(Received for publication, October 28, 1994; and in revised form, December 9, 1994)
From the
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.
Retinol (vitamin A) undergoes metabolic activation by
dehydrogenation into retinal, followed by oxidation into the hormone
all-trans-retinoic acid (RA). 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. 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.
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.
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.
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)
GXG (at Gly
), and the
putative SCAD active site Y(X)
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
17-hydroxysteroid dehydrogenase, type II (15, 16, 25) (Table 1). There is less
similarity among RoDH, 11
-hydroxysteroid dehydrogenases,
17
-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
11-hydroxysteroid dehydrogenase(24) ; human
17
-hydroxysteroid dehydrogenase, type I(17) ; human
15-hydroxyprostaglandin dehydrogenase(26) . RoDH has 6 cysteine
residues, more than others except the rat liver D-
-hydroxybutyrate dehydrogenase, which also has
6(15) .
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) .
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 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 g supernatant was indicated by an average K
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
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) .
The >5 µM of holoCRBP in normal rat liver exceeds the K
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 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
g supernatants.
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.