©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of Mouse Estradiol 17-Dehydrogenase (A-Specific), a Member of the Aldoketoreductase Family (*)

Yoshihiro Deyashiki , Kiyoshi Ohshima , Masayuki Nakanishi , Kumiko Sato , Kazuya Matsuura , Akira Hara (§)

From the (1) Biochemistry Laboratory, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Several mammalian livers contain monomeric 17-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.


INTRODUCTION

17-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) .

Livers of rabbits (8, 9) , guinea pigs (10, 11) , and mice (12, 13) contain cytosolic and monomeric 17-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.

Mouse liver 17-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.


EXPERIMENTAL PROCEDURES

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) .

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 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-HSD in E. coli

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 (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.

For the purification of r17-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) .

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 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 Rvalues with the authentic steroids, indan-1-one and catechol, as described previously (9) .


RESULTS

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-HSD

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 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 Kvalues 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.




DISCUSSION

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-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) .

Of the aldoketoreductase family proteins, three-dimensional structures of human placental aldose reductase (37) and rat liver 3-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) .

Since mouse liver 17-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).

Monomeric 17-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).

The tissue distribution of 17-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)



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D45850.

§
To whom correspondence should be addressed: Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502, Japan. Tel.: 81 58 237 3931; Fax. 81 58 237 5979.

The abbreviations used are: HSD, hydroxysteroid dehydrogenase; r17-HSD, recombinant 17-hydroxysteroid dehydrogenase; bp, base pair(s).

K. Sato and K. Ohshima, unpublished results.


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