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Endocrinology Vol. 138, No. 1 182-190
Copyright © 1997 by The Endocrine Society

ARTICLES

Expression, Purification and Characterization of the Rat Luteal 20{alpha}-Hydroxysteroid Dehydrogenase1

Jifang Mao, Rachel W. Duan, Laura Zhong, Geula Gibori2 and Salman Azhar

Department of Physiology and Biophysics (J.M., R.W.D., L.Z., G.G.), University of Illinois College of Medicine, Chicago, Illinois 60612-7342; and Geriatric Research, Education, and Clinical Centers (S.A.), Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304

Address all correspondence and requests for reprints to: Salman Azhar, Ph.D., Research Career Scientist, Geriatric Research Education and Clinical Center (182-B), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The enzyme, rat ovarian 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}HSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20{alpha}-hydroxypregn-4-ene-3-one (20{alpha}-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20{alpha}HSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20{alpha}HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20{alpha}HSD expressed as glutathione-S-transferase-20{alpha}HSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies. Both rec preparations of 20{alpha}HSD demonstrated a single polypeptide chain of 37 kDa with similar Km values for 20{alpha}-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20{alpha}HSD expressed in the insect cells. The rec 20{alpha}HSD showed preference for progesterone/20{alpha}-hydroxyprogesterone. 17{alpha}-Hydroxyprogesterone was only 30% as effective. The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency. The rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity. Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme.

In conclusion, these studies demonstrate that rat luteal 20{alpha}HSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 20{alpha}HSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e. posttranslational modification of 20{alpha}HSD may not be required for the maximal expression of enzyme activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE 20{alpha}-HYDROXYSTEROID dehydrogenase (20{alpha}HSD) enzyme plays a central role in the termination of pregnancy (1, 2, 3, 4). The enzyme catalyzes the NADPH-dependent reduction of progesterone to 20{alpha}-hydroxypregn-4-ene-3-one (20{alpha}-hydroxyprogesterone) (5, 6), which is biologically inactive and cannot maintain pregnancy (2, 7, 8, 9). It is the increase in luteal 20{alpha}HSD activity rather than decreased progesterone synthesis that contributes to the reduced systemic progesterone levels associated with the termination of pregnancy. Indeed, 20{alpha}HSD activity in the rat corpus luteum remains repressed throughout pregnancy, but it is rapidly induced to a high level before parturition (2, 3).

Ovarian 20{alpha}HSD activity is thought to be under hormonal regulation. Hormones such as PRL, which signal through the tyrosine kinase system, have been shown to down-regulate 20{alpha}HSD activity (10, 11, 12, 13), whereas hormones that activate protein kinase A (PKA; LH) and protein kinase C (PKC; GnRH and PGF2{alpha}) simulate the activity of this enzyme (4, 14, 15, 16, 17, 18). However, in general, it is not clear whether the hormone-induced changes in 20{alpha}HSD activity are due to allosteric modulation, covalent modification, or changes in enzyme concentration. Recently, we demonstrated that at least the PRL-mediated inhibition of 20{alpha}HSD activity is accompanied by down-regulation of both its 20{alpha}HSD gene expression and enzyme synthesis (19, 20).

Efforts to characterize the structural/functional aspects of the ovarian 20{alpha}HSD enzyme have been hampered by the limited availability of purified native enzyme. Additionally, such preparations are unsuitable for studies designed to evaluate the role of posttranslational events (e.g. glycosylation and phosphorylation) in enzyme catalysis. Recently, we and others have reported the cloning of a gene encoding rat luteal 20{alpha}HSD (20, 21). The deduced amino acid sequence contains potential sites for phosphorylation by PKC, PKA, and tyrosine kinase(s) and putative sites for N-glycosylation (20). Also, the complementary DNA (cDNA) and deduced amino acid sequences show putative sites for NADH/NADPH binding and exhibit a high degree of homology with the members of the aldo-keto reductase family, including, aldehyde/aldose reductases, and bovine PGF2{alpha} synthase (20, 21). Enzymes with weak 20{alpha}HSD activity have recently been cloned from bovine testes and human placenta (22, 23, 24); they differ markedly from the ovarian enzyme by their amino acid structure and the substrate they use (20, 24, 25). Therefore, the current studies were initiated to further explore the kinetic/functional properties of ovarian 20{alpha}HSD and to examine the effects of glycosylation and phosphorylation on the enzyme activity. To accomplish these goals, efforts were made to express 20{alpha}HSD in both bacterial and insect cell systems. The heterologous proteins produced in a bacterial system are generally biologically active. However, these proteins may not adopt structural conformations of native proteins (i.e. similar to those found in mammalian cells), are nonglycosylated, and are poorly phosphorylated. The advantages of the baculovirus expression system are the abundant expression of recombinant protein combined with the co- and posttranslational modifications, including N-glycosylation and phosphorylation (26, 27, 28, 29). Thus, the use of these two expression systems provides an opportunity to evaluate the roles of co- and posttranslational modifications, such as glycosylation and phosphorylation.

Here we describe the high level expression of rat luteal 20{alpha}HSD using bacterial and baculoviral expression systems and the purification and characterization of the rec enzyme. Overall, purified rec enzyme preparations from the two systems exhibit the antigenic and kinetic properties and substrate specificity of native enzyme purified from ovarian sources (6, 7, 9). In addition, rec 20{alpha}HSD can use substrates specific for aldose/aldehyde reductases. Both bacterial and baculoviral expressed enzymes, however, lack the intrinsic PG synthase activity. Finally, we have also shown that phosphorylation and/or glycosylation of enzyme protein are not necessary for enzyme catalysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
pGEx-4T-2 vector and glutathione-Sepharose 4B were purchased from Pharmacia (Piscataway, NJ). pBlueBacIII vector and liposome-mediated transfection kit with Autographa californica nuclear polyhidrosis virus (AcNPV) and Spodoptera frugiperda 9 (Sf9) insect cell line were obtained from Invitrogen Corp. (San Diego, CA). Grace’s insect culture medium was supplied by Life Technologies (Gaithersburg, MD). The DNA sequencing kit was the product of U.S. Biochemical Corp. (Cleveland, OH). 20{alpha}-[1,2-N-3H]Hydroxypregn-4-ene-3-one (SA, 51.2 Ci/mmol; 1.9 tetrabecquerels/mmol) and [5,6,8,9,11,12,14,15-N-3H]arachidonic acid (SA, 60–100 Ci/mmol; 2.22–2.370 tetrabecquerels/mmol) were obtained from DuPont-New England Nuclear Research Products (Boston, MA). Various restriction enzymes and T4 ligase were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Polyclonal antibodies against the 20{alpha}HSD were prepared by us. All others reagents used were of analytical grade.

Expression of 20{alpha}HSD in Escherichia coli and its purification
Plasmid construction and expression. Standard procedures of DNA manipulation and transfection were followed (30). A DNA fragment of 1.2 kilobases containing the entire coding region of 20{alpha}HSD was removed from pBluescript vector by EcoRI and XhoI digestion, purified, and ligated into the EcoRI and XhoI cloning sites of the pGEX-4T-2 vector, a glutathione-S-transferase (GST) fusion protein expression vector. Correct orientation of the translational reading frame for the GST-20{alpha}HSD fusion protein was confirmed by DNA sequencing.

Purification of expressed 20{alpha}HSD. Two hundred microliters of overnight culture of E. coli DH5{alpha} transfected with GST-20{alpha}HSD cDNA was inoculated into 200 ml fresh, prewarmed, 2 x YTG medium (16 g/liter tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl) containing 100 mg/ml ampicillin. The culture was incubated at 37 C with shaking to achieve an absorption reading between 1.0–2.0 at 600 nm. Subsequently, isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 0.1 mM. The incubation was continued for an additional 4 h. After centrifugation, the pelleted bacteria were resuspended in PBS, sonicated, made 1% with respect to Triton X-100, and mixed gently at room temperature for 30 min. The suspension was then centrifuged at 1200 x g for 10 min. The supernatant containing recombinant (rec) 20{alpha}HSD GST was further processed to obtain pure 20{alpha}HSD using standard protocols (31). In brief, a 50% slurry of glutathione-Sepharose 4B beads was mixed with supernatant (~2 ml of the 50% slurry of beads to each 100 ml supernatant) and agitated gently at room temperature. At the end of incubation, the beads were collected by centrifugation and washed several times with 10 vol PBS. After final centrifugation, the glutathione beads (~2 ml) with bound 20{alpha}HSD-GST fusion protein were resuspended in 1 bed volume PBS and digested with 500 cleavage units of bovine plasma thrombin at room temperature for 16 h. After thrombin cleavage of the fusion protein, the beads with bound GST were separated from the rec 20{alpha}HSD protein by centrifugation at 10,000 x g for 10 min at 4 C. The supernatant was adjusted to 50% (vol/vol) glycerol, aliquoted, frozen in liquid N2, and stored at -70 C. Usually, 500 µg rec 20{alpha}HSD was obtained from 200 ml bacterial cell culture.

Expression of 20{alpha}HSD in insect cells using the baculovirus system
Construction of a 20{alpha}HSD cDNA-containing baculovirus transfer vector. A 1.2-kilobase BamHI fragment consisting of the entire coding region of rat luteal 20{alpha}HSD was ligated into the pBlueBacIII vector immediately downstream of the polyhedrin promotor to create pBlueBacIII-20{alpha}HSD. Correct orientation was confirmed by DNA sequencing.

Transfection and isolation of recombinant baculovirus. Recombinant baculovirus, AcNPV, containing the 20{alpha}HSD sequence under the transcriptional control of the polyhedrin promotor was produced by in vivo homologous recombination according to Webb and Summers (32) as described in Invitrogen’s instruction manual. In brief, 2 µg of the pBlueBacIII-20{alpha}HSD were mixed with 1 mg linearized wild-type AcNPV viral DNA and cotransfected into Sf-9 insect cells by cationic liposome-mediated gene transfer according to the protocol suggested by the manufacturer (Invitrogen). One hundred and twenty hours postinfection, the medium was collected, centrifuged, diluted 10- to 10,000-fold, and used to infect a fresh monolayer of Sf-9 cells. To facilitate the identification of viral plaques, a layer of 0.625% agarose containing 75 µg/ml X-Gal (5-bromo-4-chloro-3-inolyl-ß-D-galactopyranoside) was applied to the transfected cells. After 6–8 days, recombinant viruses, designated AcNPV-20{alpha}HSD, were detected by the formation of blue plaques. Several blue plaques were picked and subjected to three cycles of plaque purification until cells with inclusion bodies were not detected. After purification, several strains of recombinant AcNPVs were obtained, and four strains (designated as AcNPV-20{alpha}HSD 1, 2, 3, and 4) were used in further studies.

Insect cell culture. Sf-9 insect cells were maintained in Grace’s medium supplemented with 10% FBS, supplemented with yeastolate (3.3 g/liter), lactalbumin hydrolysate (3.3 g/liter), gentamicin (50 µg/ml), and fungizone (2.5 µg/ml) in monolayer or suspension culture. Cells were transfected or infected in log phase of growth at 2 x 106 cells/ml.

Expression and purification of recombinant 20{alpha}HSD. Sf-9 cells were seeded at a density of 2–2.5 x 106/60-mm dish or 9 x 106 cells/75-cm2 flask. After the cells were attached, the medium was removed, and a volume of virus inoculum, sufficient to just cover the cells at a multiplicity of infection of 3 or 4 to 1, was added. After incubation at 27 C for 1.5 h, the inoculum was replaced with fresh Grace’s medium and incubated at 27 C for up to 5 days; infected insect cells were collected and used for purification of recombinant 20{alpha}HSD (9, 33).

Immunoblot analysis of recombinant proteins
Immunoblotting of bacterial and insect cell-expressed 20{alpha}HSD was performed using polyclonal antirat 20{alpha}HSD antibody (34). The fusion protein expressed in either E. coli or Sf9 cells were subjected to SDS-PAGE under reducing conditions on a gel containing 7.5% polyacrylamide and then transferred to a nitrocellulose membrane. After transfer, the blots were blocked with 3% BSA and then probed with the anti 20{alpha}HSD antibody or preimmune serum. The immunoreactive proteins were visualized using alkaline phosphatase-conjugated antirabbit IgG as secondary antibody.

Enzyme assays
Measurement of 20{alpha}HSD activity. Enzyme activity was determined as the rate of conversion of [1,2-3H]-20{alpha}-hydroxy-pregn-4-ene-3-one (20{alpha}-hydroxyprogesterone) to [1,2-3H]progesterone in the presence of NADPH as described by Jones and Hsueh (15). One unit of enzyme activity is defined as that amount of enzyme catalyzing the formation of 1 nmol progesterone/min. Specific activity is expressed as units per min/mg protein. The protein concentration was determined by the Bradford method, using BSA as the standard (35). In some cases, enzyme activity was also measured spectrophotometrically either by following the reduction of NADP (6, 9) or by oxidation of NADPH (7, 9).

Measurement of PG endoperoxide synthase activity. To test for the PG endoperoxide synthase type catalytic activity in rec 20{alpha}HSD, both cyclooxygenase activity (that converts arachidonic acid into the hydroperoxide, PGG2) and peroxidase activity (that reduces PGG2 and other hydroperoxides to the corresponding alcohols, such as PGH2) were measured. Cyclooxygenase assay was performed by following the conversion of [3H]arachidonic acid to [3H]PGs (PGH2 and PGE2) as described by Mitchell et al. (36). Peroxidase activity was measured by monitoring the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine at 611 nm (37).

Aldo-keto reductase activity. Aldo-keto reductase activity of rec 20{alpha}HSD was assayed spectrophotometrically by measuring the rate of oxidation of NADPH at 340 nm with a Gilford DU model spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, OH) at 37 C using the saturating concentration of substrate (benzaldehyde, 4-nitrobenzaldehyde, D,L-glyceraldehyde methylglyoxal, 9,10-phenanthrequinone, or D-galactose) (38). One unit of enzyme activity is defined as that amount of enzyme that catalyzes the formation of 1 nmol NADP/min.

In vitro phosphorylation of rec 20{alpha}HSD. Purified bacterially expressed rec 20{alpha}HSD was first incubated in 500 ml 20 nM Tris-HCl (pH 8), 1 mM MgCl2, 1 mg/ml BSA, 0.5 mM dithiothreitol, and 60 U alkaline phosphatase coupled to agarose beads for 30 min at 30 C. After centrifugation, the supernatant containing 20{alpha}HSD was used as a substrate in various in vitro kinase assays. The PKA, PKC, and insulin receptor-associated tyrosine kinase activities were measured as described previously (39, 40). Src kinase (p60src) activity was assayed as described by Simonson and Herman (41). The concentration of rec dephosphorylated 20{alpha}HSD used in these assays was between 2–3 µg.

To test the relationship between phosphorylation and alteration of 20{alpha}HSD activity, aliquots of 20{alpha}HSD (1–2 µg) were incubated with 25 mM PIPES, pH 6.8, containing 10 mM Mg acetate, 100 µM ATP (or [{gamma}-32P]ATP to monitor phosphorylation), and EGTA (0.5 mM) plus purified rat liver PKC (4 µg; 10 U) or PKC, phosphatidylserine (250 µg/ml), diolein (10 µg/ml), plus CaCl2 (0.25 mM) in a final volume of 50 ml for 60 min at 30 C. Similarly, the PKA-mediated phosphorylation of rec 20{alpha}HSD was carried out in a final volume of 50 ml containing 50 mM MOPS (pH 7.0), 10 mM MgCl2, and 250 µM ATP (or [{gamma}-32P]ATP) to monitor phosphorylation and the catalytic subunit of PKA (10 U) for 60 min at 30 C. Reactions without ATP were performed under identical conditions. The catalytic activities of phosphorylated and nonphosphorylated forms of 20{alpha}HSD were determined radiochemically as described above. The phosphorylated 32P-labeled 20{alpha}HSD preparations were analyzed by SDS-PAGE followed by autoradiography (42).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and purification of rec 20{alpha}HSD in E. coli
20{alpha}HSD was expressed as GST fusion in bacteria using pGEX expression plasmids. After induction with isopropyl-ß-D-thiogalactopyranoside, a 63-kDa protein corresponding to GST-20{alpha}HSD was the most prominent band within the crude bacterial lysates, as judged by the SDS-PAGE (data not shown). The GST-20{alpha}HSD was purified by binding to glutathione-Sepharose beads (Fig. 1Go, lane 2) followed by thrombin cleavage (Fig. 1Go, lanes 3 and 4). Incubation of purified GST-20{alpha}HSD with thrombin released a 37-kDa protein (the predicted size of 20{alpha}HSD) as determined by SDS-PAGE and immunoblot analysis (Fig. 1Go, lanes 3 and 4). The purified preparation of 20{alpha}HSD showed high levels of enzymatic activity (Fig. 2Go). The average specific activity of the purified preparations of 20{alpha}HSD was 332 ± 47 nmol/min·mg protein ± SE), and activity was concentration (enzyme protein) and time dependent. No such enzyme activity was detected using the GST protein alone.



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  		Figure 1. SDS-PAGE and Immunoblotting of purified rec GST-20{alpha}HSD and purified rec 20{alpha}HSD. E. coli transfected with pGEX-4T-3-20{alpha}HSD was grown in 2 x YTG medium. 1PTG was added to induce the expression of fusion protein. Twenty micrograms of protein were used as a starting material. GST and rec GST-20{alpha}HSD were purified with glutathione beads. To release 20{alpha}HSD, the bound GST-20{alpha}HSD proteins were digested with thrombin at room temperature for 6 h. The purified rec GST-20{alpha}HSD and purified rec 20{alpha}HSD proteins (3.2 µg each) were subjected to SDS-PAGE, transferred to cellulose membranes, and immunodetected with the anti-20{alpha}HSD antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit IgG. Left panel, Gel stained for proteins with Coomassie blue. Right panel, Immunoblots. Lane 1, Purified GST; lane 2, purified GST-20{alpha}HSD; lane 3, thrombin cleavage for fusion protein bound to glutathione-Sepharose 4B; lane 4, proteins remained absorbed to column after thrombin digestion.

 


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  		Figure 2. Catalytic activity of bacterially expressed rec 20{alpha}HSD as a function of enzyme concentration and incubation time. Left panel, Various amounts of purified 20{alpha}HSD (0.025–2.5 µg 20{alpha}HSD expressed on the x-axis) were incubated with 100 µM [3H]20{alpha}-hydroxyprogesterone and 300 µM NADP in buffer for 30 min. Right panel, 0.625 µg purified 20{alpha}HSD was incubated with 100 µM [3H]20{alpha}-hydroxyprogesterone and 300 µM NADP for 5–40 min. In each case, the enzyme activity was quantified by the formation of [3H]progesterone. The purified preparation of rec 20{alpha}HSD was obtained as described in Fig. 1Go and Materials and Methods. Essentially the same results were obtained for three separate experiments.

 
Expression and purification of 20{alpha}HSD in insect (Sf-9) tissue culture cells using the baculovirus system
Insect cells infected with recombinant 20{alpha}HSD viral construct AcNPV/20{alpha}HSD 1, 2, 3, or 4 were harvested 5 days after infection, lysed, and analyzed by SDS-PAGE and immunoblotting. All four recombinants showed a prominent band in the 37-kDa region of the Coomassie blue-stained gel (Fig. 3Go, left panel); the recombinant virus AcNPV/20{alpha}HSD no. 4, however, exhibited the highest expression of 37-kDa protein. Immunoblots of these same samples using a rat luteal 20{alpha}HSD specific antibody are shown in Fig. 3Go (right panel). The antibody immunoreacted with the 37-kDa protein, confirming that the protein is indeed 20{alpha}HSD.



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  		Figure 3. SDS-PAGE and immunoblot analysis of insect cells infected with rat 20{alpha}HSD recombinant baculoviruses. Sf-9 cells were infected with recombinant viruses, and after 5 days, cell lysates (obtained from 1 x 107 cells) were subjected to SDS-PAGE. Left panel, The 10% gel was stained for proteins with Coomassie blue. Right panel, After gel electrophoresis, the proteins were transferred to nitrocellulose membrane. The blot was visualized with rabbit anti-20{alpha}HSD followed by alkaline phosphatase-conjugated goat antirabbit IgG. Lane 1, AcNPV/20{alpha}HSD 1; lane 2, AcNPV/20{alpha}HSD 2; lane 3, AcNPV/20{alpha}HSD 3; lane 4, AcNPV/20{alpha}HSD 4.

 
To assess the functional activity of recombinant 20{alpha}HSD, Sf-9 cells were harvested 5 days after infection with AcNPV/20{alpha}HSD 1, 2, 3, or 4 (or wild-type virus as a control), and cellular lysates were tested for 20{alpha}HSD activity. As shown in Fig. 4Go, only one of the four recombinant viral clones (no. 4) showed strong expression of 20{alpha}HSD activity and was used for further study. Uninfected control cells or wild-type virus exhibited cells infected with no activity (data not shown). Next, Sf-9 cells infected with the recombinant baculovirus containing 20{alpha}HSD cDNA (AcNPV/20{alpha}HSD 4) were assayed for 20{alpha}HSD activity as a function of days after infection (Fig. 5Go). Enzyme activity in the total cell lysates was maximal (range, 750–893 pmol/min·mg protein) at 2 days postinfection. Between days 3–6 there was a steady and substantive decline in 20{alpha}HSD activity.



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  		Figure 4. Demonstration of catalytic function of 20{alpha}HSD in Sf-9 cells infected with recombinant viruses. Sf-9 insect cells were infected with recombinant viruses AcNPV/20{alpha}HSD 1, 2, 3, and 4 as described in Fig. 3Go. After 5 days, the cells were harvested, and total cell extracts were assayed for 20{alpha}HSD activity radiochemically. The data are expressed as the mean of duplicate determinations from a single experiment.

 


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  		Figure 5. Time course of expression of 20{alpha}HSD activity in insect cells infected with 20{alpha}HSD recombinant baculovirus. Sf-9 cells were infected with a recombinant baculovirus vector (AcNPV/20{alpha}HSD 4) and then harvested 1, 2, 4, 6, and 7 days postinfection, as indicated. Total cell lysates were prepared and assayed for 20{alpha}HSD activity.

 
To purify 20{alpha}HSD to homogeneity, 5.3 g insect cell extract were subjected to several steps of purification, as shown in Table 1Go. The purification procedure sequentially used ion exchange chromatography on diethylaminoethyl cellulose and affinity chromatographies on Matrex Gel Green dye and Red-Sepharose (9, 33). The protocol resulted in 238-fold purification of rec 20{alpha}HSD (Table 1Go). On SDS-PAGE, the purified rec 20{alpha}HSD showed a single protein band with a mol wt of 37 kDa (data not shown).


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  		Table 1. Purification of recombinant 20{alpha}HSD from insect tissue culture cells

 
Properties of rec 20{alpha}HSD
The purified preparations of recombinant 20{alpha}HSD expressed in E. coli and insect cells were employed to examine the enzyme characteristics.

To determine the substrate specificity of 20{alpha}HSD, we examined the oxidation/reduction of several steroid substrates (Table 2Go). In addition to its capacity for 20{alpha}-hydroxyprogesterone oxidation, 20{alpha}HSD reduced progesterone and 17{alpha}-hydroxyprogesterone. However, compared to 20{alpha}-hydroxyprogesterone, 20{alpha}HSD activity toward 17{alpha}-hydroxyprogesterone was weaker, and its activity toward progesterone was relatively stronger. In contrast, corticosterone and 5{alpha}-dihydrotestosterone were not used by the enzyme. Recombinant 20{alpha}HSD showed an absolute requirement for NADP when using 20{alpha}-hydroxyprogesterone as a steroid substrate; 20{alpha}HSD activity with NAD as a cofactor was less than 1% of that with NADP.


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  		Table 2. Substrate specificity of recombinant 20{alpha}HSD

 
Steady state kinetic analysis of rec 20{alpha}HSDs
As the structure of 20{alpha}HSD contains a putative N-glycosylation site, we sought to determine whether glycosylation was required for catalytic activity. To accomplish this, we compared the catalytic parameters of rec enzyme preparations expressed in bacteria (nonglycosylated) and insect cells (presumably glycosylated protein). Kinetic constants were determined with 20{alpha}-hydroxyprogesterone as a substrate and NADP as a cofactor. The results obtained with different amount of 20{alpha}-hydroxyprogesterone (1–100 µM) and a fixed concentration of NADP (1 mM) using bacterially expressed rec enzyme shown in Fig. 6AGo indicate that enzyme activity reached a saturation level around 10 µM 20{alpha}-hydroxyprogesterone. The data were transformed into a reciprocal plot [Lineweaver and Burk plot to calculate the Km and maximum velocity (Vmax) values; Fig. 6AGo, inset]. The initial velocity experiments were also performed under conditions where NADP concentrations varied (1–1000 µM) at a fixed saturating concentration of 20{alpha}-hydroxyprogesterone (100 µM). Such data using bacterially expressed enzyme are shown in Fig. 6BGo. The maximal 20{alpha}HSD activity was around 100 µM NADP. Similar data were obtained using the purified preparations of 20{alpha}HSD expressed in insect cells, and the kinetic parameters obtained from bacteria and insect cell-derived 20{alpha}HSD are summarized in Table 3Go. The calculated Km values of bacterially expressed enzyme for 20{alpha}-hydroxyprogesterone and NADP were 5.9 and 9.5 µM, respectively. The corresponding values for baculovirus-expressed 20{alpha}HSD were 5.8 and 9.6 µM, respectively. Thus, the enzyme preparations from two different sources had similar values. However, the rec 20{alpha}HSD expressed in E. coli had Vmax values that were 20–30% higher than those of the rec 20{alpha}HSD expressed in insect cells when measured using similar assay conditions. These results suggest that glycosylation of 20{alpha}HSD may not be required for the expression of catalytic activity.



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  		Figure 6. 20{alpha}-Hydroxyprogesterone and NADP dependence of purified rec 20{alpha}HSD expressed in bacteria. A, Initial velocities vs. 20{alpha}-hydroxyprogesterone concentrations. Each reaction mixture contained 200 ng rec 20{alpha}HSD, the indicated concentrations of 20{alpha}-hydroxyprogesterone, and 1 mM NADP. The assays were conducted at 37 C for 10 min. B, Initial velocities vs. NADP concentrations. Each assay mixture contained 200 ng rec 20{alpha}HSD, the indicated concentration of NADP, and a saturating concentration of 20{alpha}-hydroxyprogesterone. The assays were carried out at 37 C for 7.5 min. The double reciprocal plot for each substrate is shown in the inset. Essentially the same results were obtained for three separate experiments.

 

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  		Table 3. Steady state kinetic parameters of recombinant 20{alpha}HSD

 
Aldose/aldehyde reductase and PG synthase activities of rec 20{alpha}HSD
As the cDNA and predicted amino acid sequence of rat luteal 20{alpha}HSD are related to bovine lung PGF2{alpha} synthase and aldose/aldehyde reductase (20, 21, 43), we sought to determine whether 20{alpha}HSD was active as an aldose/aldehyde reductase and/or PG synthase. As shown in Table 4Go, the purified rec enzyme preparations were quite active against the general substrate of aldose/aldehyde reductase. The enzyme reduced benzaldehyde, 4-nitrobenzaldehyde, and D,L-glyceraldehyde very efficiently, whereas it was only weakly reactive toward methylglyoxal, 9,10-phenanthrenquinone, and D-galactose. Maximal activity was observed with either benzaldehyde or D,L-glyceraldehyde, which was roughly one third of that observed with 20{alpha}-hydroxyprogesterone (Table 4Go). Also, the rec bacteria- and baculovirus-expressed proteins showed similar reactivities. In contrast, no PG synthase type of activity (measured by following the conversion of [3H]arachidonic acid to PGs or the production of H2O2) was detected in the purified preparations of rec bacterial and baculoviral 20{alpha}HSDs.


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  		Table 4. Aldo-Keto reductase activity of recombinant 20{alpha}HSD

 
Phosphorylation and catalytic function of 20{alpha}HSD
To test the possibility that phosphorylation modulates 20{alpha}HSD activity, the purified rec bacterial 20{alpha}HSD was incubated with various kinases after pretreatment with alkaline phosphatase to ensure the protein was in a nonphosphorylated state. After treatment with various kinases the rec enzyme was assayed for 20{alpha}HSD activity. As shown in Table 5Go and Fig. 7Go, A and B, rec 20{alpha}HSD was readily phosphorylated by in vitro incubation with purified PKA or PKC in the presence of [{gamma}-32P]ATP. Phosphoamino acid analysis of phosphorylated 20{alpha}HSD confirmed that [32P]phosphorylation was restricted to serine and threonine residues, and label was predominantly on serine residues (data not shown). In contrast, members of the tyrosine kinase family, including Src kinase and insulin receptor-associated tyrosine kinase, failed to phosphorylate 20{alpha}HSD (Table 5Go). All kinases tested, however, rapidly phosphorylated their respective artificial substrates (Table 5Go). As shown in Fig. 7CGo, when rec 20{alpha}HSD was phosphorylated with PKC in the presence of ATP-Mg2+, DAG, and phosphatidylserine, 20{alpha}HSD activity was unaffected. Similarly, the enzyme activity was not significantly affected by phosphorylation with PKA. These results suggest that phosphorylation of 20{alpha}HSD is not essential for catalytic activity.


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  		Table 5. Effects of various serine/threonine and tyrosine kinases on the phosphorylation of recombinant 20{alpha}HSD

 


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  		Figure 7. Phosphorylation and catalytic function of 20{alpha}HSD. A, PKC-mediated 32P phosphorylation of rec, dephosphorylated, bacterially expressed 20{alpha}HSD was performed as described in Materials and Methods and analyzed by SDS-PAGE and autoradiography. Left lane, Basal (EGTA); right lane, Ca2+, DAG, phosphatidylserine, and PKC. B, Aliquots of rec dephosphorylated 20{alpha}HSD (0.9 µg) were phosphorylated with [{gamma}-32P]ATP-Mg2+ with or without PKC, DAG, and Ca2+ in vitro as described in Materials and Methods. The incorporation of 32P into 20{alpha}HSD was quantified by liquid scintillation spectrometry. C, Aliquots of rec dephosphorylated 20{alpha}HSD were phosphorylated as described in B, except [{gamma}-32P]ATP was replaced with a similar concentration of unlabeled ATP. The catalytic activities of both phosphorylated and dephosphorylated forms were quantified by following the conversion of [3H]20{alpha}-hydroxyprogesterone to [3H]progesterone. Other details are given in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although 20{alpha}HSD plays a central role in luteolysis and parturition (1, 2, 3, 4), the limited availability of the purified form of enzyme has restricted its biochemical and functional studies. The recent cloning of 20{alpha}HSD gene (20, 21) revealed a high degree of homology to members of the NADP aldo-keto reductase family such as aldose/aldehyde reductase and PGF2{alpha} synthase and has helped to identify several important structural features that may be critical to enzyme function. These included potential sites for phosphorylation by serine/threonine and tyrosine kinases and sites for adenine nucleotide binding and glycosylation (20). To evaluate the functional significance of these features, we first directed our efforts at establishing conditions that would allow for high level 20{alpha}HSD protein expression through the use of bacterial and baculoviral expression systems (31, 32). This was then followed by purification of rec enzyme to apparent homogeneity and the detailed characterization of the kinetic, biochemical, and functional properties of the enzyme.

Our results show that these two expression systems provide large quantities of rec 20{alpha}HSD for biochemical studies and for examination of the various catalytic functions performed by the enzyme. Also, these results may provide insights into the roles of glycosylation and phosphorylation in enzyme catalysis. In general, rec 20{alpha}HSD had catalytic properties similar to those reported for 20{alpha}HSD purified from rat ovarian tissues (6, 7, 9). Immunoblotting experiments using purified rec 20{alpha}HSD preparations from bacterial and insect cells and specific 20{alpha}HSD antibody resulted in the detection of a 37-kDa protein. This agrees closely with the calculated molecular mass of the encoded protein (20) and with that of the native ovarian enzyme found in the corpus luteum (34).

Data presented in this report also demonstrate that rec 20{alpha}HSD was able to use (reduce) substrates specific for aldose/aldehyde reductases, although these substrates were 3–5 times less active relative to progesterone/20{alpha}-hydroxyprogesterone. Although these findings could be interpreted as indicating that rec enzyme might be an aldose/aldehyde reductase, the cDNA sequence information does not support this possibility (20). The cDNA encoding rat luteal 20{alpha}HSD shows less than 50% nucleotide sequence homology with that of rat aldose/aldehyde reductase (20, 21). We believe that the observed aldose/aldehyde reductase activity for the rec 20{alpha}HSD represents an additional intrinsic catalytic function of the enzyme protein. Recently, 20{alpha}HSD activity in bovine testis has been shown to be due to aldose reductase (22). In this case, however, the cDNA encoding bovine testicular 20{alpha}HSD is 100% identical with that encoding bovine lens aldose reductase, and that deduced amino acid is identical with that of bovine aldose reductase (22). In addition, the testicular enzyme, unlike the ovarian 20{alpha}HSD, favors 17{alpha}-hydroxyprogesterone over progesterone (24). Also, the purified enzyme can reduce glucose, benzaldehyde, and glyceraldehyde (the classic aldose reductase substrates) with the same high efficiency as that of 17{alpha}-hydroxyprogesterone (22).

Despite extensive efforts, we were unable to demonstrate PG synthase activity using rec 20{alpha}HSD preparations purified from bacterial and baculoviral expression systems. This lack of PG synthase activity is surprising, as the cDNA deduced amino acid sequence of 20{alpha}HSD shows a great degree of sequence homology (~67%) with bovine lung PG synthase (20). The reason for this unexpected finding is not clear, but one could consider several possibilities. One of the simplest possibilities could be that despite a close sequence homology, the rec 20{alpha}HSD does not possesses PG synthase-type activity. Other possibilities might include that rec 20{alpha}HSD lacks proper protein folding and configuration, is not properly glycosylated, or is not adequately processed. Indeed, glycosylation per se is necessary for the catalytic function of both the constitutive and inducible forms of the cyclooxygenase component of PG synthases (44, 45). Clearly, more experimental work is needed to determine whether rec 20{alpha}HSD has any intrinsic PG synthase activity.

Because the activity of ovarian 20{alpha}HSD is regulated by hormones that activate tyrosine kinase(s), PKC and PKA, we considered the possibility that phosphorylation may affect enzyme activity. We first compared the catalytic activities of rec bacterial and insect enzymes, which should differ in their phosphate content. In general, the proteins expressed in bacteria are presumed to be nonphosphorylated, whereas the rec phosphoproteins of baculoviral origin are considered to be sufficiently phosphorylated (28, 29). However, no difference in enzyme activity was noted between these two preparations. To further examine the effects of phosphorylation on 20{alpha}HSD activity, the dephosphorylated rec enzyme was phosphorylated with serine/threonine- or tyrosine-specific kinases. 20{alpha}HSD was a good substrate for both PKA and PKC. In contrast, Src- and insulin receptor-associated tyrosine kinases failed to act on 20{alpha}HSD, and no phosphorylation on tyrosine residues was detected. Interestingly, neither PKA nor PKC induced phosphorylation of rec 20{alpha}HSD had any significant effect on its activity. These findings together with the results we previously reported (18, 20, 34) suggest that hormonal regulation of 20{alpha}HSD activity is not through a phosphorylation/dephosphorylation mechanism, but, rather, through regulation of 20{alpha}HSD gene expression. PRL inhibits the activity of 20{alpha}HSD and markedly down-regulate 20{alpha}HSD gene expression and the level of its protein, whereas LH stimulates 20{alpha}HSD enzyme activity, which is accompanied by increased levels of 20{alpha}HSD messenger RNA.

In the current studies, we failed to detect any difference in activity between bacterially expressed (nonglycosylated) and insect cell-expressed (presumably glycosylated) forms of 20{alpha}HSD. These indirect studies led us to conclude that glycosylation of 20{alpha}HSD may not be required for the catalytic function of the enzyme. The follow-up studies are in progress to more directly examine the glycosylation of 20{alpha}HSD and further assess the role of glycosylation in enzyme catalysis.

In summary, this report describes the high level expression, purification, and characterization of 20{alpha}HSD using E. coli and baculovirus expression systems. The rec enzyme preparations are essentially indistinguishable from that isolated from rat ovary, except that bacterially expressed enzyme is nonglycosylated and possibly poorly phosphorylated. The kinetic parameters of both bacterially expressed and insect cell expressed rec enzymes have been determined and suggest that phosphorylation and glycosylation of 20{alpha}HSD may not be required for enzyme catalysis. Finally, the fact that the rec 20{alpha}HSD can be expressed and readily purified, indicates that the bacterial and viral systems are excellent choices for exploring the detailed mechanism of action of this multifunctional enzyme protein.


   Footnotes
 
1 This work was supported by NIH Research Grant HD-11119 (to G.G.). This material is based upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. Back

2 Merit Awardee (HD-11119). Back

Received June 10, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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