Download Characterization of Structural and Functional Properties of Human

Characterization of Structural and
Functional Properties of Human
17b-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
Human 17b-hydroxysteroid dehydrogenase (17HSD) 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 17HSD 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. (Molecular Endocrinology 11: 77–86, 1997)
INTRODUCTION
17b-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 17HSD enzymes have been described (1–5). 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 (11–13)
and in certain estrogen target tissues, such as healthy
and malignant breast and endometrial tissues (14–17).
It is therefore suggested that the enzyme might have a
significant role in the regulation of estrogen exposure
0888-8809/97/$3.00/0
Molecular Endocrinology
Copyright © 1997 by The Endocrine Society
77
MOL ENDO · 1997
78
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 (18–21). 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
(24–28). Similarly, substituting arginine, isoleucine,
glutamine, or leucine in place of the largely conserved
lysine residue always abolishes the enzymatic activity
totally (25–27, 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 3a,20b-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 17HSD 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
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. 1A), native PAGE
(Fig. 1B), and Superdex 75 gel filtration column chromatography (Fig. 4C) confirmed that similar to human
Vol 11 No. 1
Fig. 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.
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 b-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 (aE and aF), forming a four-helix
bundle. In the present study, two substitutions
(Leu111GluVal113Phe and Ala170Glu1Phe172) 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 1) and in cultured Sf9
cells (data not shown).
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
Characterization of Human 17b-HSD Type 1
79
Table 1. Activity Measurements of 17-HSD Type 1 Enzymes in Vitro
Estradiol to Estrone
Purified Enzymes
Wild type
Ser134Ala
Cell Homogenates
Wild type
Ser142Ala
Lys159Ala
Glu282Ala
His221AlaGlu282Ala
Glu282Gln
His221AlaGlu282Gln
Leu111GluVal113Phe
Ala170Glu 1 Phe172
Estrone to Estradiol
Km (mM)
Kcat (U/mg)
Kcat/Km
Km (mM)
Kcat (U/mg)a
Kcat/Km
2.11 6 0.56
2.53 6 0.59
109.17 6 10.96
116.60 6 17.31
51.74
46.09
0.90 6 0.13
0.78 6 0.01
206.52 6 14.06
102.12 6 9.12
229.47
130.92
2.29 6 0.33
5.26 6 1.26
5.39 6 1.64
1.63 6 0.13
3.74 6 0.24
1.80 6 0.12
4.28 6 0.34
86.66 6 5.56
0.39 6 0.10
0.36 6 0.05
64.37 6 8.84
8.54 6 0.41
62.06 6 2.94
14.92 6 0.85
Nondetectable
Nondetectable
37.84
0.07
0.07
39.49
2.28
34.48
3.49
1.38 6 0.11
1.66 6 0.21
1.14 6 0.21
1.13 6 0.11
5.11 6 1.00
0.66 6 0.12
3.75 6 0.58
122.76 6 8.39
0.64 6 0.03
0.36 6 0.03
142.77 6 11.15
44.08 6 5.23
90.04 6 9.06
59.87 6 3.86
Nondetectable
Nondetectable
88.96
0.39
0.32
126.35
8.63
136.42
15.97
a
a
One micromole of product per minute was defined as one unit of enzyme activity. Concentrations of the purified 17-HSD type
1 enzymes were measured by Bio-Rad protein assay, and the enzyme concentrations in the cell homogenates were measured
by immunofluorometric assay (47).
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. 1C). The primary structure of the human
17-HSD type 1 enzyme contains a potential cAMPdependent 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.
2). Furthermore, destroying the potential phosphorylation site by substituting alanine in place of Ser134
totally abolished phosphorylation (Fig. 2). The substitution, however, did not have any significant effects on
the catalytic properties (Table 1) or microheterogeneity
(Fig. 1C) 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. 3). Identical properties were
also observed using MCF-7 human breast cancer cells
transiently transfected with the cDNAs for Ser134Alasubstituted 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.
Fig. 2. Phosphorylation of Purified Wild Type and Ser134AlaSubstituted 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 mg) were thereafter separated by
SDS-PAGE and visualized by staining with Coomassie Blue
(A) or by autoradiography (B).
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. 4A), and gel filtration analysis revealed
that the truncated enzyme was still present as a dimer
(Fig. 4C) 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. 3), and that
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Fig. 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 (6SD) of triplicate specimens.
the truncated enzyme possessed similar microheterogeneity to that detected in the native type 1 enzyme
(Fig. 4B).
Substitutions at the Active Site of Human 17-HSD
Type 1
Recently, a model for the catalytic mechanism of 17HSD type 1 has been proposed by utilizing the threedimensional 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. 5). 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
1 and Fig. 5), and simultaneous substitutions of His221
and Glu282 resulted in similar inactivation of the enzyme in vitro (Table 1) as observed after the substitution of His221 alone (28). However, in cultured Sf9
insect cells (Fig. 5), all the His221 and/or Glu282-sub-
stituted 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.
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 1) and in
cultured Sf9 cells (Fig. 5). 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 7 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
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
Characterization of Human 17b-HSD Type 1
Fig. 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.
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 Ala170Glu1Phe172 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
81
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 bD, 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 aF-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 aE and strand bE 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
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Fig. 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 (6SD) 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.
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 posttranslational modifications were not detected. Furthermore, three isoforms resolved by IEF showed identical
densities after silver staining (Fig. 1C). 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 threedimensional 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 pro-
cedures 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 sub-
Characterization of Human 17b-HSD Type 1
strate 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 substrateprotein 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 (24–26, 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.
83
using the BaculoGold Transfection System (Pharmingen, San
Diego, CA) as previously described (28). Sf9 cells were grown
at a density of 2.0 3 106 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 3 g for 10 min,
washed once with PBS, and stored at 270 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 3
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 3 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 mM NADP1, 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 3 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 270 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 ml 70% trifluoroacetic acid. Thereafter, a
sample of 1 ml was subjected to MALDITOF-mass spectrometric analysis (KOMPACT MALDI III, Kratos Analytical,
Manchester, UK).
Modeling of Human 17-HSD Type 1 Proteins
MATERIALS AND METHODS
Chemicals and Reagents
[g-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-3,17dione (110 Ci/mmol) were purchased from Amersham Life
Science (Little Chalfont, UK). 17b-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
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 17HSD 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 (Ala170Glu1Phe172). 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
(59-TTATATTAGCGGCCGCACCATGGCCCGCACCGTG-39 and
59-TATATGAATTCAGGAAGCCTTTACTGCGGGGC-39) and the
internal primers presented in Table 2. In PCR reactions, wild type
cDNA was used as a template except in the cases of
MOL ENDO · 1997
84
Vol 11 No. 1
Table 2. Internal Primers Used for Construction of Substituted Human 17-HSD Type 1 Enzymes
Human 17-HSD Type 1 Substitution
Primer
Sequence
Reverse
Forward
59-CGCGTCCGGCACCGCGCCT-39
59-GCGCGGTGCCGGACGCGTG-39
Deletion mutation
Forward
59-TATATGAATTCTTAGGCCTTTGCCGGAACGT-39
Ser142Ala
Reverse
Forward
59-TCCCACGGCCCCGGTCACCAA-39
59-GTGACCGGGGCCGTGGGAGGA-39
Lys159Ala
Reverse
Forward
59-CGCGAACGCGCTGGCGCAAT-39
59-CGCCAGCGCGTTCGCGCTCGA-39
Leu111GluVal113Phe
Reverse
Forward
59-TACATTGAAGTCCTCCACAGAGG-39
59-TCTGTGGAGGACTTCAATGTAGTA-39
Ala170Glu 1 Phe172
Reverse
Forward
59-GCAGCAGGAAAACCTCCAGACT-39
59-AGTCTGGAGGTTTTCCTGCTGC-39
Glu282Ala
Reverse
Forward
59-GAACACTGCCCGGTGCATG-39
59-GCACCGGGCAGTGTTCGGC-39
Glu282Gln
Reverse
Forward
59-GAACACTTGCCGGTGCATG-39
59-GCACCGGCAAGTGTTCGGC-39
134
Ser
Ala
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.
The substituted proteins were produced in Sf9 insect cells
in 600-ml tissue culture flasks at a cell density of 30 3 106
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 3 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-ml sample was then applied,
the system was operated at a flow rate of 60 ml/min, and
30-ml 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 mg/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 mmol of substrate/liter). The reactions were started
by adding a cofactor (NAD1/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 reversephase 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 3 150 mm) connected to a HPLC
system (Waters, Milford, MA). Radioactivity was measured by
Characterization of Human 17b-HSD Type 1
an on-line b-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 6 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 3 106 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 mM [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 220 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 mCi [g-32P]ATP, and 20
U of the catalytic subunit of PKA in a final reaction volume of
50 ml. The amount of substrate was 15 mg of protein per
reaction. After 30 min incubation at 30 C, the reactions were
stopped by keeping them on ice for 10 min. [g-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.
Received June 3, 1996. Re-revision received and accepted
October 4, 1996.
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.
85
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