From the
Several mammalian livers contain monomeric
17
17
Livers of rabbits
(8, 9) , guinea pigs
(10, 11) , and mice
(12, 13) contain cytosolic and monomeric 17
Mouse liver 17
The cDNA inserts from
the positive clones were subcloned into restriction sites (see
Fig. 1
) of pBluescript II plasmids, and the nucleotide sequences
of the cDNAs were determined with a Taq dye primer cycle
sequencing kit and Taq DyeDeoxy
For the
purification of r17
Total RNA was extracted from the mouse tissues by the method of
Chomczynski and Sakki
(27) . Twenty µg of total RNA was
size-fractionated by electrophoresis through a 1.0% agarose gel in the
presence of 6.3% (v/v) formaldehyde
(24) and blotted onto a
noncharged nylon membrane (Pall BioSupport) using 20
Recent progress on the isolation and cloning of HSDs
indicates that they are classified into two protein families, the short
chain dehydrogenases, and aldoketoreductases, except that mammalian
3
Of the aldoketoreductase family
proteins, three-dimensional structures of human placental aldose
reductase
(37) and rat liver 3
Since mouse
liver 17
Monomeric 17
The tissue
distribution of 17
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
-hydroxysteroid dehydrogenase (17
-HSD) with
A-stereospecificity in hydrogen transfer, which differs from the
B-specific dimeric enzyme of human placenta in its ability to catalyze
the oxidoreduction of xenobiotic trans-dihydrodiols of
aromatic hydrocarbons and carbonyl compounds. Here, we report the
isolation and characterization of a mouse cDNA clone encoding monomeric
17
-HSD of the liver. This clone had an entire coding region for a
protein of 323 amino acid residues with a molecular weight of 37,055.
The deduced sequence of the protein aligned with a high degree of
identity with rat and rabbit 20
-HSDs, rat and human
3
-HSD/dihydrodiol dehydrogenases, and bovine prostaglandin F
synthase, which are members of the aldoketoreductase family, but was
distinct from human 17
-HSD and carbonyl reductase, members of the
short chain dehydrogenases. The expression of the cDNA in
Escherichia coli resulted in synthesis of a protein that was
active toward androgens, estrogens, and xenobiotic substrates. The
recombinant and mouse liver 17
-HSDs also exhibited low 20
-HSD
activity toward progestins, which is similar to bifunctional activity
of human placental 17
-HSD. Therefore, the mouse enzyme was given
the designation of estradiol 17
-dehydrogenase (A-specific).
Northern analysis of mouse tissues revealed the existence of a single
1.7-kilobase 17
-HSD mRNA species in the liver, kidney, testis, and
stomach. The liver mRNA content was considerably more abundant than
those found in the other tissues, as 17
-HSD protein was mainly
detected in the liver by Western analysis.
-Hydroxysteroid dehydrogenase (17
-HSD)
(
)
catalyzes the reversible oxidation of 17
-hydroxy group
of estrogens and androgens via a pyridine nucleotide-dependent reaction
and is implicated in the biosynthesis and metabolism of the steroid
hormones. The enzyme activity is distributed in both microsomal and
cytosolic fractions of various mammalian tissues including endocrine
tissues and is mediated by enzymes that differ in cofactor and
substrate preferences and ratios of oxidation and reduction
(1) . For example, human placenta contains soluble and
microsomal 17
-HSD isoenzymes. Although the two isoenzymes favor
NAD
-dependent oxidation of the hydroxysteroids and
exhibit 20
-HSD activity for progestins, the soluble enzyme is a
68-kDa dimeric protein highly specific for estrogens
(2, 3) , whereas the microsomal enzyme accepts both
estrogens and androgens as substrates
(4) . Recently, another
microsomal 17
-HSD isoenzyme, which catalyzes the NADPH-dependent
reduction of androst-4-ene-3,17-dione to testosterone, has been
identified in human testis
(5) , and a cDNA encoding porcine
endometrial and renal 17
-HSD with a molecular mass of 80 kDa has
been cloned
(6) . Despite the differences of their catalytic
and/or molecular properties, the amino acid sequences deduced from
cDNAs for these human and porcine 17
-HSDs are structurally related
to one another
(4, 5, 6) and belong to the
short chain dehydrogenase superfamily
(7) .
-HSDs
with molecular masses around 35 kDa, which are distinct from the
enzymes of men and pigs. First, the monomeric enzymes prefer NADP(H) to
NAD(H) as the cofactor and androgens to estrogens as the substrates,
and show A-stereospecificity in hydrogen transfer between the cofactors
and substrates
(14, 15) , which differs from the
B-stereospecific enzyme of human placenta
(2) . Second, they
exhibit additional activities of dihydrodiol dehydrogenase and carbonyl
reductase toward nonsteroidal substrates
(9, 11, 13, 16) . Third, monomeric
17
-HSD exists in multiple forms with different charges in these
animal liver cytosols
(8, 9, 10, 11, 12, 13) .
Rabbit liver 17
-HSD shows an age-dependent change in its
multiplicity
(17) , which is distinct from that of the kidney
enzyme
(1) . The appearance of the multiple forms of the guinea
pig enzyme is also different in the liver and kidney
(10, 18) , and the enzyme activity in the kidney is
altered by administration of androgens
(19) . In mouse tissues,
the cytosolic enzyme activity was detected in the liver and testis
(20) , and three multiple forms of the enzyme have been isolated
from the liver
(13) . These findings have suggested that the
expression of monomeric 17
-HSD undergoes hormonal and/or
tissue-specific regulation.
-HSD cDNA was cloned
in the present study to determine its relationship to the human and
porcine 17
-HSDs and functionally related oxidoreductases,
dihydrodiol dehydrogenase and carbonyl reductase (previously
sequenced), and to facilitate future work on its multiplicity,
structure-function analysis, and gene regulation. Its successful
cloning and expression show that this mouse enzyme is a
17
(20
)-HSD with primary structure distinct from the human and
porcine enzymes.
Materials
The gt11 mouse liver cDNA library
was obtained from Clontech Laboratories; salmon sperm DNA, and lysyl
endopeptidase were from Wako Pure Chemical Industries (Osaka, Japan);
and Escherichia coli cells and pBluescript II SK(+) were
from Stratagene. Restriction and DNA-modifying enzymes were purchased
from Nippon Gene (Tokyo, Japan) and Takara Shuzou (Kyoto, Japan).
Steroids and protein standards were obtained from Sigma, and
zearalenone was from Makor Chemicals (Jerusalem, Israel).
trans-Benzene dihydrodiol was synthesized as described by
Platt and Oesch
(21) . The three multiple forms (ED1, ED2, and
ED3) of mouse liver 17
-HSD were purified to homogeneity, and
rabbit antibodies against the purified ED2 were prepared as described
previously
(13) . All other chemicals were of the highest grade
that could be obtained commercially, unless otherwise specified.
Library Screening and DNA Sequencing
The anti-ED2
IgG was used as the probe for cDNA cloning from the gt11 mouse
liver cDNA library. Recombinant phages coding for ED2 antigen were
identified by the plaque screening method
(22) using E.
coli Y1090 as the host, and immunopositive phage plaques were
detected as described previously
(23) .
terminator cycle
sequencing kit (Applied Biosystems, Japan) according to the dideoxy
chain termination method
(24) .
Figure 1:
Partial restriction map and sequencing
strategy for cDNA clones of C1, C9, and C10. The darkbox and solidline indicate the open reading frame
and 3`-untranslated region, respectively. The arrows denote
the length and direction of DNA sequencing.
Expression of 17
The cDNA
(C10) was subcloned into expression vector pKK223-3 (Pharmacia
Biotech Inc.) at the EcoRI and HindIII restriction
sites, and the construct, pKKML17DH, was then transfected into the host
strain E. coli JM109 by the heat-shock method
(24) . To
express recombinant 17-HSD in E. coli
-HSD (r17
-HSD), the E. coli cells transfected with pKKML17DH were cultured in LB medium
containing ampicillin (50 µg/ml) at 37 °C until the absorbance
at 600 nm reached 0.4. Then
isopropyl-1-thio-
-D-galactopyranoside (1 mM) was
added to induce transcription, and growth of the culture continued for
4 h at 37 °C. For determining time-dependent expression of
r17
-HSD, aliquots (3 ml) were taken from a 100 ml of culture of
the cells at time intervals, and the cells were collected by
centrifugation at 12,000
g for 5 min at 4 °C. The
cell paste was suspended in 0.5 ml of 50 mM Tris-HCl, pH 8.0,
containing 2 mM EDTA, 0.1% Triton X-100, and lysozyme (0.1
mg/ml); incubated for 15 min at 30 °C; and then sonicated for 90 s
at 4 °C. The sample was centrifuged at 12,000
g for 15 min at 4 °C, and the supernatant (cell extract) was
analyzed for 17
-HSD activity and protein. The protein
concentration was determined by the method of Lowry et al.(25) using bovine serum albumin as the standard.
-HSD, the cell extract was prepared from a
2-liter culture of the expression induced E. coli JM109 cells
as described above. r17
-HSD was purified by the method for the
purification of mouse liver 17
-HSD
(13) , except that
Matrex Red A (Amicon) was employed instead of blue Sepharose. The
enzyme fraction was applied to a Matrex Red A column (0.8
5 cm)
equilibrated with 5 mM Tris phosphate buffer, pH 7.4,
containing 5 mM 2-mercaptoethanol and 0.5 mM EDTA.
The column was washed with 20 mM Tris-HCl, pH 8.5, containing
5 mM 2-mercaptoethanol, 0.5 mM EDTA, and 0.1
M NaCl, and eluted with the buffer plus 0.5 mM
NADP
.
Enzyme Assay
17-HSD activity was assayed with
0.1 mM testosterone and 0.25 mM NADP
as the substrate and cofactor, respectively, by measuring the
NADPH fluorescence at 445 nm (excitation at 340 nm)
(12, 13) . The reductase activity of the enzyme was
determined spectrophotometrically by recording the NADPH oxidation in
the reaction mixture (2.0 ml), which consisted of 0.1 M
potassium phosphate buffer, pH 6.0, various concentrations of steroids,
0.1 mM NADPH, and the enzyme. The reaction was initiated by
the addition of the enzyme solution. One unit of the enzyme activity
was defined as the amount that catalyzes the production or oxidation of
1 µmol of NADPH/min at 25 °C.
Western and Northern Analyses
Brain, lung, heart,
liver, spleen, stomach, small intestine, kidney, and testis were
excised from 5-week-old male ddY mice, and the 105,000 g supernatants of the tissue homogenates were prepared as described
previously
(13) . The supernatant was subjected to Western
immunoblot analysis using the antibodies against ED2
(26) .
SSC (1
SSC: 0.15 M NaCl, 15 mM sodium citrate, pH
7.0). Prehybridization was carried out in 10 mM sodium
phosphate, pH 6.5, containing 50% (v/v) formamide, 0.5% SDS, 5
SSC, 10
Denhardt's solution, and 0.1 mg/ml sonicated and
denatured salmon sperm DNA at 65 °C for 4 h. The blot was then
hybridized with the digoxigenin-labeled RNA probe, which corresponded
to the cDNA clone (C10), in the solution at 65 °C for 18 h. The
preparation of the digoxigenin-labeled RNA probe using a digoxigenin
luminescent detection kit (Boehringer Mannheim) and detection of the
probe were performed as described by the manufacturer.
Protein Sequencing
The purified mouse liver
17-HSD was digested with lysyl endopeptidase after the reductive
pyridylethylation
(23) . The peptide fragments were separated by
reverse phase high pressure liquid chromatography, and then the
isolated peptides were sequenced by automated Edman degradation using a
473A protein sequencer (Applied Biosystems, Japan) as described
previously
(23) .
Sequence Comparison
DNA sequence data were stored
and analyzed by a computer program, DNASIS (Hitachi Soft Engineering,
Osaka, Japan). The amino acid sequence deduced from the cDNA sequence
was compared against all the sequences in the NBRF (PIR R35.0) and EMBL
(SWISS-PROT R24.0) data bases using the program.
Other Analytical Methods
SDS-polyacrylamide gel
electrophoresis on a 12.5% polyacrylamide slab gel
(28) and
isoelectric focusing on a 7.5% polyacrylamide disc gel
(29) were performed as described. The proteins in the gels were
stained with Coomassie Brilliant Blue R-250. The molecular mass of
r17-HSD was estimated by gel filtration on a Sephadex G-100 column
in 5 mM Tris phosphate buffer, pH 7.4, containing 0.15
M KCl. The products of the enzymatic reaction were extracted
with ethyl acetate from the reaction mixture, analyzed on thin-layer
chromatography, and identified by comparing their
R
values with the authentic steroids,
indan-1-one and catechol, as described previously
(9) .
Cloning and cDNA Sequence
Approximately 4.5
10
plaques of the
gt11 mouse liver cDNA
library were screened using the polyclonal antibodies against one of
multiple forms of mouse liver 17
-HSD, ED2, that cross-reacted with
the other multiple enzyme forms
(13) . Fourteen immunopositive
clones were purified from the cDNA library, but only nine clones with
cDNA inserts of 0.9-4.3 kilobases could be subcloned into the
pBluescript plasmids. When about 250 base pairs (bp) of both the 5`-
and 3`-ends of cDNA fragments of the clones were sequenced, three
clones (C1, C9, and C10) had similar coding regions for internal amino
acid sequences of ED2, which were known by sequencing the peptides
derived from the purified enzyme (). The three clones were
subjected to sequence determination (Fig. 1). The C10 covered 969
bp of the open reading frame, starting with an ATG codon and
terminating at a TAA stop codon, with a 125-bp 3`-noncoding region
where neither a polyadenylation hexamer nor poly(A) tail was observed.
Although the other clones, C1 and C9, lacked 363 and 459 bp,
respectively, of the 5`-end of open reading frame of C10 clone, their
remaining sequences of the coding region and the 125-bp 3`-noncoding
region were identical to those of C10 clone, and their 3`-noncoding
regions, longer than that of C10 clone, were the same sequences, which
contained one polyadenylation hexamer and poly(A) tail. The nucleotide
sequence for mouse liver 17
-HSD cDNA (Fig. 2) is shown by
combining the two sequences of C10 and C9. The coding region of the
cDNA translates to a 323-residue protein chain that predicted a
molecular weight of 37,055. All of the sequences obtained by sequencing
the peptide fragments of ED2 () perfectly matched the
regions of the amino acid sequence deduced from the cDNA.
Figure 2:
Nucleotide and deduced amino acid
sequences of the cDNA inserts of clone C9 and C10. The amino acid is
placed below the central nucleotide of its codon. The stop codon (TAA)
is labeled with an asterisk. The 3`-noncoding region of C10
terminated at nucleotide 1094, after the sequence from C9 (indicated by
brokenunderlining) was combined. EcoRI
linkers located at either end of these inserts are not shown. The
polyadenylation signal sequence is indicated by boldface letters. There are several putative phosphorylation sites for
tyrosine kinase (= = = =), protein kinase C
(
), and Ca
-calmodulin protein
kinase (# # # #).
Expression of r17
The cDNA (C10) was
expressed in E. coli to determine which of the three multiple
forms (ED1, ED2, and ED3) of mouse liver 17-HSD
-HSD is encoded in this
cDNA. The extract of E. coli transfected with the pKKML17DH
construct contained a 36-kDa immunoreactive band when tested with the
antibodies against ED2 (Fig. 3 A), and demonstrated
17
-HSD activity that was not detected in the extract of E.
coli transfected only with the vector (pKK223-3). The enzyme
activity reached the maximum level (5 ± 1 milliunits/mg) at 4 h
of incubation of the cells.
Figure 3:
Western blot analysis and
SDS-polyacrylamide gel electrophoresis of the cell extracts and the
purified r17-HSD. A, Western analysis using the
antibodies against mouse liver ED2. The extract (47 µg of protein)
of E. coli cells transfected with pKKML17DH ( lane1) and the purified r17
-HSD (1 µg, lane2) were analyzed. B, SDS-polyacrylamide gel
electrophoresis of the cell extract ( lane1), the
purified r17
-HSD ( lane2), and mouse liver ED2
( lane3). The positions of the molecular mass
standards were indicated at the left of the respective
panels.
r17-HSD was purified to
electrophoretic homogeneity (), showing a single 36-kDa
protein on SDS-polyacrylamide gel electrophoresis
(Fig. 3 B). r17
-HSD also had a molecular mass of 35
kDa on gel filtration and a pI value of 7.1 by gel isoelectric focusing
analysis, which was lower than those of native 17
-HSDs (pI 7.8,
8.1, and 8.5 for ED1, ED2, and ED3, respectively, Ref. 13). The
N-terminal sequence of 12 residues determined for r17
-HSD was the
same as that deduced from the cDNA. The specific activity of
r17
-HSD was comparable with those of ED1 and ED2 (2.3 and 2.2
units/mg, respectively) but was much lower than that of ED3 (13
units/mg). r17
-HSD, ED1, and ED2 showed similar kinetic constants
for steroidal and xenobiotic alcohols, which differed from those of ED3
(I). r17
-HSD was inhibited by hexestrol, stilbestrol,
and zearalenone, giving IC
(concentrations required for
50% inhibition) values of 4, 18, and 40 µM, respectively.
The IC
values are comparable with those obtained with ED1
and ED2 but higher than those of ED3
(13) .
Sequence Homologies
The amino acid sequence
deduced from the cDNA for mouse liver 17-HSD did not show
significant similarity with those of the human and porcine 17
-HSDs
(4, 5, 6) , but it shared 79, 69, 76, 75, 71,
and 71% identity with rabbit ovary 20
-HSD
(30) , rat ovary
20
-HSD
(31) , human bile acid binder/dihydrodiol
dehydrogenase
(32) , human liver 3
-HSD/dihydrodiol
dehydrogenase
(27, 33) , rat liver 3
-HSD
(34) , and bovine lung prostaglandin F synthase
(35) ,
respectively (Fig. 4). These highly homologous proteins are
members of the aldoketoreductase family, and a slightly low degree of
identity (49%) was also observed between mouse 17
-HSD and human
aldose reductase
(36) . The mouse 17
-HSD sequence conserved
several functionally important amino acid residues that have been
suggested or demonstrated by site-directed mutagenesis and
crystallographic studies of aldose reductase
(37, 38, 39) and rat liver 3
-HSD
(40) . Although mouse liver
17
-HSD exhibits dihydrodiol dehydrogenase and carbonyl reductase
activities
(13) , there is no significant homology between the
17
-HSD and human carbonyl reductase
(41) or bacterial
cis-dihydrodiol dehydrogenases
(42) , which are members
of the short chain dehydrogenase family.
Figure 4:
Comparison of amino acid sequences among
mouse 17-HSD and other oxidoreductases. The deduced amino acid
sequence of rat liver 3
-HSD ( rl 3hd) is aligned with
those of mouse liver 17
-HSD ( ml 17hd), human liver bile
acid binder/dihydrodiol dehydrogenase ( hl bbdd), human liver
3
-HSD/dihydrodiol dehydrogenase ( hl 3hd), rat ovary
20
-HSD ( ro 20hd), and rabbit ovary 20
-HSD ( rb
20hd). Identical residues are enumerated with dashes. The
boldfaceitalic residues on the rat liver 3
-HSD
have been reported to be implicated in substrate binding
(40).
Since mouse 17-HSD
exhibited high homology with the 20
-HSDs, 3
-HSDs, and
prostaglandin F synthase, we examined 3- and 20-hydroxysteroids,
prostaglandin E
, and prostaglandin D
as
substrates for r17
-HSD and native 17
-HSDs of mouse liver. Of
the substrates, 5
-pregnan-20
-ol-3-one and
pregn-4-en-20
-ol-3-one were slowly oxidized by the mouse enzymes
(I). In the reverse reaction, r17
-HSD and ED2 reduced
progesterone to pregn-4-en-20
-ol-3-one. The
K
values of the recombinant and native
enzymes were 2.2 and 2.5 µM, respectively, and the
respective k
values were 2.5 and 1.6
min
.
Tissue-specific Expression
By Northern blot
analysis of total RNAs of several mouse tissues, 17-HSD mRNA was
detected as a single 1.7-kilobase mRNA species in liver, kidney,
testis, and stomach. Although not quantified, the amount of 17
-HSD
mRNA in the liver was substantially greater than that in the kidney
(Fig. 5 A). Of the tissues examined by Western blot
analysis using the antibodies against ED2, an immunopositive 36-kDa
protein was detected highly in the liver and faintly in the kidney,
testis, and stomach, of which kidney showed an additional 38 kDa band
(Fig. 5 B). No immunoreactive band was detected in brain,
heart, lung, spleen, and small intestine (results not presented).
Figure 5:
Tissue
distribution of mouse 17-HSD. The samples from brain ( a),
heart ( b), lung ( c), liver ( d), kidney
( e), testis ( f), spleen ( g), small intestine
( h), and stomach ( i) were analyzed. A,
Northern hybridization with the digoxigenin-labeled RNA probe. The
amount of total RNA analyzed was 20 µg. Mobility of RNA size
markers is indicated at the left of this panel. B,
Western blot analysis with the anti-ED2 IgG. The 105,000
g supernatants (each 5 µg) of the selected tissues, ED2 (1
µg, laneE) and the recombinant 17
-HSD (1
µg, laneR) were
analyzed.
-HSD/
-
-isomerases comprise
another protein family
(43) . While human and porcine
17
-HSDs
(4, 5, 6) , human and rat
11
-HSDs
(44) , pig testis 20
-HSD
(45) ,
Streptomyces hydrogenans 3
(20
)-HSD
(46) , and
Pseudomonas testosteroni 3
-HSD
(47) belong to the
former family, human and rat 3
-HSDs
(23, 33, 34) and 20
-HSDs of some mammals
(30, 31, 48) are members of the latter family. The present study
demonstrated that mouse liver 17
-HSD with A-stereospecificity in
hydrogen transfer between cofactor and substrate belongs to the
aldoketoreductase family. This suggests that the classification of HSDs
based on their structural bases may reflect the difference in their
stereospecificity in hydrogen transfer rather than that in their
regiospecificity for steroidal substrates, because most HSDs of the
short chain dehydrogenase family are B-specific
(2, 49, 50) and the HSDs of the aldoketoreductase family are all
A-specific
(49, 51) .
-HSD
(40) have been
determined. The two enzymes, despite their sequence identity of 58%,
have been shown to contain almost the same
/
barrels, and
residues present at their active sites have been identified. The mouse
17
-HSD sequence conserves 14 of 18 residues involved in binding
NADPH to human aldose reductase and catalytically important residues
(Tyr-55, Lys-84, and Asp-50) of rat 3
-HSD. Therefore, the reaction
catalyzed by mouse 17
-HSD may follow the same mechanism of
catalysis proposed for rat 3
-HSD. On the other hand, despite high
sequence similarity among 3
-, 17
-, and 20
-HSDs in this
family, their specificity for steroid substrates are clearly distinct.
This may result, in part, from the differences in residues, which
recognize the steroidal molecules, among the HSDs. Eight residues have
been proposed to be implicated in binding substrate to rat liver
3
-HSD
(40) . Although mouse liver 17
-HSD conserves
five of the eight residues, Leu-54, Phe-128, and Phe-129 of rat liver
3
-HSD sequence are replaced by Met, Tyr, and Leu, respectively, in
the 17
-HSD sequence. The residues at positions 128 are especially
diverse among rat and human 3
-HSDs, rat and rabbit 20
-HSDs,
and mouse 17
-HSD (Fig. 4). In addition, a marked residue
difference among the HSDs is seen in the region composed of residues
306-310. The C-terminal part has been shown to be implicated in
substrate binding for aldose reductase
(39) .
-HSD accepts androgens and estrogens as substrates, it has
been called estradiol 17
-dehydrogenase
(13) . The present
observation that the mouse enzyme is 17
(20
)-HSD is similar to
the bifunctional activity of human placental 17
-HSD, which is also
designated as estradiol 17
-dehydrogenase (EC 1.1.1.62). However,
the mouse and human enzymes clearly differ from each other with respect
to the primary structure and stereospecificity in hydrogen transfer.
The steroid specificity of the mouse 17
-HSD is different from
those of NAD
- and NADP
-dependent
testosterone 17
-dehydrogenases (EC 1.1.1.63 and EC 1.1.1.64),
20
-HSD (EC 1.1.1.149), 3
(20
)-HSD (EC 1.1.1.210), and
3
(17)
-HSD (EC 1.1.1.51). Thus, the mouse liver 17
-HSD
cannot be classified into the HSDs listed in the most recent
recommendation of the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology
(52) . In our opinion, the
trivial name of estradiol 17
-dehydrogenase (A-specific) is
suggested for this mouse liver enzyme to distinguish it from the known
estradiol 17
-dehydrogenase (B-specific) (EC 1.1.1.62).
-HSD exists as multiple forms in guinea pig,
rabbit, and mouse livers. Seven multiple forms of the guinea pig enzyme
have been shown to comprise at least two isoenzymes with different
amino acid compositions
(10) , whereas four multiple forms of
the rabbit enzyme are thought to result from post-translational
modification of the expressed enzyme
(8, 17) . The three
multiple forms (ED1, ED2, and ED3) of mouse 17
-HSD differ in
charges, kinetic constants for the substrates, and inhibitor
sensitivity but are immunologically indistinguishable
(13) .
Although the present cDNA probably encodes ED1 or ED2, the Northern
blot analysis revealed the existence of a single 1.7-kilobase
17
-HSD mRNA species in mouse liver. In addition, our sequence
analysis of ED3-derived peptides showed that its partial sequence (of
total 75 amino acids) matched the regions of the amino acid sequence
deduced from the isolated cDNA.
(
)
These results
suggest that the multiplicity of the mouse enzyme is due to the
post-translational modification of one enzyme protein from a single
gene. Of various reactions in the post-translational modification of
proteins
(53) , phosphorylation might be involved in the
formation of the multiple forms of the mouse enzyme, because the
sequence deduced from the cDNA has nine potential sites of
phosphorylation by protein kinase C, tyrosine kinase, and
Ca
-calmodulin kinase II (Fig. 2).
-HSD mRNA and its protein, together with that of
its activity in mice
(20) , suggests that the expression of
17
-HSD activity is regulated at the transcriptional level and/or
by tissue-specific effects on mRNA stability. In addition, the
expression of 17
-HSD might be regulated at the translational level
because the enzyme protein content in the kidney was very low despite
the considerable amount of the enzyme mRNA in the tissue. The tissue
distribution of monomeric 17
-HSD is different from those of human
and porcine 17
-HSDs
(4, 5, 6) . Considering
the previous works on the functions and structures of 17
-HSD, the
present study provides a supposition that two structurally distinct
types of 17
-HSD are tissue-specifically expressed in mammals; the
short chain dehydrogenase type of the enzyme may be expressed in
endocrine tissues and implicated in the steroid metabolism, whereas the
aldoketoreductase type of the enzyme may be expressed mainly in liver
and functions in the metabolism of both steroids and xenobiotics.
Table:
Sequence data of the peptides derived from
lysylendopeptidase digestion of a multiple form (ED2) of mouse liver
17-HSD
Table:
Purification
of r17-HSD
Table:
Comparison of kinetic constants for steroids
and xenobiotics among r17-HSD and native mouse liver 17
-HSDs
(ED1-ED3)
/EMBL Data Bank with accession number(s)
D45850.
-HSD, recombinant
17
-hydroxysteroid dehydrogenase; bp, base pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.