiolabeled substrate in the aqueous phase was verified by counting an aliquot of the suspension. The 250-µl reactions were started with the addition of cofactor and stopped after
15 min by addition of 3.5 ml of methylene chloride. The aqueous phase
was removed and methylene chloride was evaporated under stream of
N2. Steroids were dissolved in 50 µl of ethanol; 10 µl
were spotted on alumina oxide thin layer chromatography plates (Sigma)
and resolved by development in chloroform/ethyl acetate (3:1),
according to Biswas and Russell (18). The RF values for
steroids under these conditions were 0.26 for 3
-adiol, 0.48 for
androsterone, 0.53 for dihydrotestosterone, and 0.82 for
5
-androstan-3,17-dione (androstanedione). The lanes were cut into
pieces ~1 cm wide and counted in scintillation liquid (Bio-Safe II).
For determination of apparent Km values, five
concentrations between 0.6 and 1 µM were used for
androsterone, 0.25-4 µM for dihydrotestosterone, and
0.1-2.5 µM for adiol. Initial velocities (nmol of
product formed/mg of protein) were obtained by linear regression. The
amount of product formed was less than 10% within the 15-min reaction
time and was linearly proportional to the amount of microsomes added.
The Km values for oxidation of alcohols were
determined at a fixed NAD+ (1 mM)
concentration; for reduction of dihydrotestosterone, values were
determined at a fixed NADH (0.5 mM) concentration. Each
Km determination was repeated at least three times.
A control without added cofactor was included with each experiment. The
apparent Km values for cofactors were determined
with six concentrations between 0.05-6.4 µM for
NAD+, 0.25-8 mM for NADP+,
15-1000 µM for NADPH, and 0.15-10 µM for
NADH.
Retinol Assays and HPLC Analysis of Reaction Products-- Assays of RoDH-4-catalyzed oxidation and reduction of retinoids were performed in 90 mM potassium phosphate, pH 7.3, and 40 mM KCl at 37 °C in siliconized glass tubes. Retinoid stock solutions in Me2SO were added to the reaction buffer along with equimolar bovine serum albumin and sonicated for 10 min. Experiments with tritiated retinol showed that this procedure improved solubilization of retinol. The concentration of Me2SO in the reaction mixture did not exceed 1%. The 500-µl reactions were started with the addition of cofactor and stopped after 30 min by addition of an equal volume of cold ethanol supplemented with 100 µg/ml butylated hydroxytoluene and an internal standard, retinol acetate. Reactions were placed on ice and extracted twice with 7 volumes of hexane. The aqueous phase was removed, and hexane was evaporated under a stream of N2. Retinoids were dissolved in 200 µl of mobile phase, and an aliquot was analyzed by HPLC.
All HPLC procedures were performed using an automatic injector 710WIS from Waters and a Varian 9010 pump. Elution was monitored at 370 nm with a variable wavelength Varian 9050 detector connected to a Hewlett Packard P3390 integrator. The stationary phase was a Beckman ultrasphere ODS column (4.6 mm x 15 cm). The mobile phase consisted of 0.05 M ammonium acetate, pH 7.0:acetonitrile:tetrahydrofuran (70:168:12). The flow rate was 1 ml/min. Under these conditions, all-trans-retinol, all-trans-retinaldehyde, and retinol acetate eluted at 13, 17, and 34 min, respectively. All-trans-retinal was quantitated by comparing its peak height to a calibration curve of the amount of pure retinal injected onto the column versus the resulting peak heights. ![]() |
RESULTS |
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To isolate human homologs of rat RoDH isoenzymes, we prepared a
RoDH-1 cDNA probe by reverse transcription-PCR amplification of rat
liver mRNA with gene-specific primers. The radiolabeled RoDH-1
cDNA was used to screen the human liver gt10 cDNA library. We applied low stringency conditions for screening the library, because
our goal was to find an enzyme with similar (recognition of
all-trans-retinol) but not identical properties (preference for NAD+, not NADP+). Four positives were
identified during the first round of screening. Two of these positive
clones were purified and sequenced. The longest clone contained a
1308-base pair cDNA (Fig. 1), which exhibited 78% nucleotide sequence identity with RoDH-1 cDNA in the
coding region. Similar to RoDH-1, the human cDNA encoded a 317-amino acid protein including a starting Met. The deduced protein product exhibited features characteristic of the short chain
dehydrogenase/reductase family of enzymes, such as putative consensus
sequence for the cofactor binding site,
G(X)3GXG, at Gly-36, and the active
site consensus sequence, Y(X)3K, at Tyr-176. The
new human short chain dehydrogenase/reductase showed the highest
sequence identity with the recently reported
cis-retinol/androgen dehydrogenase from mouse (73%), rat
RoDH forms 1 and 3 (72%), and rat RoDH-2 (71%). Because rat RoDHs
share 98% sequence identity with their mouse homologs, we assumed that
the human cDNA cloned in this study encoded a previously unknown
form of short chain dehydrogenase/reductase, and we called the new
isoenzyme RoDH-4.
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The tissue distribution of RoDH-4 mRNA was analyzed by Northern blot analysis (Fig. 2). A very strong signal was observed in human liver, similar to the expression patterns of rat all-trans-retinol dehydrogenases (3, 4, 12). In addition, relatively high levels of hybridizing message were detected in fetal liver and lung (Fig. 2). The size of the message in fetal lung was somewhat smaller than in fetal liver. This could be due to cross-hybridization with a closely related gene product or to a different processing of the mRNA. The tissue distribution of RoDH-4 was distinctively different from that of the mouse cis-retinol/androgen dehydrogenase (6) and the two human cis-retinol dehydrogenases that are not active toward all-trans-retinol (2, 5).
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Next, we tested whether the mRNA for RoDH-4 is translated into RoDH-4 protein in the human liver by Western blot analysis. Antibodies were raised against the N-terminal and the C-terminal fragments of RoDH-4 expressed in E. coli as described under "Materials and Methods." A sample of frozen human liver was homogenized and fractionated by centrifugation into cytosol and microsomes. RoDH-4 protein was detected by incubation with either anti-N-terminal or anti-C-terminal antiserum (Fig. 3). A single band of 35 kDa appeared in the 105,000 × g membrane fraction using either antiserum (Fig. 3, lane P), indicating that RoDH-4 message is translated into a protein in human liver, and the protein is associated with membranes. This is similar to the subcellular localization of other RoDH isoenzymes.
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To obtain a catalytically active enzyme, the cDNA for RoDH-4 was expressed in insect cells using the BaculoGold Baculovirus system. The full-length cDNA was subcloned into the pVL1392 transfer vector and cotransfected with linearized BaculoGold DNA into Sf9 cells. The recombinant virus was amplified and used to produce recombinant RoDH-4. The identity of the band was confirmed by Western blotting using polyclonal antiserum raised against partial RoDH-4. Our attempts to solubilize the recombinant enzyme and purify it from the membranes using Triton X-100 led to complete inactivation of the enzyme. Thus, intact membranes were used for kinetic characterization of RoDH-4.
Human RoDH-4 was most similar to the mouse
cis-retinol/androgen dehydrogenase (CRAD) (6) and rat
all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3
(3, 4). Both CRAD and RoDH were recently found to oxidize the
3-hydroxysteroids 3
-adiol and androsterone to dihydrotestosterone
and androstanedione, respectively (6, 18). Thus, we tested whether
RoDH-4 could function as a 3
-hydroxysteroid dehydrogenase.
RoDH-4-containing microsomes were incubated with tritiated 3
-adiol
and androsterone in the presence of NAD+ or
NADP+. The product of 3
-adiol oxidation comigrated with
dihydrotestosterone on alumina oxide thin layer plate (18). The product
of androsterone oxidation comigrated with androstanedione. RoDH-4 also
catalyzed the reverse reaction, reduction of tritiated
dihydrotestosterone in the presence of NADH, similar to the human
3
-hydroxysteroid dehydrogenase characterized by Biswas and Russell
(18). No activity was detected in the microsomes from mock-transfected
Sf9 cells, or in the controls without cofactor that were
included with each experiment.
The apparent Km values for oxidation of 3-adiol
and androsterone were determined with saturating concentration of NAD+, and the Km value for the
reduction of dihydrotestosterone was determined with saturating NADH.
All three steroids exhibited Km values of under 1 µM (Table I), similar to
RoDH-1 and CRAD. The Vmax values of RoDH-4 were
in the range of nmol/min/mg of microsomes (Table I). The apparent
Km values for cofactors NAD+ and
NADP+ were determined with saturating androsterone, and the
Km values for NADH and NADPH were determined with
saturating dihydrotestosterone (Table
II).
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RoDH-4 oxidized all-trans-retinol and 13-cis-retinol to corresponding retinaldehydes (Fig. 4). The reaction products were analyzed by HPLC. Microsomes that contained RoDH-4 oxidized all-trans-retinol to all-trans-retinal (Fig. 4, A and B) in the presence of NAD+. No activity was detected in the absence of cofactor or with microsomes isolated from mock-transfected cells. The reaction rate increased linearly with the amount of enzyme in the reaction mixture (Fig. 4A). About 400 pmol of all-trans-retinaldehyde were produced from 10 µM all-trans-retinol by 1.9 µg of RoDH-4-containing microsomes in 30 min. More product was formed in the presence of NAD+ compared with NADP+ from both all-trans-retinol (not shown) and 13-cis-retinol (Fig. 4C), consistent with the cofactor preference of RoDH-4 determined in the experiments with steroids. Thus, human RoDH-4 is similar to rat RoDH-1 in that it also recognizes all-trans-retinol as a substrate. However, unlike the rat enzyme, human RoDH-4 prefers NAD+ over NADP+ and is likely to function in the oxidative direction in vivo. Human RoDH-4 is also different from the NAD+-dependent mouse CRAD, which is specific for cis-retinols and does not oxidize all-trans-retinol. Hence, RoDH-4 is clearly a new isoenzyme with a distinctively different primary structure and catalytic properties.
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We tested whether stereoisomers of retinol can inhibit oxidation of androsterone, because both retinol and sterols were substrates for RoDH-4. Both all-trans-retinol and 13-cis-retinol acted as competitive inhibitors of androsterone oxidation (Table III). Most all-trans-retinol is bound to CRBP in liver cells. RoDH-1 recognizes CRBP-bound retinol as substrate (3); therefore, it was very interesting to see whether retinol bound to CRBP could act as efficiently as free retinol in inhibiting steroid oxidation. Surprisingly, the apparent Ki value for CRBP-retinol inhibition was almost the same as the Ki value for free retinol (Table III). Apo-CRBP alone had no effect on the reaction. The almost identical Ki values for the free and bound all-trans-retinol suggest that CRBP binding does not alter the affinity of RoDH-4 for all-trans-retinol.
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Other inhibitors of RoDH-4 activity toward steroids included citral and acyclic isoprenoids. The concentration of androsterone in these experiments was at the Km value of 0.14 µM. 20 µM citral (3,7-dimethyl-2,6-octadienal) inhibited RoDH-4 activity with androsterone about 50% (Table IV). Some of the acyclic isoprenoids, perillyl alcohol, geraniol, farnesol, and geranyl geraniol, were even more potent inhibitors of steroid oxidation (Table IV). Ethanol at 100 mM had no effect on RoDH-4 activity with androsterone.
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DISCUSSION |
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We have cloned and characterized a new short chain
dehydrogenase/reductase, RoDH-4, which represents the first human
microsomal enzyme capable of oxidizing all-trans-retinol to
all-trans-retinaldehyde and the second human enzyme to
oxidize 3-hydroxysteroids with NAD+ as the preferred
cofactor. The primary structure of RoDH-4 is about 70% identical to
the rodent retinol/sterol dehydrogenases.
Currently, four types of retinol-oxidizing short chain dehydrogenases/reductases can be distinguished. The first type are the NADP+-preferring isoenzymes that utilize free and CRBP-bound all-trans-retinol in rat (RoDH-1 and RoDH-2) (3, 4) and mouse (RoDH-1 and RoDH-2) (6). The second type is the NAD+-dependent CRAD, recently cloned from mouse (6). CRAD has 80-85% sequence identity to RoDHs, and it oxidizes 9-cis- retinol and 11-cis-retinol but not all-trans-retinol. The third type is the eye-specific retinol dehydrogenase in retinal pigment epithelium that recognizes only 11-cis-retinol (2). The fourth type shares 95% amino acid sequence identity with the eye 11-cis-retinol dehydrogenase but apparently prefers 9-cis-retinol as substrate and is found in multiple tissues (5). The last two cis-retinol dehydrogenases prefer NAD+ over NADP+, and have only 50 to 55% identity with RoDHs and CRAD.
Recently, a cDNA for the first human RoDH-like short chain
dehydrogenase/reductase has been isolated from the human prostate cDNA library by screening with rat RoDH-1 cDNA (18). Analysis of the substrate specificity of the prostate RoDH-like enzyme, transiently expressed in embryonic kidney 293 cells, showed that it
oxidizes 3-hydroxysteroids efficiently and exhibits higher affinity
for NAD+ than for NADP+. Whereas reductive
NADP+-dependent 3
-hydroxysteroid
dehydrogenases have been described, the prostate RoDH-like
dehydrogenase represents the first known oxidative
NAD+-dependent 3
-hydroxysteroid
dehydrogenase. Whether it is also active on retinols was not reported.
This prostate 3
-hydroxysteroid dehydrogenase has only 62% amino
acid sequence identity with human RoDH-4 reported in this study and is
less similar to retinol-oxidizing RoDH isoenzymes (61-63% amino acid
sequence identity) than RoDH-4 (72% identity).
Similar to the prostate RoDH-like steroid dehydrogenase, RoDH-1 and
CRAD were found to oxidize the 3-hydroxysteroids 3
-adiol and
androsterone to dihydrotestosterone and androstanedione, respectively (3, 6). We tested whether the new human RoDH-4 was active against
steroid alcohols. As expected, human RoDH-4 oxidized 3
-adiol to
dihydrotestosterone and androsterone to androstenedione in the presence
of either NAD+ or NADP+. Accordingly,
3
-dihydrotestosterone was reduced to 3
-adiol in the presence of
NADH or NADPH. The catalytic efficiency of RoDH-4 in the reductive
direction was about 2.5 times lower than in the oxidative
direction.
The nonphosphorylated cofactors NAD+ and NADH were clearly preferred by RoDH-4: the apparent Km value for NAD+ was almost 1500 times lower than that for NADP+ (Table III). Similarly, the apparent Km value for NADH was 250 times lower than that for NADPH.
The Km values of RoDH-4 for steroid substrates
(under 1 µM) were in the same range as those of other
microsomal dehydrogenases (3, 6). The reported
Vmax values (nmol/min·mg of cell lysates) for
steroid dehydrogenases transiently expressed in eukaryotic cells varied
depending on the transfection efficiency of each experiment; thus, no
direct comparisons can be made. The relative catalytic efficiencies of
all steroid-oxidizing isoenzymes toward each substrate, however, were
similar. In general, the catalytic efficiencies were about twice higher
for oxidation of 3-adiol than for oxidation of androsterone.
Attempts to purify this and similar enzymes out of microsomal membranes resulted in either loss or significant decrease in enzymatic activity. The only retinol/sterol dehydrogenase ever purified directly from rat tissues, RoDH-1, had to be reconstituted with phosphatidylcholine to regain its activity (21). The specific activity reported for the partially purified RoDH-1 was 307 pmol/min·mg with free retinol and 96 pmol/min·mg for CRBP-bound retinol in the presence of NADP+ (21). RoDH-1 activity with free retinol was lower in the presence of NAD+: 1.7 times with free retinol and 5.6 times if CRBP-retinol was a substrate.
Similar to RoDH-1, RoDH-4 converted all-trans-retinol to all-trans-retinal in the presence of either NAD+ or NADP+; however, the reaction rate was about five times higher with NAD+. Enzymatic activity similar to RoDH-4 has been described in the liver microsomes of rat and ADH-negative deermouse (22). The rat liver enzyme in the above study was more active with NAD+ than with NADP+, was similarly insensitive to ethanol inhibition, and was strongly inhibited by Triton X-100. Thus, it is likely that homologs of human RoDH-4 exist in rats and mice.
If the catalytic site of RoDH-4 faces the cytosol, then the cytosolic
ratios of cofactors (NAD+/NADH = 1000) should drive
the reaction in the oxidative direction. However, it is not known how
RoDH and similar enzymes are inserted into the microsomal membrane. We
analyzed the primary structures of retinol-oxidizing short chain
dehydrogenases/reductases for the presence of membrane-spanning domains
using programs HELIXMEM (23), RAOARGOS (24), and SOAP (25, 26). These
programs predict the existence of membrane-associated helices in a
protein sequence, evaluate the hydropathic index along the sequence,
and predict whether a membrane protein is peripheral or integral. Although the number and exact location of the membrane-spanning domains
predicted by the different programs differed somewhat, all
retinol-oxidizing short chain dehydrogenases/reductases were classified
as integral membrane proteins (Fig.
5A). Comparative analysis of
the primary structures of the isoenzymes suggested that RoDH-4 contains
four potential transmembrane segments: the N-terminal segment 1 (amino
acids 1-21), the two closely positioned central segments 2 and 3 (amino acids 105-125 and 130-150), and the C-terminal segment 4 (amino acids 288-309) (Fig. 5, boxed). The hydrophobic
N-terminal segment 1 is followed by the two highly conserved arginines
at positions 19 and 21. The hydrophobic domain 3 is also followed by
two conserved arginines at positions 156 and 158. The topological rules
for membrane protein assembly in eukaryotic cells imply that the
positively charged amino acids, such as arginine and lysine,
immediately downstream of the first hydrophobic domain prevent
translocation across the microsomal membrane and that there is a clear
tendency for highly charged internal loops to remain on the cytoplasmic
side (27). The two neighboring hydrophobic segments, 2 and 3, which are
connected by a short loop, can be inserted according to a helical
hairpin mechanism (28).
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Based on the analysis of the primary structure of RoDH-4, we propose a model of transmembrane insertion of RoDH-4 in the microsomal membrane (Fig. 5B). According to this model, the two loops between the hydrophobic segments will remain on the cytosolic side. Because the concentration of NAD+ in the cytosol greatly exceeds that of NADH, RoDH-4 will likely function as a dehydrogenase, not reductase, in the cells. Interestingly, the cofactor binding consensus sequence is located on the N-terminal loop and is highly conserved among short chain dehydrogenases/reductases. The substrate binding consensus sequence is located on the less conserved C-terminal loop, which is consistent with the different substrate specificity of the isoenzymes. Based on our model of transmembrane insertion of RoDH-4, the cofactor binding domain will face the cytosol, and RoDH-4 will function in the oxidative direction.
This model is consistent with our observation that CRBP-retinol inhibits oxidation of androsterone catalyzed by RoDH-4 with the same efficiency as free retinol, suggesting that both forms of retinol have equal access to the active site. CRBP is a cytosolic protein, and this is consistent with the suggestion that the active site of RoDH-4 faces cytosol. Several microsomal enzymes were found to metabolize bound forms of retinoids with the same catalytic efficiency as free. For example, the intestinal retinal reductase does not distinguish between free and CRBP-II-bound all-trans-retinal (29). It exhibits similar affinity (0.78 µM for free and 0.46 µM for bound) and almost identical rates (~300 pmol/min/mg of microsomal protein) with both forms of retinal. In the pigment epithelium of the eye, 11-cis-retinaldehyde reductase is about equally efficient in reducing free 11-cis-retinal and 11-cis-retinal complexed with cellular retinal-binding protein (30).
Citral is often used to inhibit oxidation of retinol and retinaldehyde to retinoic acid in cell culture experiments (31). Human keratinocytes incubated in the presence of 100 µM citral for 4 h show 75% reduction in retinoic acid synthesis from all-trans-retinol (32). In our experiments, 20 µM citral inhibited RoDH-4 45%. This also distinguishes RoDH-4 from RoDH-1, which is not inhibited by citral.
RoDH-4 can oxidize the carbon 3 alcohol group on 3-adiol, producing
dihydrotestosterone, a powerful androgen with high affinity for
androgen receptor. RoDH-4 is the second isoform of human
NAD+-dependent 3
-hydroxysteroid
dehydrogenases; it shares 62% sequence identity with the first
isoform, human RoDH-like 3
-hydroxysteroid dehydrogenase cloned from
prostate. In prostate, dihydrotestosterone is produced mainly from
testosterone by steroid 5
-reductases (reviewed in Ref. 33).
Dihydrotestosterone is inactivated by NADPH-dependent
cytosolic 3
-hydroxysteroid dehydrogenases, which reduce
dihydrotestosterone to adiol (reviewed in Ref. 34). The presence of the
NAD+-dependent 3
-hydroxysteroid
dehydrogenase in prostate explains the so-called back reaction, in
which adiol is oxidized back to dihydrotestosterone. Both RoDH-4 and
RoDH-like steroid dehydrogenase are abundant in the liver.
Dihydrotestosterone is inactivated in the liver cytosol by multiple
reductive 3
-hydroxysteroid dehydrogenases. The role of
dihydrotestosterone producing RoDH-4 in the non-steroidogenic liver is
not clear yet.
Dietary acyclic isoprenoids d-limonene, perillyl alcohol, geraniol, farnesol, and geranyl geraniol are currently undergoing clinical trials as chemopreventive therapeutical agents against mammary, liver, lung, stomach, skin, and pancreas cancers (35). Geraniol and farnesol are present in the essential oils of lemongrass, perillyl alcohol is present in cherry and spearmint, and d-limonene is present in orange peel oil, caraway, and dill. The primary metabolites of limonene, perillic acid and dihydroperillic acid, are more potent inhibitors of tumor cell proliferation than is perillyl alcohol (36). The enzymes that oxidize acyclic isoprenoids to their corresponding acids in vivo are not known. We hypothesized that acyclic isoprenoids may be recognized by RoDH-4 along with all-trans-retinol and steroid alcohols. In this study, we have established that acyclic alcohols can function as effective inhibitors of RoDH-4.
To our knowledge, RoDH-4 is the first human retinol-oxidizing short chain dehydrogenase/reductase that is active toward all-trans-retinol and prefers NAD+ over NADP+. This implies that in vivo RoDH-4 functions in the oxidative direction and can contribute to the biosynthesis of all-trans-retinoic acid in the adult and fetal liver, and possibly in fetal lung. The contribution of this microsomal enzyme to retinoic acid biosynthesis relative to cytosolic ADH isoenzymes is a subject of future studies. It is clear, however, that microsomal RoDH isoenzymes may have a broader substrate specificity than was initially thought. Additional isoforms of all-trans-retinol dehydrogenases may be responsible for retinol oxidation in extrahepatic tissues.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ting-Kai Li, the Principal Investigator on NIAAA Grant AA02342 and the Preceptor on the Research Career Development Award (to N. Y. K.) for his continuous support and guidance, Dr. William F. Bosron and Robert A. Harris for helpful discussions and critical reading of the manuscript, and Natividad Dumanal for technical assistance with retinol assays and HPLC analysis.
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FOOTNOTES |
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* This work was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA00221 and AA02342.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF057034.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS 405, Indianapolis, IN 46202-5122. Tel.:
317-278-0296; Fax: 317-274-4686.
1
The abbreviations and trivial names used are:
ADH, alcohol dehydrogenase; CRBP, cellular retinol-binding protein;
RoDH, retinol dehydrogenase; HPLC, high performance liquid
chromatography; Me2SO, dimethyl sulfoxide; 3-adiol,
5
-androstan-3
,17
-diol; androsterone, 5
-androstan-3
-ol-17one; dihydrotestosterone,
5
-androstan-17
-ol-3-one; androstanedione,
5
-androstan-3,17-dione; CRAD, cis-retinol/androgen dehydrogenase; PCR, polymerase chain reaction.
2 N. Y. Kedishvili, W. H. Gough, W. I. Davis, S. Parsous, T.-K. Li, and W. F. Boston, unpublished observations.
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REFERENCES |
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