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(Received for publication, June 26, 1995; and in revised form, August 16, 1995) From the
A retinol dehydrogenase, RoDH(I), which recognizes holo-cellular
retinol-binding protein (CRBP) as substrate, has been cloned,
expressed, and identified as a short-chain dehydrogenase/reductase
(Chai, X., Boerman, M. H. E. M., Zhai, Y., and Napoli, J. L.(1995) J. Biol. Chem. 270, 3900-3904). This work reports the
cloning and expression of a cDNA encoding a RoDH isozyme, RoDH(II). The
predicted amino acid sequence verifies RoDH(II) as a short-chain
dehydrogenase/reductase, 82% identical with RoDH(I). RoDH(II)
recognized the physiological form of retinol as substrate, CRBP, with a K
Metabolic activation of retinol provides the hormone
all-trans-retinoic acid (RA). ( A microsomal RoDH
has been partially purified, its active site has been identified with a
34-kDa polypeptide by chemical cross-linking with holo-CRBP, and its
cDNA has been cloned and
expressed(16, 17, 18) . This RoDH, hereafter
termed RoDH(I), belongs to the family of short-chain
dehydrogenase/reductase(19) . By Northern blot analysis, mRNA
expression of RoDH(I) was detected only in rat liver, despite the well
established occurrence of CRBP-recognizing RoDH activity in multiple
tissues. These results suggested occurrence of multiple RoDHs. This
work reports the cDNA cloning and expression of a second RoDH,
RoDH(II), shows that it is a previously unknown short-chain
dehydrogenase/reductase that can catalyze the first step in RA
synthesis with CRBP as substrate, and compares the expression of
RoDH(II) mRNA in rat tissues with that of RoDH(I) by RNase protection
assays.
A single
open reading frame in PBSK/RoDH(II) predicts an RoDH(II) of 317 amino
acids with a calculated molecular mass of
Figure 1:
Nucleotide and deduced amino acid
sequences of RoDH(II). The first line of amino acid sequence
immediately below the nucleotide sequence shows the predicted
amino acid sequence of RoDH(II). The underlined residues in
this line identify the 21 amino acids in RoDH(II) conserved in >70%
of the 57 members of the extended short-chain dehydrogenase/reductase
superfamily(19) . The second line of amino acid
sequence represents RoDH(I). The blanks indicate residues
identical to RoDH(II). The underlined residues in this second
line are those that have been determined by microsequencing of
RoDH(I)(18) .
Figure 2:
Hydropathy plot and predicted secondary
structures of RoDH(II). Top, the hydropathy plot was
calculated as the average of the Kyte and Doolittle (27) values
with a seven-residue window. Hydrophobic areas are indicated by
positive values. Bottom, secondary structure predictions were
made according to Garnier et
al.(28) .
Figure 3:
Characteristics of RoDH(II) transiently
expressed in P19 cells. RoDH activity was assayed with 5 mM holo-CRBP/2 mM apo-CRBP in the 10Ksup from P19 cells
transfected on two separate occasions (A and B). Bars 1-4, pcDNA3 (mock transfected); bars
5-12, transfected with pcDNA3/RoDH(II). Assays were done
with 150 mg of protein, and the complete assay medium was done without
phosphatidyl choline (bars 1, 2, 5, and 6) and with phosphatidyl choline (bars 3, 4,
and 7-12). Bars 1, 3, 5, 7 and 8-12 represent assays done with 2 mM NADP. Bars 2, 4 and 6 were done with 2 mM NAD. Other additions were: bar 9, 1 mM ethanol; bar 10, 500 mM carbenoxolone; bar 11, 1
mM PAO; bar 12, 500 mM 4-methylpyrazole. The solid bars at the base line indicate that no retinal was
detected; blanks indicate that data were not
obtained.
The
average K
Figure 4:
RoDH(II) activity versus holo-CRBP concentration. RoDH activity was measured from holo-CRBP
composed of total CRBP/retinol in the ratio 1.4 with 175 mg of protein
from the 10Ksup of P19 cells transfected with pcDNA3/RoDH(II). The
Michaelis-Menten data were fit with Enzfitter(35) . The curve shown is from one of two experiments, each done with a
separate transfection.
Figure 5:
Distribution of RoDH(I) and RoDH(II) in
adult rat tissues. RNase protection assays are shown. Top,
RoDH(II); bottom, RoDH(I). RNA was isolated from: lanes
3, brain; lanes 4, kidney; lanes 5, liver; lanes 6, lung; lanes 7, testis. Lanes 1 and 2 show digested and undigested yeast RNA, respectively. The
expected protected fragments of 341 nucleotides are
indicated.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) RODHII and
U33500[GenBank].
Volume 270,
Number 47,
Issue of November 24, 1995 pp. 28408-28412
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EXPRESSION OF ITS mRNA RELATIVE TO TYPE I (*)
of 2 mM. Similar to microsomal
RoDH and RoDH(I), RoDH(II) had higher activity with NADP rather than
NAD, was stimulated by ethanol and phosphatidyl choline, was not
inhibited by the medium-chain alcohol dehydrogenase inhibitor
4-methylpyrazole, but was inhibited by phenylarsine oxide and the
short-chain dehydrogenase/reductase inhibitor carbenoxolone. Northern
blot analysis detected RoDH(I) and RoDH(II) mRNA only in rat liver, but
RNase protection assays revealed RoDH(I) and RoDH(II) mRNA in kidney,
lung, testis, and brain. These data indicate that short-chain
dehydrogenases/reductase isozymes expressed tissue-distinctively
catalyze the first step of retinoic acid biogenesis from the
physiologically most abundant substrate, CRBP.
)RA produces a
variety of biological responses by modulating the expression of genes
that regulate the state of differentiation or entry into apoptosis of
diverse cell types in numerous
organs(1, 2, 3, 4, 5) . A
model of RA biosynthesis postulates that the enzyme(s) that catalyze RA
synthesis physiologically recognize as substrate the predominant form
of retinol in vivo, viz. retinol bound within
CRBP(6) . The CRBP concentration exceeds that of retinol
(7 versus
5 mM, respectively(7) ),
and CRBP has a high affinity interaction with retinol (K
= 0.1-1
nM), much higher than substrate-enzyme
interactions(8, 9) . Binding of retinol within CRBP
shields the prohormone from the cellular environment and would confer
specificity on RA biosynthesis by restricting access of retinol to
enzymes capable of recognizing and interacting with the CRBP-retinol
``cassette.'' This would prevent opportunistic oxidation by
dehydrogenases/oxidases with inexact substrate tolerances, thereby
contributing to precise spatial-temporal control over RA biogenesis.
CRBP binding would also protect retinol from nonenzymatic oxidation and
cells from the membrane-altering faculty of unbound
retinol(10, 11, 12) . A pathway of RA
biosynthesis consistent with this hypothesis involves as the first and
rate-limiting step production of retinal in microsomes with holo-CRBP
as substrate, i.e. RoDH(13, 14) . Retinal
generated in microsomes from holo-CRBP by RoDH supports RA biosynthesis
by cytosolic retinal dehydrogenases(15) .
Library Screening
A rat liver gt11
cDNA library (Clontech) was screened through three rounds with probe A
(nucleotides 653-975 of RoDH(I)), as described(18) . DNA
from one of the three plaques obtained was cloned into P-Direct to
provide p-DirectRo3, which contained a 1.5-kilobase cDNA insert
distinct from RoDH(I) but with no initiation codon. Probe B
(nucleotides 587-909 of the final product) was prepared from
p-DirectRo3 by AvaII digestion and used to identify 36 plaques
by screening the same library through three rounds. The inserts of
these plaques were amplified by PCR and analyzed by Southern blot with
a synthetic 32-base pair oligonucleotide (probe C, nucleotides 974-1005
of the final cDNA). Six PCR products hybridized at 42 °C to probe C
and were washed at 68 °C in 0.5% SDS in 0.1
SCC (SSC
= 0.15 M NaCl and 15 mM sodium citrate). The
longest was cloned into pBluescript to provide PBSK/RoDH(II). The
insert in PBSK/RoDH(II) was sequenced by dideoxy chain termination with Taq DNA polymerase.Expression of RoDH
The coding region of
RoDH(II) of PBSK/RoDH(II) was amplified with the sense primer
5`-CGCGGATCCCCTCTGCTTGTCTTCTAC-3` (nucleotides 206-223) and the
antisense primer 5`-CGGAATTCCTCCCTAACACTGTACCA-3` (nucleotides 1233 to
1216) containing BamHI and EcoRI cleavage sites,
respectively (underlined). The PCR product was ligated into pcDNA3
(Invitrogen) to yield pcDNA3/RoDH(II). pcDNA3/RoDH(II) was transfected
by calcium phosphate/DNA precipitation into semi-confluent P19 cells as
described(18) . Mock transfections were done with pcDNA3. 24 h
after transfection cells were harvested, the pellets were suspended in
10 mM Hepes, 10% sucrose, pH 7.5, on ice for 15 min and
homogenized with a Dounce homogenizer, and a 10Ksup was
prepared(13, 16) .Northern Blot Analysis
Northern blot
analysis was done as described (18) with 5 mg of
poly(A) mRNA hybridized at 65 °C for 16 h to a
RoDH(II) probe of the 841 nucleotide FokI/SphI
product (nucleotides 1148-1988) from the 3`-untranslated region
of PBSK/RoDH(II). The blot was reprobed with a glyceraldehyde
dehydrogenase cDNA probe.
RNase Protection Assays
The
RoDH(I)-specific probe was obtained by amplifying a 341-base pair
fragment from p-DirectRo2 (18) by PCR with the sense primer
5`-CGCGGATCCCTTCTCAGACTCCCTCA-3` (nucleotides 849-865) and the
antisense primer 5`-CGGAATTCGTGGGAAGGTAGCTCATG-3` (nucleotides
1190-1173) containing BamHI and EcoRI cleavage
sites, respectively (underlined). The RoDH(II)-specific probe was
obtained by amplifying a 341-base pair fragment from PBSK/RoDH(II) by
PCR with the same sense primer used for RoDH(I) and the antisense
primer 5`-CGGAATTCTCCCGTAGGTGTTCTTTCCA-3` (nucleotides 1140-1123)
containing an EcoRI cleavage site (underlined). The fragments
were subcloned into pcDNA3 in the antisense orientations, and the
plasmids were linearized with BamHI. P-labeled
antisense riboprobes were transcribed with SP6 RNA polymerase (Ambion)
for 1 h at 37 °C in 10 mM dithiothreitol, 0.5 mM
each ATP, CTP, and GTP, 50 mM UTP, and 50 mCi
[
-P]UTP. DNA templates were removed with
DNaseI. The 450-base pair transcripts, which included 109 base pairs of
plasmid, were purified with a 5% polyacrylamide/8 M urea gel.
A 250-base pair KpnI and XbaI fragment of mouse
-actin was used to generate an antisense probe for -actin
mRNA. RNase protection assays were done with a ribonuclease protection
kit (Ambion). Probes (1.2 10
cpm) were
co-precipitated by 0.5 M ammonium acetate with total RNA,
extracted with guanidinium thiocyanate/phenol/chloroform from the
tissues of adult male Sprague-Dawley rats (30 µg for
extra-hepatic tissues and for
-actin in all tissues and 15 mg
for liver with RoDH probes). The precipitates were resuspended in 20 ml
of 80% deionized formamide, 100 mM sodium citrate, 300
mM sodium acetate, and 1 mM EDTA, pH 6.4, and were
incubated at 45 °C for 20 h. The same amounts of probes were
hybridized with 10 mg of yeast RNA as control. After digestion with
RNase A (2.5 units/ml) and RNase T1 (100 units/ml) for 30 min at 37
°C, the protected fragments were resolved on 5% polyacrylamide/8 M urea gels and visualized at -80 °C overnight with
one intensifying screen. Quantification was done with a Bio-Rad Model
GS-670 densitometer.
RoDH Assay
Assays for retinol metabolism
were done in duplicate with retinol bound to excess CRBP for 30 min at
37 °C in 0.5 ml of 10 mM Hepes, 150 mM KCl, 2
mM EDTA, and 2 mM NADP, pH 8, with 2 mM egg
yolk L--phosphatidyl choline (added in 2 µl of
ethanol). Retinal was quantified by high performance liquid
chromatography(18, 20, 21) . Holo- and
apo-CRBP were generated in Escherichia coli from pMONCRBP (22) and were purified (15) .
cDNA and Amino Acid Sequence
During
screening of a cDNA library with a probe from RoDH(I), a 1.5-kilobase
partial cDNA was identified with sequence distinct from RoDH(I). A
probe from this cDNA was used to re-screen the library. The longest
clone identified (2 kilobase) was subcloned to create
PBSK/RoDH(II) and was sequenced in both directions to reveal a cDNA
with a nucleotide sequence distinct from that of RoDH(I).
35 kDa; the same as
RoDH(I) (Fig. 1). Of the 23 amino acid residues conserved in
>70% of the extended short-chain dehydrogenase/reductase family, 21
have been conserved in RoDH(II)(19) . These include the
residues typical of an short-chain dehydrogenase/reductase: the
G(X)
GXG cofactor binding site
(Gly), N-terminal to the active site; the sequence
LXNNAG (Leu
); and the active site
Y(X)
K (Tyr), C-terminal to the
cofactor binding site. One of the two substitutions, D107W, represents
a common substitution in short-chain dehydrogenase/reductase; the
second, A191R, also occurs in the short-chain dehydrogenase/reductases
11
-hydroxysteroid and D--hydroxybutyrate
dehydrogenases of rat(23, 24) . RoDH(II) differs from
RoDH(I) in 57 amino acid residues; most are nonconservative
substitutions. Seven of these had been verified in RoDH(I) by
microsequencing(18) . Yet the predicted primary sequence of
RoDH(II) diverges less from RoDH(I) compared with a bovine
11-cis-retinol dehydrogenase, a short-chain dehydrogenase/reductase
expressed only in retinal pigment epithelium (25) , or to other
rat short-chain dehydrogenase/reductases (Table 1). A second
Y(X)K pattern 15 residues closer to the C terminus
from the probable active site Y(X)K, presents a
curious feature of both RoDH(I) and RoDH(II). The first
Y(X)K most likely serves as the active site
because of its position in the primary sequence and because it
resembles the Y(C/G/S)(A/I/S)(T/S)K sequence of other short-chain
dehydrogenase/reductase active sites(26) .
Secondary Structure Predictions
RoDH(II)
and RoDH(I) have identical N-terminal sequence through residue 103. The
first 18 amino acids have an average hydropathy of
1.6(27) , consist mostly of helix(28) , and are
juxtaposed to four of the most hydrophilic amino acids, RERK (Fig. 2). This probably serves a membrane-anchoring function,
with the positioning of hydrophobic amino acids next to hydrophilic
amino acids fastening RoDH in the membrane. Two other hydrophobic
sections of RoDH(II) seem long enough to span membranes: the 22
residues 131 through 152 and the 18 residues 160 through 177. The
former has an average hydropathy of
1.2, and the latter, which
includes the putative active site, has an average hydropathy of
0.8. Presumably, because of insufficient hydrophobicity, neither
spans a membrane. Thus, RoDH(II) seems to lack transmembrane helices
similar to RoDH(I), a microsomal enzyme, and human heart (R)-3-hydroxybutyrate dehydrogenase, an inner mitochondrial
membrane short-chain dehydrogenase/reductase(29) . These
secondary structure predictions suggest anchoring of both RoDHs to the
endoplasmic reticulum through N-terminal sequence, with the bulk of the
polypeptide extending into the cytoplasm, accessible to CRBP.
Transient Transfection in P19
Cells
RoDH(II) was expressed transiently in P19 cells to
determine its catalytic characteristics. In two different
transfections, mock transfected cells had no RoDH activity in the
absence of phosphatidyl choline and low activity in its presence (Fig. 3, bars 1-4). In contrast, the 10Ksup of
cells transfected with the expression vector pcDNA3/RoDH(II) produced
retinal from holo-CRBP in the absence of phosphatidyl choline (compare lane 5 with lane 1) and much more retinal in its
presence than in mock transfected cells (compare lane 7 with lane 4). The other characteristics of the expressed enzyme
also paralleled those of RoDH(I) and the rat liver microsomal RoDH
activity, using microsomes or semi-purified
RoDH(13, 16) . High concentrations of the alcohol
dehydrogenase inhibitor 4-methylpyrazole had little effect on RoDH(II)
(compare lane 7 with lane 12). RoDH(II) had higher
activity with NADP than NAD (compare lane 7 with lane 8 and lane 5 with lane 6). Ethanol enhanced
RoDH(II) activity (compare lanes 7 and 9).
Carbenoxolone and PAO inhibited RoDH(II) (compare lane 7 with lanes 10 and 11, respectively). PAO inhibits by
forming covalent heterocyclic adducts between the reagent and spatially
proximal sulfhydryl groups(30, 31, 32) . The
six cysteine residues of RoDH(II) and RoDH(I) occur in the same
positions, with Cys in the putative cofactor binding site
and Cys
in the putative active site. Binding of PAO to
either one or both of these and another cysteine residue close in the
secondary or tertiary structure could inhibit RoDH. The steroidal
aglycone of glycyrrhizin, carbenoxolone, inhibits short-chain
dehydrogenase/reductase besides RoDH(II) and RoDH(I), including
11
-hydroxysteroid dehydrogenase(33, 34) .
value of RoDH expressed in the P19 cell
10Ksup for holo-CRBP was 2 mM. This value was obtained from
two transfections: 1.6 ± 0.2 and 2.4 ± 0.4, each
determined by fitting data with the nonlinear regression program
Enzfitter (35) (Fig. 4). This K compares well with the previously determined values for holo-CRBP
of 1.6 mM for rat liver microsomal RoDH, 0.6 mM for
partially purified RoDH(I), and 0.9 mM for recombinant
RoDH(I)(13, 16, 18) .
Tissue Distribution of RoDH mRNA
Northern
blot analyses of RoDH(II) revealed a 1.8-kilobase mRNA in liver but did
not detect mRNA in brain, kidney, lung, or testis (data not shown),
just as had occurred with RoDH(I)(18) . RNase protection assays
of RoDH(I) and RoDH(II), however, showed wider tissue distribution of
both mRNAs (Fig. 5; Table 2). Liver was the major site of
expression of both RoDHs. Expression of RoDH(I) in the extra-hepatic
tissues screened was <2% of liver. RoDH(II), in contrast, had
relatively abundant expression in kidney and more abundant expression
in brain and lung than RoDH(I). Testis had equivalent expression of
RoDH(II) and RoDH(I). RNase protection assays under high stringency
conditions revealed many protected fragments that could not be
rationalized from the nucleotide sequences of RoDH(I) and RoDH(II) and
the sequences of the probes, consistent with occurrence of closely
related mRNAs, possibly from additional isozymes of RoDH. This
expression of RoDH mRNA among multiple tissues reflects the ability of
multiple tissues to biosynthesize RA (12, 13, 36, 37) and the widespread
tissue distribution of CRBP(38, 39, 40) .
Concluding Summary
This work identifies a
second RoDH as a another isozyme that catalyzes the first step in RA
biosynthesis. Occurrence of this heretofore unknown short-chain
dehydrogenase/reductase indicates the importance of tissue-distinctive
expression of RoDHs to RA biogenesis. The >5 mM
concentration of holo-CRBP in normal rat liver exceeds the K value for RoDH(I) and (II), consistent with
physiological roles for them in RA biogenesis(7) . Their
ability to recognize CRBP provides a mechanism for accessing the major
pool of retinol in vivo while allowing CRBP to control the
availability and distribution of retinol. Two other classes of proteins
important to retinoid function are expressed in definite
temporal-spatial patterns, the receptors RAR and RXR and the binding
proteins CRBP and cellular retinoic acid-binding protein. These
proteins also belong to superfamilies of sterol/lipid hormone receptors (41, 42, 43) and sterol/lipid-binding
proteins(44, 45) , respectively. Thus, RoDHs represent
a third class of proteins potentially important to retinoid action that
belong to a larger superfamily of steroid/lipid-specific proteins.
) g supernatant of a cell homogenate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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X.-F. Huang and V. Luu-The Molecular Characterization of a First Human 3(alpha right-arrowbeta )-Hydroxysteroid Epimerase J. Biol. Chem., September 15, 2000; 275(38): 29452 - 29457. [Abstract] [Full Text] [PDF]
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K. Tryggvason, A. Romert, and U. Eriksson Biosynthesis of 9-cis-Retinoic Acid in Vivo. THE ROLES OF DIFFERENT RETINOL DEHYDROGENASES AND A STRUCTURE-ACTIVITY ANALYSIS OF MICROSOMAL RETINOL DEHYDROGENASES J. Biol. Chem., May 25, 2001; 276(22): 19253 - 19258. [Abstract] [Full Text] [PDF]
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C. M. Soref, Y.-P. Di, L. Hayden, Y. H. Zhao, M. A. Satre, and R. Wu Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol J. Biol. Chem., June 22, 2001; 276(26): 24194 - 24202. [Abstract] [Full Text] [PDF]
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