Characterization of Structural and Functional Properties of Human 17ß-Hydroxysteroid Dehydrogenase Type 1 Using Recombinant Enzymes and Site-Directed Mutagenesis

Terhi Puranen, Matti Poutanen, Debashis Ghosh, Pirkko Vihko and Reijo Vihko

Biocenter Oulu and Department of Clinical Chemistry (T.P., M.P., P.V., R.V.) University of Oulu FIN-90220 Oulu, Finland
Hauptman-Woodward Medical Research Institute, Inc. (D.G.) Buffalo, New York 14203
Roswell Park Cancer Institute (D.G.) Buffalo, New York 14263


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human 17ß-hydroxysteroid dehydrogenase (17-HSD) type 1 catalyzes the conversion of the low activity estrogen, estrone, into highly active estradiol, both in the gonads and in target tissues. The present study was carried out to characterize the dimerization, microheterogeneity, and phosphorylation of human 17-HSD type 1 and to evaluate the current model of hydride transfer and substrate recognition of the enzyme, based on its x-ray structure. 17-HSD type 1 is a homodimer consisting of noncovalently bound subunits, and the data in the present study indicate an exceptionally strong association between the monomers [dissociation constant (Kd) < 5 pmol/liter]. Furthermore, substitutions constructed at the hydrophobic dimer interface always resulted in inactive aggregates of the protein. The enzyme was shown to be phosphorylated by protein kinase A exclusively at Ser134 only in vitro. However, in contrast to previous suggestions, phosphorylation of Ser134 was shown to play no role in the activity or microheterogeneity of human 17-HSD type 1. The presence of microheterogeneity in the recombinant enzyme also indicates that it does not result from the frequent protein polymorphism previously found for the enzyme. In line with the x-ray structure and the proposed catalytic mechanism of the enzyme, our results indicate that Ser142, Tyr155, and Lys159 are all critical for hydride transfer in human 17-HSD type 1. In contrast, the proposed interaction between His221, Glu282, and the 3-OH group of the steroid at the substrate recognition helix could not be shown to exist. Neither of these residues plays a critical role in the catalytic action of the enzyme in cultured cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
17ß-Hydroxysteroid dehydrogenase (17-HSD) enzymes catalyze the interconversions between 17-keto- and 17-hydroxysteroids, thus playing an important role in the biosynthesis as well as metabolism of steroid hormones. Currently, four different human 17-HSD enzymes have been described (1, 2, 3, 4, 5). The enzymes differ from each other in their tissue distribution, substrate specificities, and in subcellular localization, indicating that they possess different physiological functions in humans. However, all the enzymes belong to the short-chain dehydrogenase/reductase (SDR) protein family (6), also called the short-chain alcohol dehydrogenase superfamily (7, 8).

Human 17-HSD type 1 mainly catalyzes the reduction of estrone to a biologically more active estrogen, estradiol (9, 10), both in steroidogenic tissues (11, 12, 13) and in certain estrogen target tissues, such as healthy and malignant breast and endometrial tissues (14, 15, 16, 17). It is therefore suggested that the enzyme might have a significant role in the regulation of estrogen exposure and estrogen-dependent growth of breast cancer tissue. Hence, 17-HSD type 1 inhibitors could be a new potential approach in blocking estradiol biosynthesis, both in the gonads and in target tissues.

It has been shown that human 17-HSD type 1 protein is a dimer consisting of identical subunits of 35 kDa in size (18, 19, 20, 21). The protein has been purified in several laboratories (11, 18, 20, 22), but the microheterogeneity (23) and the possible role of phosphorylation in regulating enzyme activity have not been studied in detail. Based on the x-ray structure of the enzyme (21), a model for the reaction mechanism of human 17-HSD type 1 has been recently proposed. According to this model, the side chains of Ser142, Tyr155, and Lys159 residues are involved in hydride transfer by the enzyme. These amino acids are strictly conserved in the SDR family members, and results of several studies indicate that the tyrosine residue corresponding to Tyr155 in human 17-HSD type 1 is directly involved in the catalytic action of SDR members (24, 25, 26, 27, 28). Similarly, substituting arginine, isoleucine, glutamine, or leucine in place of the largely conserved lysine residue always abolishes the enzymatic activity totally (25, 26, 27, 29). The role of the conserved serine residue (Ser142 in human 17-HSD type 1) in the catalytic activity of any of the enzyme family members has not been studied so far. The x-ray structure of the bacterial 3{alpha},20ß-hydroxysteroid dehydrogenase-carbenoxolone complex, however, indicates that Ser139 forms a hydrogen bond with the hydroxyl group of strictly conserved Tyr152 (30), and could, therefore, directly facilitate proton transfer (21, 31). Structural data have also suggested that in human 17-HSD type 1, the His221 residue could have an important role in substrate recognition. The data, furthermore, indicate that the side chain of Glu282 forms a salt bridge with the side chain of His221, which could further interact with the 3-hydroxyl group of the substrate (21). In the present study, several central aspects concerning the biochemical and catalytical properties of human 17-HSD type 1, including dimerization, microheterogeneity, and phosphorylation, were characterized. In addition, site-directed substitutions were constructed to evaluate the current model of hydride transfer and substrate recognition by human 17-HSD type 1, based on the x-ray structure of the enzyme.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dimerization of Human 17-HSD Type 1
In the first stage of this study, we characterized the dimerization of purified human 17-HSD type 1 produced in Spodoptera frugiperda (Sf9) insect cells. The protein was purified to apparent homogeneity from the cytosolic fraction of Sf9 cell lysate in a two-step procedure. Analysis by SDS-PAGE (Fig. 1AGo), native PAGE (Fig. 1BGo), and Superdex 75 gel filtration column chromatography (Fig. 4CGo) confirmed that similar to human 17-HSD type 1 purified from placental tissue, the resulting recombinant protein exists as a dimer of 70–78 kDa. The structural data and the fact that a monomeric stage of the protein was observed in SDS-PAGE without ß-mercaptoethanol, indicated that the enzyme monomers were not covalently bound. Hence, to characterize the affinity between the noncovalently bound enzyme monomers, a series of human type 1 protein dilutions were analyzed by Superdex 75 gel filtration chromatography. At all the protein concentrations used, the enzyme migrated in an elution volume corresponding to a homodimer, and therefore the exact dissociation constant (Kd) of human 17-HSD type 1 could not be measured. The lowest amount of protein measured in the eluted fractions by immunofluorometric assay was 0.35 ng/ml, indicating that the Kd is less than 5 pmol/liter. Hence, it is evident that the enzyme exists predominantly as a dimer, and there is no equilibrium between monomer and dimer in vitro. The previously resolved x-ray structure of the enzyme dimer indicated that there are two dimerization helices in each monomer ({alpha}E and {alpha}F), forming a four-helix bundle. In the present study, two substitutions (Leu111GluVal113Phe and Ala170Glu+Phe172) were generated to analyze the role of the dimeric interface in the folding of an active protein. The substituted proteins eluted in the void volume in Superose 12 gel filtration column chromatography. The data, therefore, indicate that disrupting the formation of one of the two helices results in an aggregated protein of more than 300 kDa, while dimeric or monomeric forms of the enzyme could not be detected. Both of the enzymes generated were found to be inactive both in vitro (Table 1Go) and in cultured Sf9 cells (data not shown).



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Figure 1. Biochemical Properties of Purified Wild Type (lane 1) and Ser134Ala-Substituted (lane 2) Human 17-HSD Type 1 Proteins Analyzed by SDS-PAGE (A), Native PAGE (B), and IEF (C)

The electrophoreses were carried out on a PhastSystem (Pharmacia Biotech) using PhastGel gradient media of 10–15% for SDS-PAGE and 8–25% for native PAGE. IEF was performed in a pH gradient of 3–9. After electrophoreses or IEF, the proteins were visualized by silver staining.

 


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Figure 4. Biochemical Properties of Wild Type Human 17-HSD Type 1 and an Enzyme Lacking the Last 36 C-Terminal Amino Acids

Lanes 1 and 2 contain the wild type and truncated enzyme, respectively. Cytosolic proteins from the Sf9 cell homogenates were resolved by SDS-PAGE (A) and in an IEF gel with a pH gradient from 3 to 9 (B). Thereafter, the proteins were transferred onto a nitrocellulose membrane and immunostained as described by Poutanen et al. (17). Evaluation of the molecular masses of the 17-HSD type 1 proteins was carried out by measuring the Kav values using a Superdex 75 gel filtration column connected to a SMART System (C). Standards used were ribonuclease A (13.7 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), and BSA (67 kDa) with corresponding Kav-values of 0.36, 0.27, 0.16, and 0.11, respectively.

 

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Table 1. Activity Measurements of 17-HSD Type 1 Enzymes in Vitro

 
Phosphorylation of Human 17-HSD Type 1
Previous studies have shown that 17-HSD type 1 purified from the human placenta consists of three different isoforms that can be resolved by isoelectric focusing (IEF) (23). The purified recombinant enzyme yielded an identical pattern of three bands upon silver staining with isoelectric point (pI) values of 4.9, 5.0, and 5.1 (Fig. 1CGo). The primary structure of the human 17-HSD type 1 enzyme contains a potential cAMP-dependent phosphorylation site (Ser134). We therefore investigated whether phosphorylation could contribute to the catalytic and/or biochemical properties of human 17-HSD type 1, including its microheterogeneity. The data indicated that the protein was efficiently phosphorylated by protein kinase A (PKA) in vitro (Fig. 2Go). Furthermore, destroying the potential phosphorylation site by substituting alanine in place of Ser134 totally abolished phosphorylation (Fig. 2Go). The substitution, however, did not have any significant effects on the catalytic properties (Table 1Go) or microheterogeneity (Fig. 1CGo) of the purified enzyme. Our studies further indicated that the Ser134Ala-substituted and the wild type enzymes had identical enzymatic properties in cultured Sf9 cells (Fig. 3Go). Identical properties were also observed using MCF-7 human breast cancer cells transiently transfected with the cDNAs for Ser134Ala-substituted and wild type enzymes (data not shown). Furthermore, in mass spectrometric (MALDITOF) analysis of the purified recombinant enzyme, only one fragment with the correct molecular mass was observed. This indicates that no significant mass heterogeneity, resulting, for example, from differential phosphorylation states, was present. In addition, no electron density appropriate for a phosphate moiety at the Ser134 side chain was detected in the crystal structure of the enzyme. The results, therefore, indicate that human 17-HSD type 1 is phosphorylated by PKA exclusively at Ser134 in vitro, but this phosphorylation has no effect on the catalytic properties of 17-HSD type 1, and most likely it does not occur in vivo.



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Figure 2. Phosphorylation of Purified Wild Type and Ser134Ala-Substituted Human 17-HSD Type 1 Proteins by PKA in Vitro

Lanes 1 and 2 contain the wild type and Ser134Ala-substituted 17-HSD type 1 enzymes, respectively. Purified proteins were incubated with PKA, as described in Materials and Methods. The proteins (3.6 µg) were thereafter separated by SDS-PAGE and visualized by staining with Coomassie Blue (A) or by autoradiography (B).

 


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Figure 3. Estrogenic (A) and Androgenic (B) Activity of Wild Type and Substituted Human 17-HSD Type 1 Enzymes in Cultured Sf9 Insect Cells

Both estrogenic and androgenic [estrone (E1) to estradiol (E2), androstenedione (A-dione) to testosterone (T)] activities were measured in cultured Sf9 insect cells. The activities represent typical reaction curves after subtraction of the small endogenous 17-HSD activity present in Sf9 cells. The results are given as average (±SD) of triplicate specimens.

 
C-Terminal Deletion of Human 17-HSD Type 1
The structure of the C terminus of human 17-HSD type 1, consisting of the last 43 amino acids, could not be determined by x-ray crystallography (21). To find out whether the carboxy terminus of human 17-HSD type 1 plays a role in the catalytic mechanism or the microheterogeneity present in the human enzyme, we analyzed the properties of the enzyme lacking the last 36 amino acid residues (amino acids 291–327) of the C terminus. Western blot analysis showed the correct molecular mass for the monomer of the constructed protein (Fig. 4AGo), and gel filtration analysis revealed that the truncated enzyme was still present as a dimer (Fig. 4CGo) of approximately 54 kDa. The data, furthermore, indicated that deleting the last 36 amino acids did not have a dramatic effect on the catalytic properties measured in cultured Sf9 cells (Fig. 3Go), and that the truncated enzyme possessed similar microheterogeneity to that detected in the native type 1 enzyme (Fig. 4BGo).

Substitutions at the Active Site of Human 17-HSD Type 1
Recently, a model for the catalytic mechanism of 17-HSD type 1 has been proposed by utilizing the three-dimensional structure of the enzyme (21). In this model the side chains of Ser142, Tyr155, and Lys159 are involved in hydride transfer between the cofactor and 17-keto group of the substrate, and the His221 side chain is responsible for substrate recognition by the enzyme. Our previous activity measurements in vitro showed that a His221Ala substitution resulted in a marked (11-fold) decrease in reductive enzyme activity (28). Unexpectedly, our present data indicate that in cultured Sf9 cells a His221Ala substitution does not result in a decreased conversion of E1 to E2, compared with the wild type enzyme (Fig. 5Go). The Glu282 residue is in close proximity to His221 and forms a salt bridge with the histidine residue. Hence, we suggested that Glu282 could replace the function of His221, and additional substitutions at the putative substrate recognition helices (His221AlaGlu282Ala, His221AlaGlu282Gln, Glu282Ala and Glu282Gln) were constructed. After substituting alanine or glutamine in place of Glu282, no major effects on the catalytic properties of the enzyme were observed in vitro or in intact cultured cells (Table 1Go and Fig. 5Go), and simultaneous substitutions of His221 and Glu282 resulted in similar inactivation of the enzyme in vitro (Table 1Go) as observed after the substitution of His221 alone (28). However, in cultured Sf9 insect cells (Fig. 5Go), all the His221 and/or Glu282-substituted proteins catalyzed the reduction of E1 to E2 in a manner similar to that observed for the wild type enzyme, which indicates that neither His221 nor Glu282 is critical for substrate recognition in vivo.



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Figure 5. Estrogenic 17-HSD Activity of Wild Type and Substituted 17-HSD Type 1 Enzymes Measured in Cultured Sf9 Insect Cells

Conversion of estrone to estradiol was measured in cultured Sf9 insect cells, by the wild type (wt) and substituted enzymes. Substitutions were generated at the potential substrate recognition site (Glu282Ala, Glu282Gln, His221Ala, His221AlaGlu282Ala and His221AlaGlu282Gln) and at the amino acids putatively involved in hydride transfer (Tyr155Ala, Lys159Ala, and Ser142Ala). The results are given as the average (±SD) of triplicate specimens, with a reaction time of 20 min, after subtraction of the small endogenous 17-HSD activity present in the cells. Conversion of E1 to E2 was calculated according to the amount of enzyme expressed.

 
We have previously shown the critical importance of Tyr155 for the activity of the enzyme in vitro, and similarly, the results of the present study indicated that substituting alanine in place of either Ser142 or Lys159 results in almost inactive enzyme, both in vitro (Table 1Go) and in cultured Sf9 cells (Fig. 5Go). The catalytic efficiencies [turnover rate (kcat)/Michaelis-Menten constant (Km)] of the substituted enzymes were more than 200-fold lower for both reduction and oxidation (E1 {leftrightarrow} E2) compared with the values measured for the wild type enzyme. This indicates that these amino acids have important roles in the catalytic mechanism of human 17-HSD type 1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent results suggest that in addition to being essential for glandular estradiol production, 17-HSD type 1 is also involved in the process leading to relatively high estradiol concentrations in some estrogen target tissues, such as breast cancer tissues (32, 33). Hence, much interest has been focused on the possible use of 17-HSD type 1 inhibitors in decreasing both endocrine and intracrine estradiol production, and further, the use of inhibitors in the prevention and/or treatment of estrogen-dependent breast cancer (34). Recently, the x-ray structure of human 17-HSD type 1 has been resolved, giving the possibility of rationally designing structure-based inhibitors (21). In the present study the structural data have been used together with site-directed mutagenesis to further resolve the structural and functional properties of recombinant human 17-HSD type 1. These studies are part of our work concerned with evaluating the properties of 17-HSD enzymes, which, in turn, facilitates the design of 17-HSD inhibitors.

In line with the results of previous studies (20, 35), the human 17-HSD type 1 protein was found to be expressed exclusively as a homodimer. Our present results further indicate a strong association between the subunits, resulting in a stable dimerization state of the enzyme. Structural data (21) and the activity measurements reported in the present study indicate that the substitutions Leu111GluVal113Phe and Ala170Glu+Phe172 disrupt the dimer interface of human 17-HSD type 1. When these substitutions were introduced in a three-dimensional structural model of human 17-HSD type 1 (21), Leu111Glu substitution was seen to result in a position in which two negatively charged side chains approach each other in close proximity, and Val113Phe substitution generated steric conflict with the region between residues Ala91 to Leu93 of strand ßD, as well as with the adenosine moiety of the cofactor. Ala170 resides in a hydrophobic pocket that contains Val276 from a neighboring subunit and the Leu251 side chain. The pocket is at a region close to the dimer interface, stabilizing the interior of the molecule. Hence, both of the substitutions generated are harmful with respect to the activity and folding of the enzyme. The fact that disrupting the dimerization of these helices results in totally inactive aggregates and no monomeric forms were observed is in line with the highly hydrophobic nature of their outer surfaces. Construction of a monomeric form of the enzyme might require a change in the character of the outer surfaces of these helices, which in turn might result in improper folding of the enzyme. In addition, it has been suggested that the neighboring amino acids of the conserved tyrosine and lysine residues in the active site of SDR family members might interact with the hydrophobic outer surface of the {alpha}F-helix and hence stabilize the dimer interface of the proteins (36). The close proximity of these residues to the catalytic site responsible for hydride transfer in the enzymes, together with our results, suggests that monomerization of the dimeric or tetrameric proteins in the SDR family most likely abolishes their enzyme activity.

Purified recombinant human 17-HSD type 1 yielded a pattern of three isoforms resolved by IEF. This disproves the assumption that the microheterogeneity previously also detected in type 1 enzyme purified from human placenta (23) would be a result of the frequent protein polymorphism reported in 17-HSD type 1 (37, 38). The primary structures of human, rat (39), and mouse (40) 17-HSD type 1 proteins contain a potential cAMP-dependent phosphorylation site (Ser134). The three-dimensional structure of the human enzyme revealed that Ser134 is exposed at the turn between helix {alpha}E and strand ßE and could be conducive to phosphorylation. The results of the present study, together with previous findings (41), indicate that human 17-HSD type 1 is phosphorylated exclusively at Ser134 by PKA in vitro. However, in our study no indication of a phosphate moiety at Ser134 was detected by x-ray diffraction. Furthermore, destroying the potential phosphorylation site by substituting alanine in place of Ser134 totally abolished phosphorylation but did not affect the enzymatic properties of the enzyme. These data are in line with the fact that Ser134 is not near the catalytic site or at the dimer interface. We therefore conclude that phosphorylation of Ser134 does not have a critical role in the function of human 17-HSD type 1. The results also clearly indicate that microheterogeneity of the enzyme does not result from the different phosphorylation states of the Ser134 residue. The reason for the discrepancy between our detailed data and those previously described by Barbieri et al. (41) is not known. Their data indicated that treating BeWo cell lysates with alkaline phosphatase decreased 17-HSD enzyme activity and that, in addition to serine, threonine residues were also slightly phosphorylated. However, in the mass-spectrometric analysis carried out in the present study, such post-translational modifications were not detected. Furthermore, three isoforms resolved by IEF showed identical densities after silver staining (Fig. 1CGo). This indicates a similar concentration of each of the isoforms, suggesting that they are not a result of weak phosphorylation.

The last 43 amino acids of the C terminus of human 17-HSD type 1 could not be resolved in the three-dimensional structure of the enzyme (21). We therefore hypothesized that the C terminus of the enzyme is highly flexible and could form several conformations with differential charges at the outer surface, and thereby could be responsible for the microheterogeneity observed. However, the results showed that the last 36 amino acids of the enzyme do not play any role in the microheterogeneity or catalytic activity of human 17-HSD type 1. In addition, the results of the present study exclude the possibility that fast purification procedures could eliminate the microheterogeneity of the protein, as suggested by Yang et al. (42). In addition, the absence of carbohydrates in the enzyme (18) rules out this source of heterogeneity. The reasons for and functional importance, if any, of the charge differences in human 17-HSD type 1 remain to be clarified. Asparagine and glutamine residues may, for example, undergo deamination, which might not be detectable by mass spectrometry.

In the present study we also evaluated the model for substrate recognition and hydride transfer proposed from the x-ray structure of the enzyme. We constructed several site-directed substitutions at the potential substrate recognition helices of the enzyme. Even though the x-ray structure of the enzyme suggests that the side chain of His221 is involved in substrate recognition, our results indicate that the residue does not have a critical role in substrate binding in vivo. Since the three-dimensional structure of 17-HSD type 1 was determined from the solubilized enzyme in vitro, interaction of the 3-hydroxy group of the substrate with the His221 side chain could possibly occur in vitro only, which is in agreement with our activity measurements in vitro. The nature of the substrate-protein interaction may, however, be modified in vivo, by a membrane association near the active site region. The fact that glycerol or other ampholytes are needed to retain the catalytic activity of the enzyme in vitro is an additional indication of the hydrophobic interaction needed for an active enzyme. Based on the structure of the enzyme, one of the most probable candidate residues able to replace the function of His221 was Glu282. However, the present results indicate that there is no significant interaction between Glu282 and the 3-hydroxy group of the substrate either in vitro or in intact cultured cells.

The current data indicate that Ser142, Lys159, and Tyr155 residues are all essential for the activity of human 17-HSD type 1, which perfectly matches the proposed hydride transfer mechanism (21). With respect to the tyrosine and lysine residues, the results of several studies (24, 25, 26, 29), together with our own, indicate that these strictly conserved residues are essential for the activity of all the members of the SDR family. Our present results also show that the highly conserved serine residue (Ser142 in human 17-HSD type 1) has a significant role in the catalytic action of SDR family members. The side chain of Ser142 forms a hydrogen bond with the hydroxyl oxygen of the Tyr155 residue by donating a proton (21, 30, 31). This could lower the pKa of the Tyr155 side chain proton, thereby allowing it to approach the nucleophilic carbonyl oxygen of the substrate. The role of Ser142 could be similar to that suggested for the positively charged side chain of Lys159, whose proximity to Tyr155 could also have a pKa-lowering effect.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals and Reagents
[{gamma}-32P]ATP (3000 Ci/mmol), [2,4,6,7-3H]estradiol (120 Ci/mmol), [2,4,6,7-3H]estrone (120 Ci/mmol), [1,2,6,7-3H]testosterone (105 Ci/mmol), and [1,2,6,7-3H]androst-4-ene-3,17-dione (110 Ci/mmol) were purchased from Amersham Life Science (Little Chalfont, UK). 17ß-Estradiol, estrone, testosterone, and androstenedione were the products of Steraloids Inc. (Wilton, NY). Spodoptera frugiperda insect cell line (Sf9) was obtained from Invitrogen (San Diego, CA). All the media, buffers, supplements, and reagents for cell culture were obtained from GIBCO BRL-Life Technologics (Grand Island, NY) and the Sigma Chemical Co. (St. Louis, MO). Other reagents not mentioned in the text were obtained from the Sigma Chemical Co., Boehringer Mannheim (Mannheim, Germany), New England Biolabs (Beverly, MA), and Merck A. G. (Darmstadt, Germany) and were of the highest purity grade available.

Purification of Recombinant Human 17-HSD Type 1 from Sf9 Insect Cells
Recombinant Autographa californica nuclear-polyhedrosis viruses (AcNPVs) for human 17-HSD type 1 were generated using the BaculoGold Transfection System (Pharmingen, San Diego, CA) as previously described (28). Sf9 cells were grown at a density of 2.0 x 106 cells per ml in 500- and 1000-ml spinner flasks (Techne, Cambridge, UK) in complete TNM-FH insect medium containing 10% FCS. Exponentially growing cells were infected with recombinant 17-HSD type 1-AcNPVs at a multiplicity of infection of 1–10 (43). An optimal level of expression was reached after about 70 h, and the cells were then harvested by centrifugation at 1000 x g for 10 min, washed once with PBS, and stored at -70 C before purification of the enzyme.

Harvested cells were suspended in 6 volumes (vol/vol) of 10 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 0.02% NaN3, and 20% glycerol (vol/vol) and disrupted by sonication (4 x 20 sec at 0.5-min intervals) in an ice bath. Disruption of the cells was controlled by microscopic observation. The cell suspension was centrifuged for 1 h at 100,000 x g. Human 17-HSD type 1 protein was then purified using a cofactor analogy affinity chromatography column (Reactive Red-agarose, Sigma) as previously described (11). The bound proteins were eluted with 250 µM NADP+, and the fractions containing 17-HSD type 1 protein were pooled. Thereafter, the protein was dialyzed against 0.02 M sodium acetate buffer, pH 6.3. The dialyzed sample was loaded onto a Mono Q anion-exchange chromatography column (0.5 x 5 cm) connected to an fast protein liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden), and the proteins were eluted with a linear gradient of sodium acetate (0.02–1.35 M, pH 6.3) at a flow rate of 0.5 ml/min. Fractions containing purified human 17-HSD type 1 were pooled and stored at -70 C. Protein concentrations of the purified samples were measured by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA), using BSA as a standard.

Mass Spectrometry
Purified human 17-HSD type 1 (0.6 nmol) was evaporated and dissolved in 20 µl 70% trifluoroacetic acid. Thereafter, a sample of 1 µl was subjected to MALDITOF-mass spectrometric analysis (KOMPACT MALDI III, Kratos Analytical, Manchester, UK).

Modeling of Human 17-HSD Type 1 Proteins
Model building was performed on an SGI Elan workstation with the refined 2.20 Å x-ray structure of human 17-HSD type 1 (21). The software used for this purpose was CHAIN, a modified version of FRODO (44). Substitutions in human 17-HSD type 1 were modeled by replacing side chains of the amino acids with the substituted ones and orientating them in favorable conformations. The environments of the newly introduced side chains were examined for possible steric conflicts and van der Waals and polar/charge interactions.

Site-Directed Mutagenesis of Human 17-HSD Type 1 Proteins
Ser134Ala, Ser142Ala, Lys159Ala, His221AlaGlu282Ala, His221AlaGlu282Gln, Glu282Ala, Glu282Gln, and Leu111GluVal113Phe substitutions in human 17-HSD type 1 were generated using the overlap-extension method (45). The method was also used to construct an enzyme that contained an Ala170Glu substitution and insertion of Phe at position 172 (Ala170Glu+Phe172). Furthermore, the method was used to generate human 17-HSD type 1 lacking the last 36 C-terminal amino acids. The modifications in human 17-HSD type 1 cDNA were constructed by using flanking primers (5'-TTATATTAGCGGCCGCACCATGGCCCGCACCGTG-3' and 5'-TATATGAATTCAGGAAGCCTTTACTGCGGGGC-3') and the internal primers presented in Table 2Go. In PCR reactions, wild type cDNA was used as a template except in the cases of His221AlaGlu282Ala and His221AlaGlu282Gln substitutions, which were amplified with a template of previously constructed human His211Ala-substituted 17-HSD type 1 cDNA (28). All the constructs generated were confirmed by sequencing.


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Table 2. Internal Primers Used for Construction of Substituted Human 17-HSD Type 1 Enzymes

 
The substituted proteins were produced in Sf9 insect cells in 600-ml tissue culture flasks at a cell density of 30 x 106 cells per flask (28, 46). The concentrations of the wild type and substituted 17-HSD type 1 proteins in the cell homogenates were measured using a time-resolved immunofluorometric assay (47). The Ser134Ala-substituted protein was further purified from Sf9 cells in a manner similar to that described for the wild type human enzyme. Catalytic properties of all the other proteins were analyzed in 1000 x g fractions of cell homogenates in vitro. Activity measurements were also performed in cultured cells as described below (see Measurement of 17-HSD Type 1 Activity In Vitro and In Cultured Cells).

Gel Electrophoresis, IEF, and Immunoblotting
The purified recombinant 17-HSD type 1 enzymes were analyzed by SDS-PAGE, native PAGE, and IEF. The electrophoreses were carried out on a PhastSystem (Pharmacia Biotech, Uppsala, Sweden) using acrylamide gradient gels (10–15% for SDS-PAGE and 8–25% for native PAGE), and IEF was performed on a pH gradient of 3–9. After electrophoreses or IEF, the proteins were detected by silver staining. Properties of the wild type and deleted human 17-HSD type 1 proteins were characterized by immunoblotting. Cytosolic proteins from the Sf9 cell homogenates were separated by SDS-PAGE (Mini-PROTEAN II, Bio-Rad Laboratories) or IEF (pH gradient of 3–9, PhastSystem). Thereafter, the proteins were transferred onto a nitrocellulose membrane and immunostained as previously described by Poutanen et al. (17) using polyclonal antibodies raised against the wild type human 17-HSD type 1 enzyme.

Gel Filtration Chromatography
The molecular masses of human 17-HSD type 1 proteins were determined using Superdex 75 (wild type enzyme and enzyme lacking 36 C-terminal amino acids) and Superose 12 (enzymes having substitutions at the dimerization helices) gel filtration columns connected to a SMART System (Pharmacia Biotech). The columns were equilibrated with 0.15 M potassium phosphate, pH 7.2. A 50-µl sample was then applied, the system was operated at a flow rate of 60 µl/min, and 30-µl fractions were collected. Migration of the purified native protein was followed by UV spectrometry at 280 nm, whereas migration of the substituted human 17-HSD type 1 proteins in the cytosolic fractions of Sf9 cell lysates were followed by measuring 17-HSD type 1 concentrations in the collected fractions using an immunofluorometric assay (47).

Identical conditions were used to determine the Kd value of the monomers of purified human 17-HSD type 1 using a Superdex 75 gel filtration column. The purified enzyme was applied to the column at different concentrations (10, 5, 1, 0.5, 0.1, and 0.05 µg/ml), and the 17-HSD type 1 concentrations in the collected fractions were measured by immunofluorometric assay (47).

Measurement of 17-HSD Type 1 Activity In Vitro and In Cultured Cells
The activity of 17-HSD was measured in vitro as previously described by Puranen et al. (28). Briefly, samples were diluted in 10 mM potassium phosphate, pH 7.5, containing 0.01% BSA and were then mixed with [3H]estradiol or [3H]estrone (0.73–7.3 µmol of substrate/liter). The reactions were started by adding a cofactor (NAD+/NADH, Boehringer Mannheim) to a final concentration of 1.3 mmol/liter, and the samples were incubated for 20 sec at 37 C. After incubation, the reactions were stopped by quickly freezing the reaction mixtures in an ethanol-dry ice bath. The steroids were extracted into diethyl ether-ethyl acetate (9:1). The substrates and the products were then separated in a Sephasil C18 reverse-phase chromatography column connected to a SMART System (Pharmacia Biotech) using an acetonitrile-water solution as a mobile phase, as previously described (28, 48). Alternatively we used an acetonitrile/water (48:52, vol/vol) solution as a mobile phase in a Symmetry C18 reverse-phase chromatography column (3.9 x 150 mm) connected to a HPLC system (Waters, Milford, MA). Radioactivity was measured by an on-line ß-counter (150TR, FLO-ONE Radiomatic, Packard, Meriden, CT) connected to the HPLC system, using Ecoscint A scintillation solution (National Diagnostics, Atlanta, GA). Km and kcat values for the 17-HSD type 1 enzymes were calculated by using a GraFit-program (Erithacus Software Ltd., Staines, UK). The program fits data to the Michaelis-Menten equation using nonlinear regression analysis. The values presented represent the average ± SD of at least three independent experiments. One micromole of product formed per minute was defined as one unit of enzyme activity.

Activity measurements in cultured Sf9 insect cells were carried out by plating the cells in six-well plates at a density of 1.2 x 106 cells per well. The cells were allowed to attach for 1 h and were then infected with 17-HSD type 1-AcNPVs at a multiplicity of infection of 2. After 50 h incubation at 27 C, the culture medium was removed, and both reductive (E1 to E2, A-dione to T) and oxidative (E2 to E1, T to A-dione) activities were measured in the intact cells in culture by adding 2 ml serum-free TNM-FH insect medium, containing 10 µM [3H]substrate (20 nmol/well) to each well. To assess the linearity of the reactions, the activities were measured at four different time points (10, 20, 40, 80 min). After incubation, the media were collected, frozen in dry ice, and kept at -20 C until the steroids were extracted, and the amount of substrate converted was measured as described above.

The concentrations of the constructed enzymes in the Sf9 cell homogenates were measured using a immunofluorometric assay (47). Conversion of E1 to E2 was then calculated according to the amount of enzyme expressed.

Phosphorylation of 17-HSD Type 1 Enzymes in Vitro
Phosphorylation of wild type and Ser134Ala-substituted human 17-HSD type 1 enzymes was carried out through the use of PKA in vitro in a buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 10 µCi [{gamma}-32P]ATP, and 20 U of the catalytic subunit of PKA in a final reaction volume of 50 µl. The amount of substrate was 15 µg of protein per reaction. After 30 min incubation at 30 C, the reactions were stopped by keeping them on ice for 10 min. [{gamma}-32P]ATP was separated from the reaction mixtures by using Bio-Spin Chromatography Columns (Bio-Rad Laboratories) according to the manufacturer’s instructions, after which the reaction mixtures were subjected to electrophoresis (SDS-PAGE, Mini-PROTEAN II, Bio-Rad Laboratories). The gel was then stained with Coomassie Blue and dried. Thereafter, phosphorylated proteins were visualized by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Mrs Lea Sarvanko, Mrs Pirkko Ruokojärvi, Mrs Marja-Liisa Norrena, and Mrs Saara Korhonen for their skillful technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Professor Reijo Vihko, Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland.

This work was supported by the Research Council for Health of the Academy of Finland (project numbers 3314 and 30099), the Technology Development Center of Finland (TEKES, project number 4476), and by NIH Grant DK-26546. The Department of Clinical Chemistry, University of Oulu, is a World Health Organization Collaborating Center for Research in Human Reproduction, supported by the Ministries of Education, Social Affairs and Health, and Foreign Affairs, Finland.

Received for publication June 3, 1996. Accepted for publication October 4, 1996.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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