From the Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden
Received for publication, January 10, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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Retinoic acid is generated by a two-step
mechanism. First, retinol is converted into retinal by a retinol
dehydrogenase, and, subsequently, retinoic acid is formed by a retinal
dehydrogenase. In vitro, several enzymes are suggested to
act in this metabolic pathway. However, little is known regarding their
capacity to contribute to retinoic acid biosynthesis in
vivo. We have developed a versatile cell reporter system to
analyze the role of several of these enzymes in
9-cis-retinoic acid biosynthesis in vivo. Using
a Gal4-retinoid X receptor fusion protein-based luciferase reporter
assay, the formation of 9-cis-retinoic acid from
9-cis-retinol was measured in cells transfected with
expression plasmids encoding different combinations of retinol and
retinal dehydrogenases. The results suggested that efficient formation
of 9-cis-retinoic acid required co-expression of retinol
and retinal dehydrogenases. Interestingly, the cytosolic alcohol
dehydrogenase 4 failed to efficiently catalyze
9-cis-retinol oxidation. A structure-activity analysis
showed that mutants of two retinol dehydrogenases, devoid of the
carboxyl-terminal cytoplasmic tails, displayed greatly reduced
enzymatic activities in vivo, but were active in
vitro. The cytoplasmic tails mediate efficient endoplasmic
reticulum localization of the enzymes, suggesting that the unique
milieu in the endoplasmic reticulum compartment is necessary for
in vivo activity of microsomal retinol dehydrogenases.
Retinol (vitamin A) and its derivatives are essential dietary
compounds needed in a variety of physiological processes,
e.g. embryonic development, reproduction, cell
differentiation, postnatal growth, maintenance of the immune system,
and vision (1-3). The metabolically active retinoids are
9-cis-retinoic acid
(9cRA)1 and
all-trans-retinoic acid (atRA) in extraocular tissues,
acting as ligands for two classes of nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (4).
In ocular tissues, 11-cis-retinal serves as the chromophore of the visual pigments (2).
Most cells obtain all-trans-retinol as the main source of
retinoids, and it is known that activation of retinol into the active metabolites occur via tissue-specific isomerization and oxidation reactions. First, 9-cis-, 11-cis-, or
all-trans-retinols are oxidized by retinol dehydrogenases
(RDHs) into the corresponding retinals. For generation of the two
isomers of RA, 9-cis- and all-trans-retinal are
then further oxidized by a class of retinal dehydrogenases (Raldhs)
(for reviews, see Refs. 5 and 6). It has been suggested that oxidation
of the two isomers of retinol are the rate-limiting factors in the
pathways generating 9cRA and atRA.
Two classes of RDHs have been implicated in oxidation of the different
stereo isomers of retinol, i.e. microsomal members of the
short chain alcohol dehydrogenase/reductases, and the cytosolic medium
chain alcohol dehydrogenases (ADHs). Several members of both classes of
enzymes can oxidize different stereo isomers of retinol in
vitro. However, the physiological roles of these enzymes in
retinoid biosynthesis are not well understood.
The first identified member of the microsomal RDHs was RDH5, an enzyme
abundantly expressed in the retinal pigment epithelium with ability to
convert 11-cis-retinol (7), and other
cis-retinols (8, 9), into the corresponding retinals.
Subsequently, several other closely related members of the short chain
alcohol dehydrogenase/reductase family have been identified as RDHs
(10-15). RDH5 is a homodimeric integral membrane protein anchored
to the lipid bilayer via an amino-terminal signal anchor peptide and a
carboxyl-terminal transmembrane segment. The catalytic domain of the
enzyme is lumenal and only the extreme carboxyl-terminal 6-8
amino acid residues reside in the cytosol (16,
17) (the RDH4 enzyme has been
renamed to RDH5 to indicate that it is the murine counterpart of RDH5).
Genetic evidence for a role of the microsomal RDHs in ocular retinoid metabolism comes from the identification of mutations in RDH5 in
patients suffering from fundus albipunctatus (19, 20). These patients
have a reduced rate of synthesis of 11-cis-retinal resulting
in accumulation of white spots in the retina and stationary night
blindness. These results suggest a physiological role of RDH5 in
oxidation of 11-cis-retinol into 11-cis-retinal
in the visual cycle. Surprisingly, mice carrying targeted deletion of the RDH5 gene do not display similar symptoms (21). Studies on mice
carrying target deletions in the genes encoding ADH1 and ADH4 do not
show any obvious phenotypes related to altered metabolism of endogenous
retinoids (22, 23). Instead, these data provide compelling evidence
suggesting that ADH1 and ADH4 may not be critically required in
generation of RA in vivo.
In this study we have analyzed the ability of various enzymes,
suggested to play a role in RA biosynthesis, to contribute to
generation of 9cRA from 9cROL in a cell reporter assay. Furthermore, we
provide evidence suggesting that the unique milieu in the ER is
necessary for efficient generation of 9cRA in vivo.
Co-transfection Reporter Assay--
JEG-3 cells, COS-1 cells,
and 293 cells were maintained in either minimum essential medium (for
JEG-3; Life Technologies, Inc.) or Dulbecco's modified Eagle's medium
(for 293 and COS-1; Life Technologies, Inc.) with 10% fetal bovine
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The cells were transfected
using calcium phosphate precipitation as described previously (24). The
transfections were performed in 24-well plates, each experiment as
triplicates using either minimum essential medium or Dulbecco's
modified Eagle's medium with 10% charcoal-stripped fetal bovine
serum. The cDNAs encoding the enzymes were cloned into the
expression plasmid CMX-PL1 (a kind gift from Dr. T. Perlmann). Each
well was transfected with 100 ng of receptor Gal4-RXR plasmid, 100 ng
of reporter MH100-tk-luc plasmid (24), and 200 ng of internal control
plasmid encoding Site-specific Mutagenesis of CRAD1 and RDH5--
Mouse CRAD1 and
RDH5 cDNAs in pBluescriptSK Expression of Normal and Mutant CRAD1 and RDH5 in COS-1 Cells and
Measurement of Enzymatic Activity in Vitro by HPLC
Analysis--
Wild-type and mutant forms of CRAD1 and RDH5 were
expressed in transfected COS-1 cells essentially as described (7).
Isolated microsomes from the transfected cells, and from
mock-transfected cells, were collected and used in further analyses.
The in vitro activity analyses were done essentially as
described previously (19) using 9cROL as a substrate. A Waters Alliance
2690 HPLC system connected to a C18 reversed-phase column
(Supelco; 4.6 × 250 mm) was used. 9cROL was a kind gift from Dr.
Michael Klaus (Hoffman-La Roche AG, Basel, Switzerland).
Proteinase K Assay, SDS-PAGE, Immunoblotting, RT-PCR Analysis,
and Immunofluorescence Localization of CRAD1 and RDH5--
The
membrane topology of CRAD1 was determined using limited proteolysis as
described previously (16). Essentially, microsomes (total 10 µg of
protein) were incubated with 20 µg/µl proteinase K, with or without
1% Triton X-100 treatment. Reactions were stopped after 5, 15, 25, and
45 min with the addition of phenylmethylsulfonyl fluoride to a final
concentration of 2 mM. Samples were prepared and analyzed
on 12.5% SDS-PAGE gels and immunoblotted onto nylon membranes (Hybond
ECL, Amersham Pharmacia Biotech). CRAD1 was detected using an
affinity-purified polyclonal rabbit Ig fraction (see below). After
extensive washings, bound Ig was detected by ECL using Hyperfilm ECL
chemiluminescence film (Amersham Pharmacia Biotech) and developed.
Cellular expression of RDH5 and ADH4 was verified by immunoblotting
essentially as outlined above. Expression of CRAD2 and CRADL was
verified by RT-PCR of total RNA fractions using oligo(dT) priming. The
oligonucleotides 5'-TCT CTC TGC TTG ACT TCT ACA A and 5'-TTG GTG ACA
TCC AGG ATC were used to amplify a 282-base pair fragment corresponding
to CRAD2, and the oligonucleotides 5'-TGG GGT GAA GGT GGC TAT TAT and
5'-TCT GCA CCC TCA CAC AGC ACT were used to amplify a 418-base pair
fragment corresponding to CRADL. As negative controls aliquots of the
isolated total RNA fractions, without oligo(dT)-primed reverse
transcription, were subjected to PCR amplifications using the above
primer pairs. As positive controls, the corresponding expression
plasmids were including as templates. The amplified products were
analyzed on 1% agarose gels.
Polyclonal rabbit antisera to CRAD1 were generated by immunizing
rabbits with a glutathione S-transferase fusion protein
containing amino acids 19-288 of CRAD1, essentially as described for
the production of rabbit antisera to RDH5 (17). For affinity
purification, the rabbit antisera were passed over columns containing
the CRAD1-glutathione S-transferase fusion protein
immobilized on CNBr-activated Sepharose, and bound Ig was eluted and
characterized. The rabbit antibodies to RDH5 have been described
previously (17), and the rabbit antiserum to human ADH4 was a kind gift
from Dr. Jan-Olov Höög.
Immunofluorescence localization of CRAD1 and RDH5 was done using
transfected CHO cells as described previously (17). Texas Red-labeled
concanavalin A, Texas Red-labeled wheat germ agglutinin, and
rhodamine-labeled phalloidin were used as suggested by the supplier
(Molecular Probes).
A Reporter Assay Able to Measure the in Vivo Enzymatic Conversion
of 9cROL to 9cRA in Transfected Cells--
Three different cell lines,
JEG-3 cells, COS-1 cells, and 293 cells, were separately transfected
with expression plasmids encoding the cis-retinol-specific
dehydrogenase CRAD1 and the retinal dehydrogenase Raldh2 in an attempt
to reconstruct a metabolic pathway able to generate 9cRA from 9cROL.
Formation of 9cRA was monitored by co-transfecting the cells with
plasmids encoding a Gal4-RXR-based luciferase reporter system (24). The
induction of luciferase activity was subsequently measured following
addition of 9cROL to a final concentration of 1 µM. The
results showed that all three cell lines efficiently converted 9cROL to
9cRA when co-expressing CRAD1 and Raldh2 (data shown for JEG-3 cells only) (Fig. 1A). In cells
expressing only one of the two types of dehydrogenases, addition of
9cROL did not generate significant amounts of 9cRA. Addition of 9cRAL
induced reporter gene activation in cells expressing Raldh2, either
alone or in combination with CRAD1. Mock-transfected cells failed to
efficiently generate 9cRA upon addition of 9cROL or 9cRAL (data not
shown). The ability of the synthetic RXR agonist SR11237 to induce
reporter gene activation in cells expressing all combinations of the
two types of dehydrogenases confirmed that the transfected cells were
able to respond to the presence of ligand. These data suggest that
cellular components necessary for activity of the involved enzymes in
this coupled enzyme-reporter system are present in several cell types
and may be ubiquitously expressed.
The dose-response characteristics of transfected JEG-3 cells,
expressing both enzymes, were measured by addition of different concentrations of 9cROL, 9cRAL, atROL, and atRA (ranging between 0.1 and 50 µM for the 9-cis-retinoids and between
0.1 and 10 µM for the all-trans-retinoids).
The results showed that maximal reporter gene activation was achieved
between 1 and 10 µM 9cROL, and between 0.5 and 5 µM 9cRAL. At higher retinoid concentrations, reporter
gene activation was less effective probably due to the toxic effects of
high concentrations of retinoids. All tested concentrations of atROL
and atRAL failed to induce efficient reporter gene activation (Fig.
1B). The results are consistent with the notion that neither
of the all-trans-retinoids are efficiently converted into
the corresponding 9-cis-retinoids in vivo.
To analyze the capacity of different retinol dehydrogenases with
ability to oxidize 9cROL in vitro, RDH5 (17, 25), CRAD1 (13), CRAD2 (14), a novel CRAD-like enzyme "CRADL"3 and
the medium chain alcohol dehydrogenase ADH4 were separately expressed
in combinations with Raldh2 in JEG-3 cells. Upon addition of 9cROL, the
reporter gene activation analysis showed that efficient formation of
9cRA in vivo was achieved in cells expressing RDH5 and
CRAD1, whereas CRAD2 and CRADL were less efficient (Fig.
1C). Surprisingly, cells co-expressing ADH4 and Raldh2
displayed a low capacity to generate 9cRA from 9cROL. Addition of 9cRAL
efficiently induced reporter gene activation in transfected cells
expressing all combinations of enzymes. No obvious differences in the
capacity to oxidize 9-cis-retinal were found between Raldh1
and Raldh2 (data not shown). Immunoblot analysis of cell extracts from
cells overexpressing ADH4 and CRAD1 verified that these enzymes were produced (Fig. 1D). In addition, RT-PCR analyses of total
RNA fractions from the transfected cells demonstrated that transcripts encoding the enzymes were properly expressed. We conclude from these
experiments that the in vivo capacities of the different retinol dehydrogenases to convert 9cROL into 9cRAL differ
significantly, with CRAD1 being ~2 times as efficient in
vivo as RDH5 using this cell reporter system. In line with their
in vitro activities, 9-cis-retinol was a poor
substrate for CRAD2 (14) and CRADL.3
Membrane Topology of CRAD1 and a Structure-Activity Analysis of
CRAD1 and RDH5--
Previous analysis of RDH5, closely related in
primary structures to CRAD1, has suggested a lumenal orientation of the
catalytic domain of the enzyme and that it is membrane-anchored by a
NH2-terminal signal anchor and a transmembrane segment
close to the cytosolic COOH terminus (16, 17). To investigate the
membrane topology of CRAD1, microsomes from COS-1 cells expressing
CRAD1 were isolated and subjected to limited proteolysis using
proteinase K. In intact microsomes, CRAD1 was well protected from
degradation, whereas in Triton X-100-treated microsomes, CRAD1 was
rapidly degraded by the protease (Fig.
2A). These data are consistent
with the notion that the membrane topology of CRAD1 is similar to that of RDH5 with a lumenal catalytic domain and the COOH terminus located
in the cytosol. A schematic illustration of the membrane topology of
CRAD1 is outlined in Fig. 2B.
To analyze structure-activity relationships in CRAD1, we constructed
several mutants of the enzyme that could affect the membrane topology
and mode of membrane interaction. The mutations were introduced to
generate premature stop codons before the transmembrane segment
(L289Stop), and before the COOH-terminal cytosolic tail (R312Stop),
respectively. In addition, the active site tyrosine in CRAD1 was
mutated into a phenylalanine (Y175F) (Fig. 2B).
Expression of the CRAD1 mutants in COS-1 cells and immunoblotting
analysis of isolated microsomes from the transfected cells showed that
significant expression levels of the mutant protein was found only in
the mutant affecting the COOH-terminal tail (R312Stop). Other mutations
failed to generate detectable levels of protein, probably due to
decreased protein stability and subsequent degradation (Fig.
3A). An identical
carboxyl-terminal tail deletion was also introduced in RDH5 (R312Stop)
and the expression of wild-type RDH5 and mutant RDH5 R312Stop in
transfected COS-1 cells was verified by immunoblotting (Fig.
3B).
Activity analysis of the two truncated versions of CRAD1 and RDH5 in
the cell reporter assay showed that both mutants displayed low
capacities to catalyze the conversion of the substrate in vivo compared with the wild-type protein (Fig. 3, C and
D). Addition of 2 mM NAD to the culture medium
did not alleviate the lack of enzymatic activity in vivo
(data not shown). Separate analysis showed that the carboxyl-terminal
truncated enzymes present in isolated microsomes displayed almost full
enzymatic activity in vitro when compared with the wild-type
enzyme (Fig. 3 (E-G) for CRAD1, and data not shown for
RDH5). These results suggested that the COOH-terminal cytosolic tails
of the two enzymes are not directly involved in the catalytic
mechanism, a result consistent with the membrane topologies of the
enzymes. Instead, it may be involved in controlling intracellular
compartmentalization of the enzymes, which may indirectly effect the
ability of the enzyme to oxidize 9cROL in vivo.
To test this hypothesis, wild-type CRAD1, CRAD1 R312Stop mutant,
wild-type RDH5, and RDH5 R312Stop mutant were separately expressed in
CHO cells, and intracellular localization of the enzymes was determined
by indirect immunofluorescence microscopy. As a marker of the ER, the
cells were also stained with Texas Red-labeled concanavalin A. The
results showed that wild-type CRAD1 and RDH5 displayed typical ER
staining patterns (Fig. 4, A-C and I-K). In contrast, the CRAD1 R312Stop
and RDH5 R312Stop mutants displayed strikingly different staining
patterns. The CRAD1 mutant was localized in strongly stained elongated
perinuclear fiber-like structures, while the RDH5 mutant was localized
in unevenly distributed patchlike structures (Fig. 4, E-G
and L-N). Staining with rhodamine-labeled phalloidin, to
visualize the actin filaments, showed no obvious overlapping
localization of the actin fibers with the truncated mutant CRAD1 (Fig.
4, D and H). Similarly, staining with fluorescent
wheat germ agglutinin, to visualize the nuclear envelope and the Golgi
apparatus, showed only marginal co-localization of this marker with the
mutant enzymes (data not shown). All together, these results suggested
that the COOH-terminal tails in CRAD1 and RDH5 have a role in
compartmentalization of the enzymes and that ER localization of the
enzymes might be necessary for enzymatic activity in
vivo.
At present, three major classes of enzymes have been
implicated in synthesis of RA: the cytosolic medium chain alcohol
dehydrogenases and microsomal members of the short chain dehydrogenase
superfamily acting as retinol dehydrogenases, and the cytosolic
aldehyde dehydrogenases acting as retinal dehydrogenases. The proposed
roles of these enzymes in retinoid metabolism are mainly based on
in vitro enzymatic activities, and in some cases also
supported by the expression patterns of the enzymes. Genetic
evidences for the involvement of some of these enzymes in retinoid
biosynthesis come from the identification of patients carrying
mutations in RDH5 which effects the synthesis of
11-cis-retinal in the eye, and from the analysis of a
gene-targeted mouse strain lacking expression of Raldh2, which leads to
embryonic lethality (19, 29).
Despite the identification of several enzymes putatively involved in
the pathways generating RA, no attempts to reconstitute a metabolic
pathway able to generate RA from retinol have been reported. In the
present work we demonstrate that efficient formation of 9cRA from 9cROL
in vivo requires the concerted action of retinol dehydrogenases and retinal dehydrogenases. This finding implicates that
efficient generation of RA in other cell types, i.e. cells in adult and embryonic tissues, may also require both enzymatic activities. We anticipate that modified versions of the coupled enzyme/reporter cell system can be useful in analyzing the structural and functional requirements for efficient synthesis of atRA in transfected reporter cells.
The microsomal CRAD1 and RDH5 appeared to be the most efficient enzymes
converting 9cROL into 9cRAL of the retinol dehydrogenases tested. The
subsequent formation of 9cRA in cells co-expressing retinal
dehydrogenases shows that the two enzymatic reactions underlying
formation of 9cRA from 9cROL are coupled, despite the different
locations of the two reactions (lumenal versus cytosolic). In contrast, the cytosolic ADH4, previously reported to efficiently convert retinol into retinal in vitro, appeared as a less
efficient retinol dehydrogenase in the transfected cells. The apparent
lack of coupling of the enzymatic activities of ADH4 and the retinal dehydrogenases is surprising considering that both types of enzymes are
cytosolic. Explanations to this observation might be that the cytosol
is strongly reducing which would not favor retinol oxidation in
vivo, or that sufficient concentrations of 9cROL or cofactor are
not available to the enzyme. The latter explanation appears, however,
less likely since 9cROL is hydrophobic and would easily cross cellular
membranes and the cytosolic concentration of NAD is high. The
efficiency of some of the microsomal retinol dehydrogenases contrasts
the poor ability of ADH4 to oxidize 9cROL. Both CRAD1 and RDH5 display
a unique membrane topology with the catalytic domains in lumenal
compartments of the cells. The ER lumen is oxidative (30), and the
milieu would favor retinol oxidation provided that sufficient
concentrations of substrate and oxidized cofactors are available. CRAD1
and RDH5 have a preference for NAD as the cofactor (7, 13, 25); our
results imply that sufficient concentrations of NAD are indeed present
in the ER lumen. These conclusions are in line with previous
measurements of cofactor concentrations in isolated microsomes (31).
The origin of the lumenal NAD is currently unknown, but it may be generated inside the ER lumen by yet uncharacterized mechanisms, or it
may be accumulated from the cytosol. In the latter case, we would
predict the existence of a specific transport mechanism for the
hydrophilic pyridine nucleotides, possibly involving a specific
transmembrane transporter system.
The low in vivo activity and subcellular mislocalization of
the mutants of CRAD1 and RDH5, lacking the extreme COOH-terminal regions of 6 or 7 amino acids, respectively, implies that correct subcellular compartmentalization may be important for the activities of
these enzymes. Under the experimental conditions used by us, the
substrate 9cROL is unlikely to be the rate-limiting factor. Instead,
the mislocalized mutant enzymes may end up in a subcellular compartment
with a general milieu that does not favor retinol oxidation, or in a
compartment with insufficient concentrations of NAD, or that formed
9cRAL generated by the mutant enzyme is unable to cross the ER membrane
to reach the cytosolic Raldh2 for further oxidation to 9cRA. At
present, it is not possible to distinguish between these possibilities.
The COOH-terminal cytosolic regions of the microsomal RDHs, including
CRAD1 and RDH5, are well conserved (Fig.
5). In fact, the extreme COOH-terminal
amino acids are -A-L in all members of the enzyme family, except in
RDH5, which has an extension of one additional amino acid and ends with
a hydroxyl-containing amino acid (Y in bovine and human RDH5, and S in
mouse RDH5). Furthermore, the COOH-terminal tails of all the enzymes
have a conserved pattern of several other residues, including the
invariant proline residues at position -5 (-6 in RDH5). The conserved
features of the cytosolic tails in the RDHs, and the fact that deletion of the cytosolic tails in CRAD1 and RDH5 leads to mislocalization of
the enzymes and loss of enzymatic activities in vivo, argue for a role of the cytosolic tails in interactions with cellular components involved in retaining, or retrieving of the enzymes in the
ER. Interestingly, several fundus albipunctatus patients recently
identified in Japan have mutations in, or close to, the COOH-terminal
cytosolic tail of RDH5 (A294P (Ref. 20) and delC1085/ins GAAG (Ref.
32)), suggesting that an intact COOH-terminal tail might be necessary
for proper function of the enzyme in vivo. The
identification of putative proteins interacting with the COOH-terminal tails of the RDHs might provide further clues to the roles of these
enzymes in retinoid homeostasis in adult and embryonic tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase. In addition, 100 ng of each plasmid
encoding murine CRAD1 (13), CRAD2 (14), a CRAD-like enzyme,
CRADL,2 RDH5 (17, 25) or
murine ADH4 (a kind gift from Dr. Gregg Duester) were co-transfected
with 100 ng of expression plasmid encoding retinal dehydrogenase 1 (Raldh1) or Raldh2 in various combinations. Original retinal
dehydrogenase plasmids were kind gifts from Dr. Joseph Napoli. Unless
otherwise indicated, medium with 1 µM
9-cis-retinol (9cROL) or 9-cis-retinal (9cRAL)
was separately added to the cells after 6 h of incubation with the
calcium phosphate precipitate, and 1 µM SR11237 was used
as a positive control (26). For the titration experiment, 0.1-50
µM 9cROL, 9cRAL, all-trans-retinol (atROL), or
all-trans-retinal (atRAL) were used. Medium without added
ligand served as a negative control. After an additional 18 h, the
cells were lysed and their luciferase and
-galactosidase activities
were measured with a luminometer (Lucy-1, Anthos, Saltzburg, Austria)
as described previously (24). All luciferase values were normalized to
the internal
-galactosidase control, and the maximal relative
activation was set to 100%. The assays were performed several times
with similar results, and each data point represents the mean of
triplicate analyses.
(Stratagene) were used to produce
single-stranded DNA with the helper phage M13KO7 (27). All mutations
were made with oligonucleotide-directed site-specific mutagenesis
without phenotypic selection (28). The oligonucleotides, with mutated
nucleotide(s) underlined, were as follows; 5'-TTT GGT GGT GGT
TTC TGC ATC TCT AAG (CRAD1, changing the tyrosine in the
active site to phenylalanine, Y175F), 5'-TGG GAT GCT AAG
TGA TTC TAC CTC CCC (CRAD1, inserting a premature stop
codon before the COOH-terminal transmembrane region, L289Stop), 5'-TGG
ACT TCC CTG TGA CCT GAA AAA GCC (CRAD1,
inserting a premature stop codon 6 amino acids before the normal stop
codon thus deleting the cytosolic COOH-terminal R312Stop), and 5'-ACC
TGG ATC CTT CCC TGA CCC GCC CAG TCA GTC (RDH5,
inserting a premature stop codon 7 amino acids before the normal stop
codon thus deleting the cytosolic COOH-terminal tail, R312Stop). The
mutations were verified by sequencing. Mutated plasmids were digested
with EcoRI, the inserts were cloned into the eucaryotic
expression vector CMX-PL1, and the enzymatic activities of the mutants
were analyzed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Synthesis of 9cRA in transfected JEG3 cells
co-expressing retinol dehydrogenases and retinal dehydrogenases.
A, 9cROL and 9cRAL were separately added to JEG-3 cells
expressing CRAD1 (black bars), Raldh2
(white bars), and both CRAD1 and Ralhd2
(gray bars), and the activation of the RXR-based
luciferase gene reporter system was used to monitor formation of 9cRA.
The synthetic RXR ligand SR11237 served as a positive control.
B, dose-response curves for 9cROL ( ), 9cRAL (
), atROL
(
), and atRAL (
) added to JEG-3 cells co-expressing CRAD1 and
Raldh2. C, synthesis of 9cRA by transfected JEG-3 cells
expressing Raldh2 in combination with CRAD1 (black
bars), CRAD2 (white bars), CRADL
(dark gray bars), RDH5
(light gray bars), ADH4
(striped bars), monitored using the RXR-based
reporter system. D, expression of several retinol
dehydrogenases in the transfected JEG-3 cells. Expression of ADH4
(upper panel, left) and CRAD1
(upper panel, right) were detected by
immunoblotting using specific antibodies. Mock-transfected cells
(Mock) served as a negative control. The migration of
molecular size markers is indicated to the right.
Transcripts encoding CRAD2 (lower panel,
left) and CRADL (lower panel,
right) were detected by RT-PCR using aliquots of the total
RNA fractions isolated from the transfected cells (RT-PCR).
As positive controls the expression plasmids were used as templates
(CRAD2 and CRADL). As negative controls aliquots
of the isolated RNA fractions, without being subjected to
reverse-transcription, were used (RNA). The sizes of the
amplified fragments are shown to the right.
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Fig. 2.
Membrane topology of CRAD1.
A, limited proteolysis of CRAD1 expressed in transfected
COS-1 cells. Proteinase K was added to microsomes permeabilized with
0.1% Triton X-100 (+ Triton-X) and to intact microsomes
( Triton-X). Following the different digestions (0-45 min
of incubation), the microsomes were subjected to SDS-PAGE and
immunoblotting using antibodies to CRAD1. B, a schematic
illustration of the membrane topology of CRAD1 and with an indication
of the sites where mutations were introduced (arrows; see
text for details).
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Fig. 3.
Expression and enzymatic activity of
wild-type and mutant CRAD1 and RDH5 in vivo and
in vitro. A, immunoblotting analysis of wild-type
CRAD1 and CRAD1 mutants Y175F, L289Stop, and R312Stop expressed in
transfected COS-1 cells. B, immunoblotting analysis of
wild-type RDH5 and mutant R312Stop expressed in transfected COS-1
cells. C, in vivo enzymatic activity measured
using the reporter system in JEG-3 cells transfected with an expression
vector encoding wild-type CRAD1 (black bar), the
R312Stop mutant lacking the COOH-terminal cytosolic tail
(white bar), and background activity in
mock-transfected cells (gray bar). D,
in vivo enzymatic activity measured using the reporter
system in JEG-3 cells transfected with expression vectors encoding
wild-type RDH5 (black bar), encoding the R312Stop
mutant (white bar), and background activity in
mock-transfected cells (gray bar). In
C and D, the formation of 9cRA was assayed in
culture medium containing 1 µM 9cROL. All cell cultures
were co-transfected with plasmids encoding Raldh2 and the RXR-based
reporter system. The results were normalized to 100% activity for the
wild-type enzymes. E-G, representative reverse phase HPLC
profiles of 9cROL dehydrogenase activities in vitro in
isolated microsomes from transfected COS-1 cells expressing wild-type
CRAD1 (E), mutant CRAD1 R312Stop, lacking the COOH-terminal
cytosolic tail (F), or microsomes from mock-transfected
COS-1 cells (G).
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Fig. 4.
Localization of wild-type and mutant retinol
dehydrogenases in transfected CHO cells by indirect
immunofluorescence. A-D, expression of wild-type
CRAD1; E-H, expression of mutant CRAD1 R312Stop lacking the
COOH-terminal cytosolic tail; I-K, expression of wild-type
RDH5; L-N, expression of mutant RDH5 R312Stop lacking the
COOH-terminal cytosolic tail. The wild-type and mutant enzymes were
localized using specific antibodies to CRAD1 or RDH5, respectively,
using fluorescein isothiocyanate-labeled secondary antibodies
(panels A, C-E, G,
H and panels I, K,
L, and N, respectively, in green). The
ER was localized using Texas Red-labeled concanavalin A (B,
C, F, G, J, K,
M, and N, in red), and actin filaments
were visualized using rhodamine-labeled phalloidin (D and
H, in red). Merged images showing the wild-type and mutant
retinol dehydrogenases with the ER marker are shown in C,
G, K, and N (yellow
color indicates co-localization), and wild-type and mutant
CRAD1 with the actin filament marker are shown in D and
H. Original magnification, ×630.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 5.
Amino acid sequence alignment of the
COOH-terminal tails in microsomal RDHs. The amino acid sequences
of the extreme COOH-terminal tails in mouse CRAD1, CRAD2, and CRADL
(Refs. 13 and 14, and Footnote 3), rat RoDH1-3 (10-12), human RoDH4
(15), and RDH5 from different species were aligned (7, 17, 18, 25). The
RDH5 enzymes are one amino acid longer than the other RDHs (on
gray background). The invariant P residues are
boxed. m, mouse; r, rat; h,
human; b, bovine.
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ACKNOWLEDGEMENT |
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We thank Barbara Åkerblom for expert technical assistance.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant K99-03P-12070-03C (to U. E.) and the Karolinska Institutet for financial support.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.
To whom correspondence should be addressed. Tel.: 46-8-728-7109;
Fax: 46-8-332812; E-mail: ueri@licr.ki.se.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M100215200
2 A. Romert, K. Tryggvason, and U. Eriksson, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: 9cRA, 9-cis-retinoic acid; 9cROL, 9-cis-retinol; 9cRAL, 9-cis-retinal; atRA, all-trans-retinoic acid; atROL, all-trans-retinol; atRAL, all-trans-retinal; CRAD, cis-retinol/androgen dehydrogenase; ER, endoplasmic reticulum; RA, retinoic acid; RAR, retinoic acid receptor; RDH, retinol dehydrogenase; RT, reverse transcription; PCR, polymerase chain reaction; RXR, retinoid X receptor; Raldh, retinal dehydrogenase; ADH, medium chain alcohol dehydrogenase; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary.
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REFERENCES |
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