(Received for publication, March 2, 1995 )
From the
We have expressed in Escherichia coli functionally
active proteins encoded by two human cDNAs that were isolated
previously by using rat 3-hydroxysteroid dehydrogenase cDNA as the
probe. The expressed proteins catalyzed the interconversion between
5
-dihydrotestosterone and 5
-androstane-3
,17
-diol.
Therefore, we name these two enzymes type I and type II
3
-hydroxysteroid dehydrogenases. The type I enzyme has a high
affinity for dihydrotestosterone, whereas the type II enzyme has a low
affinity for the substrate. The tissue-specific distribution of these
two enzymes was determined by reverse transcription polymerase chain
reaction using gene-specific oligonucleotide primers. The mRNA
transcript of the type I enzyme was found only in the liver, whereas
that of the type II enzyme appeared in the brain, kidney, liver, lung,
placenta, and testis. The structure and sequence of the genes encoding
these two 3
-hydroxysteroid dehydrogenases were determined by
analysis of genomic clones that were isolated from a
EMBL3 SP6/T7
library. The genes coding for the type I and type II enzymes were found
to span approximately 20 and 16 kilobase pairs, respectively, and to
consist of 9 exons of the same sizes and boundaries. The exons range in
size from 77 to 223 base pairs (bp), whereas the introns range in size
from 375 bp to approximately 6 kilobase pairs. The type I gene contains
a TATA box that is located 27 bp upstream of multiple transcription
start sites. In contrast, the type II gene contains two tandem AP2
sequences juxtaposed to a single transcription start site.
3-Hydroxysteroid dehydrogenase (3
-HSD) (
)belongs to the aldo-keto reductase family, which includes
aldehyde reductase(1) , aldose reductase(2) ,
dihydrodiol dehydrogenase(3) , bovine prostaglandin F
synthase(4) , frog eye lens
-crystallin(5) , human
chlordecone reductase(6) , and numerous related proteins
recently identified in rats and humans in our laboratory(7) .
These enzymes catalyze the conversion of aldehydes and ketones to
alcohols by utilizing NADH and/or NADPH as the cofactor and exist in
cellular cytoplasm as monomeric 34-36-kDa proteins. These enzymes
exhibit distinct but overlapping substrate specificities and are
inhibited by a number of drugs, such as phenobarbital, pyrazole,
chlorpromazine, indomethacin, sodium valproate, quercetin, and
ethacrynic acid(8) .
3-HSD may be the most versatile
enzyme in the aldo-keto reductase family due to its ability to utilize
a large array of substrates. For example, rat 3
-HSD also carries
dihydrodiol dehydrogenase activity (9) . The importance of
dihydrodiol dehydrogenase in the detoxification of polycyclic aromatic
hydrocarbons was demonstrated by its ability to reduce
benzo(a)pyrene mutagenic activity in the Ames test (10) . 3
-HSD also converts bile acid precursors to bile
acids and may serve as a bile acid transporter in the liver and
intestine(11, 12) .
3-HSD in the liver is
responsible for inactivation of steroid hormones and maintenance of the
homeostatic balance of circulating steroid hormones. Nevertheless, not
all of the 3
-HSD metabolites are physiologically inactive. For
example, the 3
-hydroxy A-ring-reduced pregnane steroids
(allopregnanolone) have been shown to exert sedative, hypnotic, and
anesthetic effects when they were administered to animals (13) . In addition, these tetrahydrosteroids also produce a
number of other behavioral effects, such as anticonflict,
anticonvulsant, and analgesic actions(14) . These neurological
effects have been linked to the binding of the tetrahydrosteroids to
the major inhibitory
-aminobutyric acid receptor complex. It was
recently shown that the levels of tetrahydrosteroids, such as
allopregnanolone and allotetrahydrodeoxycorticosterone, were elevated
rapidly and robustly in the brain and plasma of rats after exposure to
ambient temperature swimming stress(15) . Administration of
progesterone to healthy humans induced changes in fatigue and in
delayed verbal recall; these changes correlate with the levels of
3
-tetrahydro-metabolites. These studies support the hypothesis
that 3
-tetrahydrosteroids are endogenous modulators of the
gamma-aminobutyric acid receptor(16) .
It was previously
suggested (17, 18) that multiple 3-HSD isozymes
exist in the human. In this report, we demonstrate that proteins
encoded by two of the cDNAs we previously isolated carry 3
-HSD
activity. In addition, we compare the enzyme characteristics and
describe the gene structure of these steroid hormone-metabolizing
enzymes.
Bacteria were harvested from a 10-liter batch of overnight culture
by centrifugation. The bacterial pellet was resuspended in 1 liter of
10 mM Tris-HCl buffer, pH 7.4, and then subjected to pulse
sonication. Bacterial cytosols containing the expressed enzyme were
recovered from the sonicated mixture by centrifugation at 10,000
g for 10 min.
Conversion of
5-dihydrotestosterone to 5
-androstane-3
,17
-diol by
the bacterial cytosol was determined according to a procedure
previously established(21) . The reaction mixture in a 1-ml
final volume contains 1-20 nmol of dihydrotestosterone, 10 nCi of
[
C]5
-dihydrotestosterone, 1 mM
NADPH, 300 µg of bacterial lysate, and 100 mM sodium
phosphate, pH 7.4. The reaction was performed at 37 °C for 10 min.
The metabolite and remaining substrate were extracted with methylene
chloride and were separated by thin-layer chromatography using a mixed
solvent system (chloroform/ethyl acetate/ethanol (4:1:0.2)). The
3
-hydroxyl metabolite was identified based upon the R
value previously
established(21) .
5-Androstane-3
,17
-diol was
converted from 5
-dihydrotestosterone by using rat 3
-HSD that
we had previously expressed in bacteria(19) . Assays of the
3
-dehydrogenase activity of the expressed human enzymes were
carried out in a reaction mixture that contained 3 nmol of
5
-androstane-3
,17
-diol, 10 nCi of
5
-androstane-3
,17
-diol, 300 µg of bacterial lysate,
1 mM NADP, and 100 mM sodium phosphate, pH 7.4, in a
final volume of 1 ml. Extraction and separation of metabolites were
performed according to the procedure described above.
The activity
of the expressed human 3-HSD was also determined using the
substrates chenodeoxycholic acid, acenaphthenol, and androsterone
according to a procedure established previously(19) . The
protein concentration was measured by the Lowry procedure(22) .
Figure 1:
Western blot analysis of human type I
and type II 3-HSDs. A, monoclonal antibody 3G6 was used
for the detection of the type I enzyme. B, a polyclonal
antibody raised against a purified human 3
-HSD was used for the
detection of the type II enzyme. Each lane contains 100 µg
of protein from the bacterial lysate. The lanes labeled TYPE I and TYPE II contain lysates from bacteria that
carry the type I and the type II expression vectors, respectively. The lane labeled CONTROL contains lysates from bacteria
that carry the empty expression vector. The positions and molecular
masses of protein size markers are shown on the left side of
the blots.
Both type I and type II human 3-HSDs
could convert 5
-dihydrotestosterone to
5
-androstane-3
,17
-diol in the presence of NADPH as the
cofactor (Fig. 2). The apparent affinities as determined by
double-reciprocal plots of the enzyme activities for the substrate
appear to be rather different (Fig. 3A). The K
of the type I enzyme is approximately 1
uM, whereas that of the type II enzyme is 20 uM (Fig. 3). Both enzymes were found to carry the
dehydrogenase activity when 5
-androstane-3
,17
-diol was
used as the substrate. In addition, both forms of 3
-HSD were found
to catalyze the 3
-dehydrogenation of androsterone (Table 1).
These results suggest that human type I and type II enzymes contain
both reductase and dehydrogenase activities.
Figure 2:
Thin-layer chromatography separation of
the substrate (5-dihydrotestosterone) and the 3
-HSD product
(5
-androstane-3
,17
-diol). 300 µg of protein from
bacterial lysate was used for the assay of 3
-HSD activity as
described under ``Experimental Procedures.'' Both the
C-labeled substrate and the metabolite in the reaction
mixture were extracted with methylene chloride and applied to a
thin-layer chromatography plate. Separation of the substrate and the
metabolite was performed using a mix-solvent system containing
chloroform/ethyl acetate/ethanol (4:1:0.2). The substrate and the
metabolite were identified based upon their R
values by autoradiography. The lane labeled Control contains the reaction mixture from the lysate of bacteria carrying
an empty vector. The lanes labeled Type I and Type II contain the substrate and metabolite generated by the
lysates of bacteria carrying the type I and type II expression vectors,
respectively.
Figure 3:
Conversion of 5-dihydrotestosterone
to 5
-androstane-3
,17
-diol catalyzed by type I and type
II human 3
-HSDs. A, double-reciprocal plots of the enzyme
activity. B, apparent K
and V
(mean ± standard error, n = 4) of the type I and type II 3
-HSDs. The K
and V
parameters
were determined based upon the double-reciprocal plots using an Enzfit
program. Assay conditions were identical to those described in the
legend of Fig. 2.
The type I and type II enzymes are catalytically active when 1-acenaphthenol was used as the substrate, suggesting that both enzymes carry dihydrodiol dehydrogenase activity. Much higher activities were found when chenodeoxycholic acid was used as the substrate, indicating that these two enzymes are involved in the production of bile acids in the liver (Table 1).
Figure 4:
Structures of the type I and type II human
3-HSD genes. A, regions of the type I gene contained in
the three overlapping
clones, KQ8, KQ7 and KQ26, are indicated by solid lines. B, regions of the type II gene contained
in the
clones, KQ11, KQ13 and KQ18, are also indicated by solid lines. The nine exons are numbered and represented by filled boxes. The size marker is shown on the bottom of the figure.
The exon-intron arrangements of the type I and type II genes appear to be very similar, except that the sizes of some of their corresponding introns vary considerably. The introns of the type I gene range in size from 375 bp to approximately 6 kb, whereas that of the type II gene range from 375 bp to approximately 4 kb. Three of the introns (introns 2, 3, and 5) in the type I gene are larger than that of the type II gene. Only two of the introns (introns 6 and 8) interrupt the coding sequence within codons.
The sizes and
boundaries of each of the nine exons in these two genes are identical (Fig. 4). Exon 1 contains 84 bp of the translated sequence and
some of the untranslated sequence. Exons 2-9 contain the rest of
the coding sequence. In the type I 3-HSD gene, exon 9 contains 42
bp of the translated sequence and 181 bp of the untranslated sequence.
The untranslated sequence contains an AATAAA polyadenylation signal. In
the type II 3
-HSD gene, exon 9 also contains 42 bp of the the
translated sequence, but the untranslated sequence was only partially
determined. The nine exons range in size from 77 to 223 bp. Fig. 5shows the alignment of the exons and intron-exon junctions
of these two genes. Using a DNASTAR sequence alignment program to
compare the homology between these two genes, we found high degrees of
sequence identity in exons (85%) as well as in introns (60%).
Figure 5:
Sequence comparison of the exons and
intron-exon junctions of type I and type II human 3-HSd genes. The
coding nucleotide sequence is shown in capital letters. The
amino acid sequence is displayed under the nucleotide sequence. Base
numbering is based upon the the position of the amino acid codon in the
coding sequence.
The primer extension experiment showed multiple extended products containing 109-111 nucleotides for the type I gene, indicating that the 5`-untranslated sequence of the gene consists of 25-27 nucleotides (Fig. 6A). For the type II gene, a single extended product containing 152 nucleotides was found, indicating that the 5`-untranslated sequence of the type II gene consists of 68 nucleotides (Fig. 6B).
Figure 6: Primer extension analysis. A synthetic oligonucleotide complementary to the nucleotide position 54 to 84 was used for the primer extension study. A, lane 1 shows the extension products from the type I gene. Lanes 2-5 show the sequence ladder generated with the extension primer on a PCR product. Lane 2, G; lane 3, A; lane 4, T; lane 5, C. B, lane 5 shows the extension product of the type II gene. Lanes 1-4 show the sequence ladder generated with the extension primer on a PCR product. Lane 1, G; lane 2, A; lane 3, T; lane 4, C.
Figure 7:
RT-PCR of
the expression of type I 3-HSD, type II 3
-HSD, and
-actin. RT-PCR was performed using the total RNA isolated from
human brain, kidney, liver, lung, placenta, and testis as the template.
Oligonucleotide PCR primers used in these three panels were specific
for the genes of type I 3
-HSD, type II 3
-HSD, and
-actin, respectively. 35 cycles of PCR amplification were
performed under the following conditions: 94 °C for 1 min, 60
°C for 1 min, and 72 °C for 2 min. The PCR products were
electrophoresed in a 2% agarose gel and visualized after staining with
ethidium bromide.
Figure 8:
Comparison of human type I 3-HSD and
type II 3
-HSD 5`-flanking sequences. Alignment of the two
sequences was performed using the ALIGN program (DNASTAR). Identities
between these two sequences are indicated by displaying the base in
between the two lines of sequences. Dashes are inserted for
optimal alignment. Base numbering is based upon the the position of the
first ATG start codon as +1. The transcription initiation sites
are marked with stars. The first ATG, TATA box, and potential
AP1 and AP2 sequences in the genes are underlined.
Fig. 8shows the sequence alignment of the 5`-upstream sequences of the type I and type II genes. A 60% sequence identity was found between the 5`-upstream sequences of these two genes. Several gaps were introduced into the sequences in order to obtain the best alignment. In addition, there were several regions of sequences that displayed no significant homology. These diverse regions of the 5`-upstream sequence may contain tissue-specific promoter activity that causes the difference in the tissue-specific expression of these two genes.
In this paper we report the identification of two human
3-hydroxysteroid dehydrogenases and their gene structures, which
provide an opportunity for future studies on the regulation of their
activity and tissue specificity. The existence of multiple forms of
3
-HSD in human liver has been suggested by purification of
multiple proteins exhibiting 3
-HSD activity (17) and by
molecular cloning of multiple human cDNAs encoding proteins
structurally related to rat 3
-hydroxysteroid
dehydrogenase(18) . The type I 3
-HSD shows a sequence
identical to that of the human chlordecone reductase reported by
Winters et al.(6) , except that the cDNA isolated by
us contains a full-length coding sequence. As demonstrated here, the
protein encoded by this full-length cDNA is shown to contain 3
-HSD
activity. Recently Lou et al.(29) isolated a cDNA
that was identical in sequence to one of the human cDNAs previously
identified(18) . The protein encoded by this cDNA, obtained by
expression in bacteria, exhibited dihydrodiol dehydrogenase/bile
acid-binding protein (BABP) activity but not 3
-HSD
activity(29) . The type II 3
-HSD appears to be different
from but similar to the type I enzyme (human chlordecone reductase) and
the dihydrodiol dehydrogenase/BABP. The gene structures of both human
type I and type II 3
-HSD are similar to that of the dihydrodiol
dehydrogenase/BABP as previously reported by us (24) and by Lou et al.(29) with the exception that type I 3
-HSD
gene contains several large introns. Although a high degree of sequence
homology was found between the 5`-flanking sequences of the type I and
type II 3
-HSD, the transcripts of these two genes show a marked
difference in the tissue-specific distribution. The type I 3
-HSD
appears to be a liver-specific enzyme, whereas the type II 3
-HSD
is constitutively expressed in most tissues. Future investigation into
the molecular mechanism underlying the tissue-specificity of the
multiple human 3
-HSDs and dihydrodiol dehydrogenase/BABP may yield
a model system for elucidation of the regulatory element(s) responsible
for the tissue-specificity. Our finding that multiple 3
-HSDs exist
in humans further complicates the putative role of 3
-HSD in the
production and regulation of neuroactive tetrahydrosteroids in the
human brain. It is tempting to speculate that the type I 3
-HSD may
play an essential role in the liver metabolism of steroid hormones,
whereas the type II 3
-HSD may be responsible for the production of
neuroactive steroids in the brain.
Molowa et al.(30) showed that an increase in the chlordecone reductase
activity in gerbils appeared to be due to an increase in the
transcriptional activity after the administration of chlordecone. In
studies by Ciaccio et al.(31) , transcription of
dihydrodiol dehydrogenase was induced by certain xenobiotics, such as
ethacrynic acid, dimethyl maleate, and t-butylquinone. It was
speculated that the induction of dihydrodiol dehydrogenase may be via
the regulatory sequence AP1, because these xenobiotics also induce the
DT diaphorase gene, which is controlled by the AP1 sequence within the
antioxidant responsive element(32) . Homology search of the
5`-flanking sequence of the type I and type II 3-HSD genes
revealed several AP1-like sequences near the putative promoter region.
For this reason, we are currently investigating the role of these AP1
sequences on the control of the promoter activity.
The genes
encoding two other aldo-keto reductases, human aldose reductase and the
mouse major vas deferens protein, have been recently
delineated(33, 34) . In contrast to the genes encoding
human 3-HSDs and dihydrodiol dehydrogenase/BABP, which contain 9
exons, the aldose reductase and major vas deferens protein genes
contain 10 exons. In addition, the exon-intron arrangement of the human
aldose reductase and major vas deferens protein genes differ from those
of the genes reported here. Much less homology was found in the intron
sequences and the 5`-flanking sequences between the genes encoding for
type I 3
-HSD/dihydrodiol dehydrogenase and dihydrodiol
dehydrogenase/BABP and the genes for aldose reductase and major vas
deferens protein. Furthermore, aldose reductase is located at
chromosome 7q35, whereas both 3
-HSD and dihydrodiol
dehydrogenase/BABP are located at chromosome
10p14-15(28) . Therefore, it is apparent that 3
-HSDs
and dihydrodiol dehydrogenase/BABP are more phylogenetically related to
each other. The aldo-keto reductase superfamily is involved in the
metabolism of endogenous substrates, such as steroid hormones,
prostaglandins, and bile acids, and xenobiotics, such as drugs and
environmental carcinogens. Future studies in the delineation of
structure-function relationships, regulation, and induction of the
aldo-keto reductases may shed light on the physiological and
pathological functions of this important family of enzymes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]-L43839[GenBank].