©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of a cDNA for Liver Microsomal Retinol Dehydrogenase
A TISSUE-SPECIFIC, SHORT-CHAIN ALCOHOL DEHYDROGENASE (*)

(Received for publication, October 28, 1994; and in revised form, December 9, 1994)

Xiyun Chai Manja H. E. M. Boerman Yan Zhai Joseph L. Napoli (§)

From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Retinoic acid, a hormone biosynthesized from retinol, controls numerous biological systems by regulating eukaryotic gene expression from conception through death. This work reports the cloning and expression of a liver cDNA encoding a microsomal retinol dehydrogenase (RoDH), which catalyzes the primary and rate-limiting step in retinoic acid synthesis. The predicted amino acid sequence and biochemical data obtained from the recombinant enzyme verify it as a short-chain alcohol dehydrogenase. Like microsomal RoDH, the recombinant enzyme recognized as substrate retinol bound to cellular retinol-binding protein, had higher activity with NADP rather than NAD, was stimulated by ethanol or phosphatidylcholine, was not inhibited by 4-methylpyrazole, was inhibited by phenylarsine oxide and carbenoxolone and localized to microsomes. RoDH recognized the physiological form of retinol, holocellular retinol-binding protein, with a K of 0.9 µM, a value lower than the 5 µM concentration of holocellular retinol binding protein in liver. Northern and Western blot analyses revealed RoDH expression only in rat liver, despite enzymatic activity in liver, brain, kidney, lung, and testes. These data suggest that tissue-specific isozyme(s) of short chain alcohol dehydrogenases catalyze the first step in retinoic acid biogenesis and further strengthen the evidence that the ``cassette'' of retinol bound to cellular retinol-binding protein serves as a physiological substrate.


INTRODUCTION

Retinol (vitamin A) undergoes metabolic activation by dehydrogenation into retinal, followed by oxidation into the hormone all-trans-retinoic acid (RA)^1. RA directs a variety of biological responses by modulating gene expression during development and postnatally, to control differentiation or entry into apoptosis of numerous cell types in diverse organs(1, 2, 3, 4) . Insight into RA biosynthesis has been limited by a lack of data concerning enzymes dedicated specifically to this pathway. The enzyme(s) that catalyze(s) RA synthesis physiologically should recognize the predominant form of retinol in vivo. Retinol in liver occurs bound in the protected environment of CRBP: the concentration of CRBP exceeds that of retinol, and CRBP envelops retinol in a high affinity (K 0.1 to 1 nM) binding pocket(5, 6) . CRBP may confer specificity on RA biosynthesis by restricting retinol access to enzymes capable of recognizing the retinol/retinol binding-protein ``cassette.'' This would prevent opportunistic oxidation by dehydrogenases with broad substrate tolerances, protect retinol from non-enzymatic oxidation and protect cells from the membrane-disrupting potential of free retinol (7, 8, 9) . A pathway of RA synthesis elucidated recently, consistent with this hypothesis, entails as the first and rate-limiting step an NADP-dependent microsomal RoDH, that recognizes holoCRBP as substrate (10, 11) . Retinal generated in microsomes from holoCRBP by RoDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase(12) .

Liver microsomal RoDH has been partially purified and its active site has been associated with a 34-kDa polypeptide by covalent binding with, and inactivation, by PAO and by chemical cross-linking with holoCRBP.^2 The 34-kDa polypeptide has a subunit molecular mass and other attributes typical of SCAD, including the conserved sequence WXLVNNAG, Zn independence, inhibition by carbenoxolone (IC = 55 µM), and insensitivity to inhibition by ethanol or 4-methylpyrazole. The 34-kDa polypeptide co-purified with a 54-kDa polypeptide and RoDH activity was precipitated with either anti-34-kDa or anti-54-kDa polypeptide antisera. It seems unlikely, however, that RoDH exists as a heteromultimer between the 34- and 54-kDa polypeptides, because known SCAD occur as homomultimers(13, 14, 15, 16, 17) .

This work reports the cDNA cloning and expression of the 34-kDa polypeptide from rat liver, provides new evidence that it is a previously unknown SCAD, shows that it can catalyze the first step in RA synthesis with holoCRBP as substrate, and reveals that it is expressed tissue specifically.


MATERIALS AND METHODS

Amino Acid Sequence Analysis

The N-terminal amino acid sequence of the 34-kDa polypeptide was determined to be MWLYLLALVG. Four internal peptides were obtained by digestion in situ with trypsin, isolated by high performance liquid chromatography, and sequenced: LWGLVNNAGISVPV-PNE-M, ELTYFGVK, VAIIEPGGFK, and YSPGWDAK.

Cloning

Degenerate oligonucleotide primers for reverse transcriptase PCR were synthesized based on two of the amino acid sequences described above: sense from WGLVNNA, 5`-CTCGCTCGCCCATGGGGICTIGTIAA(C/T)AA(C/T)GC-3`; antisense from PGWDAK, 5`-CTGGTTCGGCCCATTIGC(G/A)TCCCAICCIGG-3`. Nucleotides used as linkers for cloning into the vector p-Direct (Clontech) are underlined. The cDNA template was prepared by allowing 1 µg of rat liver mRNA to react with 0.5 µg of oligo(dT), 10 units of rRNase ribonuclease inhibitor, and 15 units of avian myeloblastosis virus reverse transcriptase in a total volume of 20 µl for 60 min at 42 °C. The reaction mixture was diluted one-tenth with water, and 4 µl of the diluted solution were added to a PCR reaction mixture consisting of (final concentrations): 1 µM of each primer, 1.5 mM MgCl(2), 0.2 mM of each dNTP, and 2.5 units of Taq DNA polymerase (Promega) in 0.1 ml of 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100. PCR was done using 35 cycles of 2 min at 94 °C, 2 min at 52 °C, 3 min at 72 °C, and 10 min at 72 °C after the final cycle. The 0.6-kilobase PCR product (nucleotides 612 through 1158 of the final cDNA product) was gel-purified and cloned into p-Direct to provide p-DRoDH1.

Library Screening

A 323-base pair probe (probe A, nucleotides 653-975 of the final cDNA product) was excised from p-DirectRo1 with AvaII and labeled with P by the random priming method(18) . Probe A was hybridized at 42 °C to nitrocellulose filters containing 5 times 10^5 plaques from a gt11 rat liver cDNA library (Clontech). The final wash was done at 65 °C with 0.1% SDS in 1 times SCC. Twenty-five plaques were identified after three rounds of screening. DNA from each plaque was amplified by PCR and transferred to Nylon membranes for Southern blot analyses with a 96-base pair probe (probe B, nucleotides 975-1071), also obtained by AvaII digestion of pDirectRo1. Twenty-four of these PCR products hybridized at 42 °C to probe B and were washed at 68 °C in 0.5% SDS in 0.1 times SCC. The longest, 1.8 kilobases, was cloned into p-Direct to provide p-DirectRo2. The insert in p-DirectRo2 was sequenced in both directions by dideoxy chain-termination with Taq DNA polymerase (Fig. 1).


Figure 1: Diagram of the sequencing strategy for rat liver RoDH cDNA. A 1.8-kilobase cDNA clone included the complete coding region of the 34-kDa RoDH. The middle line indicates the 1800 base pairs of the clone. The unlabeled lines show the areas sequenced. Those above the middle line were sequenced from left to right; the lines below the middle line were sequenced from right to left.



Expression of RoDH

The coding region of RoDH in p-DirectRo2 was amplified with the sense primer 5`-TGAGTCACGGCTGGGAGC-3`, nucleotides 179-196 and the antisense primer 5`ATGAGTATGGTGAACAATGG-3`, nucleotides 1328-1309. To produce pcDNA3/RoDH, the PCR product was ligated into pcDNA3 (Invitrogen), linearized with EcoRV. pcDNA3/RoDH was transfected by calcium phosphate/DNA precipitation into semi-confluent P19 cells, maintained in minimum essential medium with 10% fetal calf serum. Mock transfections were done with pcDNA3 without insert. Twenty-four h after transfection each group was harvested and used to prepare a 10,000 times g supernatant by differential centrifugation(10, 11) .

Rat Tissues and Microsomes

Male rats (250 g) fed a Chow diet were sacrificed by cervical dislocation. Kidneys and testes were decapsulated. Tissues were either used immediately for preparation of RNA or were homogenized to prepare microsomes as described elsewhere (10, 11) .

Northern Blot Analysis

RNA was prepared by guanidinium thiocyanate-phenol-chloroform extraction(19) . Five µg of poly(A) mRNA, isolated with the poly(A)Tract mRNA isolation system (Promega), were fractionated on a 1.2% agarose gel, transferred to nylon membranes, and cross-linked by UV irradiation. The probe for RoDH was the 565-nucleotide NcoI/AvaI product from the 3`-untranslated region of p-DirectRo2 (see Fig. 2). The blot was reprobed with glyceraldehyde dehydrogenase cDNA, synthesized by PCR with rat liver cDNA and the 35-60 and 994-1017-base pair fragments as the 5` and 3` primers, respectively. Probes were labeled by random priming to specific activities of 10^9 cpm/µg with [alpha-P]dCTP(18) . Hybridization was done at 65 °C for 16 h in 7% SDS, 0.25 M sodium phosphate, 1 mM EDTA, and 1 mg/ml bovine serum albumin, pH 7. Membranes were washed 2times in 5% SDS and 0.5 mg/ml bovine serum albumin in SE (40 mM sodium phosphate, 1 mM EDTA, pH 6.8) at 65 °C for 15 min, and 4times for 15 min each in 1% SDS, SE, 65 °C. Autoradiography was with Kodak XAR films and two intensifying screens at -70 °C.


Figure 2: Nucleotide and deduced amino acid sequence of RoDH. Symbols identify the 25 amino acids conserved in at least 17 of the 24 SCAD; the 6 identical in all 24 SCAD are in bold face(13, 14, 15, 16, 23) . The amino acid sequences that had been determined by microsequencing are underlined. Restriction enzyme cut sites are indicated in boldface.



Immunoblots

Microsomal protein (7-10 µg) from 10% SDS-PAGE was transferred to poly(vinylidene fluoride) (Bio-Rad) for immunoblotting with a rabbit anti-34-kDa polypeptide or preimmune serum, 1/500 dilution each.^2 The signal was developed with the Bio-Rad alkaline phosphatase kit.

RoDH Assay

Assays for retinal biosynthesis were done with retinol bound to excess CRBP for 30 min at 37 °C in 0.5 ml of 10 mM HEPES, 150 mM KCl, and 2 mM EDTA, 2 mM NADP, pH 8, with 2 mM egg yolk L-alpha-phosphatidylcholine (added in 2 µl of ethanol). Reactions were quenched and the retinal generated was quantified by high performance liquid chromatography as previously described(20, 21) .

Preparation of CRBP

CRBP saturated with all-trans-retinol was prepared and purified from CRBP generated in Escherichia coli with the vector pMONCRBP(22) , as described elsewhere(10) . ApoCRBP was prepared and purified as was holoCRBP, except for saturation with retinol. The concentration of functional apoCRBP was determined by saturating an aliquot with retinol, separating free and bound retinol by size-exclusion chromatography and determining the A/A ratio. The ratio A/A ratio of holoCRBP was not affected in the presence of the phosphatidylcholine concentration used in the RoDH assays, indicating that the integrity of the protein was not affected.


RESULTS AND DISCUSSION

cDNA and Amino Acid Sequence

The active site of rat liver RoDH was identified in a 34-kDa polypeptide by covalent binding and inactivation with PAO, and by chemical cross-linking with holoCRBP.^2 PCR amplification of rat liver cDNA, with primers based on two internal amino acid sequences of this polypeptide, yielded a 546-base pair product that encoded four internal amino acid sequences determined by microsequencing (nucleotides 613 through 1158 of the final product, Fig. 2). Two probes, obtained by AvaII digestion of this PCR product, were then used to screen a rat liver gt11 cDNA library through three rounds, followed by Southern blot of the clones. The longest of the clones identified by these procedures was subcloned to give p-DirectRo2 and was sequenced in both directions (Fig. 1).

A single open reading frame in p-DirextRo2 predicts a polypeptide with a calculated molecular mass of 34.9 kDa, comparing well to the value estimated by SDS-polyacrylamide gel electrophoresis during the isolation of the 34-kDa RoDH. A total of 54 of the 317 amino acids were verified by microsequencing. In addition to the four internal oligopeptides sequenced, the polypeptide also had the expected N-terminal amino acid sequence (Fig. 2).

The predicted RoDH is in the 25-35-kDa molecular mass range of SCAD and has amino acid residues distinctive of SCAD(13, 14, 15, 16, 17, 23) . Six amino acid residues are identical in the 24 published SCAD sequences, and all are conserved in RoDH. Nineteen other residues are identical in at least 17 of the 24 SCAD; 16 of these are conserved in RoDH, including the aforementioned sequence WXLVNNAG (at Trp), the putative SCAD cofactor binding site G(X)(3)GXG (at Gly), and the putative SCAD active site Y(X)(3)K (at Tyr). As for other SCAD, and in contrast to the medium-chain (classical) alcohol dehydrogenases, the cofactor binding site lies N-terminal to the active site. One of the three nonidentical residues is a conservative substitution, V159I; the others are not, R104G and W107D.

The closest amino acid similarity among RoDH and other SCAD is between the hydroxybutyrate dehydrogenases and 17beta-hydroxysteroid dehydrogenase, type II (15, 16, 25) (Table 1). There is less similarity among RoDH, 11beta-hydroxysteroid dehydrogenases, 17beta-hydroxysteroid, type I, and 15-hydroxyprostaglandin dehydrogenase (17, 24, 26, 27) .



SCAD generally have few cysteine residues: 14 of 24 SCAD have 2 or fewer. Several mammalian SCAD, however, contain 4: human (R)-3-hydroxybutyrate dehydrogenase(16) ; rat 11beta-hydroxysteroid dehydrogenase(24) ; human 17beta-hydroxysteroid dehydrogenase, type I(17) ; human 15-hydroxyprostaglandin dehydrogenase(26) . RoDH has 6 cysteine residues, more than others except the rat liver D-beta-hydroxybutyrate dehydrogenase, which also has 6(15) .

Secondary Structure Predictions

The first 18 N-terminal amino acid residues of RoDH have an average hydropathy of 1.6, calculated with Kyte and Doolittle (28) values. This span, consisting mostly of helix, determined according to Garnier et al.(29) , is sufficiently long and hydrophobic for membrane-anchoring (Fig. 3). A short stretch of the most hydrophilic amino acids, RERK, flanks this hydrophobic span, a typical feature at membrane junctures, presumably to aid in precise positioning in the membrane. One other area, the 22 amino acids from residues 131 through 152, also has an average hydropathy of 1.6, but consists of helix-sheet-helix. This may be a hydrophobic CRBP-interaction site: it lies close to the active site, centered about residues 176-180. The active site itself lies in a hydrophobic pocket, bounded by two very hydrophilic sections. There are no other areas sufficiently hydrophobic to be unequivocally associated with spanning a lipid bilayer. The scarcity of transmembrane helical domains in the membrane-associated 34-kDa RoDH is similar to the secondary structure of human heart (R)-3-hydroxybutyrate dehydrogenase, an inner mitochondrial membrane SCAD which lacks any transmembrane helices(16) .


Figure 3: Hydropathy plot and predicted secondary structure of RoDH. Top panel, the hydropathy plot was calculated as the average of the Kyte and Doolittle (28) values with a 7-residue window. Hydrophobic areas are indicated by positive values. Bottom panel, secondary structure predictions were made according to Garnier et al.(29) .



Transient Transfection in P19 Cells

RoDH was expressed transiently in P19 cells to determine its biochemical characteristics. The 10,000 times g supernatant of homogenates prepared from mock-transfected cells did not convert CRBP-bound retinol into retinal in four separate experiments. In contrast, the 10,000 times g supernatant of cells transfected with the expression vector pcDNA3/RoDH produced retinal from holoCRBP (Fig. 4). The characteristics of the RoDH expressed were consistent with those of the rat liver microsomal enzyme, using microsomes or semipurified RoDH(10, 11) .^2 Recombinant RoDH was not inhibited by the alcohol dehydrogenase inhibitor 4-methylpyrazole, had 3-fold higher activity with NADP compared to NAD, was stimulated by ethanol and phosphatidylcholine, and was inhibited by PAO and carbenoxolone. PAO has been considered an active-site inhibitor that forms covalent heterocyclic adducts between spatially proximal sulfhydryl groups (30, 31, 32) . One of the 6 cysteine residues of RoDH, C37, occurs in the putative cofactor binding site and another, C177, occurs in the putative active site. Binding of PAO to either one or both of these and another cysteine residue nearby in the secondary or tertiary structure may realize the requirement for inhibition. Carbenoxolone, the steroidal aglycone of licorice-derived glycyrrhizin, inhibits other SCAD, such as 11beta-hydroxysteroid dehydrogenase(33, 34) . Finally, centrifugation (100,000 times g for 1 h) of the transfected P19 cell 10,000 times g supernatant localized retinal synthesis, supported by 5 µM holoCRBP/2 µM apoCRBP, to the microsomal fraction (33 and 35 pmol of retinal/100 µg of protein), with no detectable activity in the cytosol (duplicates/180 µg of protein each).


Figure 4: Characteristics of RoDH transiently expressed in P19 cells. RoDH activity was assayed with 5 µM holoCRBP/2 µM apoCRBP in the 10,000 times g supernatant from P19 cells transfected on two separate occasions. A, 200 µg of protein; B, 125 µg of protein). 1, pcDNA3 (mock transfected); 2-8, pcDNA3/RoDH. Assays were done with the complete assay medium, 1 and 2 or with alterations: 3, + 500 µM carbenoxolone; 4, +1 mM ethanol; 5, -NADP, + 2 mM NAD; 6, -phosphatidylcholine; 7, +1 mM PAO; 8, +500 mM 4-methylpyrazole. B2 is the mean ± S.D. of four replicates. The others are averages of duplicates, each within 2 pmol of its mean.



A relatively high affinity interaction between holoCRBP and the RoDH expressed in the P19 cell 10,000 times g supernatant was indicated by an average K(m) value of 0.9 µM (mean of two separate transfections, 0.5 ± 0.1, 1.2 ± 0.5, ± S.E., determined by fitting data with the non-linear regression program Enzfitter(35) ) (Fig. 5). This K(m) value compares well with the previously determined values for holoCRBP of 1.6 µM for rat liver microsomes and 0.6 µM for partially purified RoDH(10, 11) .^2 The >5 µM of holoCRBP in normal rat liver exceeds the K(m) value for the expressed recombinant RoDH considerably, consistent with a physiological role for RoDH in RA biogenesis(7, 8, 9) .


Figure 5: Affinity of transiently expressed RoDH for holoCRBP. RoDH activity was measured from holoCRBP composed of total CRBP/retinol in the ratio 1.4/1 with 180 µg of protein from the 10,000 times g supernatant of P19 cells transfected with pcDNA3-RoDH. The Michaelis-Menten data were fit with the nonlinear regression program Enzfitter(35) . The curve shown is one of two experiments, each done with a separate transfection. In both experiments, cells mock-transfected at the same time had no RoDH activity in their 10,000 times g supernatants.



Tissue Distribution of RoDH mRNA

Many tissues and cell types convert retinol into RA (7, 9, 10, 36, 37) and CRBP has widespread tissue and cellular distribution(38, 39, 40, 41) . Northern blot analysis of liver RoDH expression in rat tissues revealed a 1.8-kilobase mRNA in liver. Unexpectedly, no mRNA was apparent in brain, kidney, lung or testis (Fig. 6). Immunoblot analysis detected this RoDH protein only in liver. Assayed with holoCRBP, microsomes from rat brain, kidney, lung and testis had RoDH activity 20-30% of that in rat liver microsomes, verifying that the holoCRBP-supported path of RA biosynthesis occurs in each, as noted previously(10) . These data indicate extra-hepatic tissues must contain isozyme(s) of hepatic RoDH which recognize holoCRBP as substrate, i.e. tissue-specific RoDH isozyme(s) occur.


Figure 6: Distribution of RoDH in rat tissues. A, RoDH mRNA. B, rat liver glyceraldehyde dehydrogenase mRNA. C, RoDH activity of microsomes from rat tissues determined with 5 µM holoCRBP/2 µM apoCRBP. Data are the means ± S.D. of six to eight replicates. Controls of no cofactor or no microsomes produced no retinal. D, immunoanalyses of rat tissue microsomal protein with anti-34-kDa polypeptide or with preimmune serum: MW, molecular weight marker; PI, preimmune serum; the lanes between MW and PI are identified at the top of the figure.



Concluding Summary

This work identifies the liver microsomal NADP-dependent RoDH, an enzyme that catalyzes the first and rate-limiting step in RA biosynthesis, as a heretofore unknown SCAD and indicates that tissue-specific isozyme(s) may be important to RA biogenesis. Retinol activation by members of a larger family of lipid metabolizing enzymes expands and complements insight developing into retinoid biology. The six ligand-activated receptors that mediate RA action and the four cellular retinol/RA binding-proteins that function in retinoid metabolism belong to superfamilies of sterol/lipid-hormone receptors (42, 43, 44) and sterol/lipid-binding proteins(45, 46) , respectively, which are expressed tissue specifically. It is also notable that the maize sex differentiation gene Tasselseed2 encodes an SCAD(23) . Perhaps SCAD enjoy widespread involvement in activating hormones.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK36870. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: School of Medicine and Biomedical Sciences, 140 Farber Hall, SUNY-Buffalo, Buffalo, NY 14214. Tel.: 716-829-2726; Fax: 716-829-2725.

(^1)
The abbreviations used are: RA, all-trans-retinoic acid; CRBP, cellular retinol-binding protein, type I; PAO, phenylarsine oxide; PCR, polymerase chain reaction; SCAD, short-chain alcohol dehydrogenase.

(^2)
M. H. E. M. Boerman and J. L. Napoli, unpublished results.


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