cDNA Cloning, Expression, and Mutagenesis Study of Liver-type
Prostaglandin F Synthase*
Toshiko
Suzuki
§,
Yutaka
Fujii¶,
Masashi
Miyano
,
Lan-Ying
Chen**,
Tomohiro
Takahashi
, and
Kikuko
Watanabe
§§
From the
Second Department, Osaka Bioscience
Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, ¶ Fukui
Medical School, 23-3 Shimoaizuki, Matsuoka-cho,
Yoshida, Fukui 910-1103,
Central Pharmaceutical Research
Institute, Japan Tobacco, Inc., 1-1 Murasaki-cho,
Takatsuki, Osaka 569-1125, Japan, the ** Department of Biochemistry,
Cardiovascular Institute and Fu-Wai Hospital, Beijing 100037, China,
and
Bioscience Research
Laboratory, Mochida Pharmaceutical Co., 1-1-1Kamiya, Kita,
Tokyo 115-0043, Japan
 |
ABSTRACT |
Prostaglandin (PG) F synthase catalyzes the
reduction of PGD2 to 9
,11
-PGF2 and
that of PGH2 to PGF2
on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme
(Km value of 120 µM for
PGD2 (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi,
O. (1985) J. Biol. Chem. 260, 7035-7041) and the
liver-type one (Km value of 10 µM for
PGD2 (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992)
Arch. Biochem. Biophys. 296, 17-26)). The liver-type
enzyme was presently found to consist of a 969-base pair open reading
frame coding for a 323-amino acid polypeptide with a
Mr of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with
the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase
(DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid
sequence of the liver-type PGF synthase was identical with that of
bovine liver DD3. The liver-type PGF synthase was expressed in COS-7
cells, and its recombinant enzyme had almost the same properties as the
native enzyme. Furthermore, to investigate the nature of catalysis
and/or substrate binding of PGF synthase, we constructed and
characterized various mutant enzymes as follows: R27E, R91Q, H170C,
R223L, K225S, S301R, and N306Y. Although the reductase activities
toward PGH2 and phenanthrenequinone (PQ) of almost all
mutants were not inactivated, the Km values of
R27E, R91Q, H170C, R223L, and N306Y for PGD2 were increased from 15 to 110, 145, 75, 180, and 100 µM, respectively,
indicating that Arg27, Arg91,
His170, Arg223, and Asn306 are
essential to give a low Km value for
PGD2 of the liver-type PGF synthase and that these amino
acid residues serve in the binding of PGD2. Moreover, the
R223L mutant among these seven mutants especially has a profound effect
on kcat for PGD2 reduction. The
Km values of R223L, K225S, and S301R for PQ were
about 2-10-fold lower than the wild-type value, indicating that the
amino acid residues at 223, 225 and 301 serve in the binding of PQ to
the enzyme. On the other hand, the Km value of
H170C for PGH2 was 8-fold lower than that of the wild type,
indicating that the amino acid residue at 170 is related to the binding
of PGH2 to the enzyme and that Cys170 confer
high affinity for PGH2. Additionally, the 5-fold increase in kcat/Km value of the
N306Y mutant for PGH2 compared with the wild-type value
suggests that the amino acid at 306 plays an important role in
catalytic efficiency for PGH2.
 |
INTRODUCTION |
F series prostaglandins
(PG)1 are widely distributed
in various organs of mammals and exhibit a variety of biological
activities including constriction of pulmonary arteries (1, 2).
PGF2 is synthesized from PGE2,
PGD2, or PGH2 by PGE 9-ketoreductase, PGD
11-ketoreductase, or PGH 9,11-endoperoxide reductase, respectively. PGF
synthase (EC 1.1.1.188) was purified from bovine lung by Watanabe
et al. (3). It forms 9
,11
-PGF2 (4) from
PGD2 (PGD2 11-ketoreductase activity) and
PGF2
from PGH2 (PGH2 9,11-endoperoxide reductase activity) on the same molecule in the
presence of NADPH (3, 4). This enzyme catalyzes the reduction of other
carbonyl compounds including 9,10-phenanthrenequinone (PQ) as well as
that of PGD2 and PGH2 but does not catalyze the reduction of PGE2. Although PGD2
11-ketoreductase activity was competitively inhibited by PQ,
PGH2 9,11-endoperoxide reductase activity was not inhibited
by PGD2 or PQ (3). PGF synthase belongs to the aldo-keto
reductase family. The bovine lung PGF synthase is a monomeric protein
with a Mr of 36,666 consisting of 323 amino
acids, and its amino acid sequence shows high homology compared with
that of other aldo-keto reductase family members (5). PGF synthase has
two isozymes, one in the lung (3) and the other one in the liver (6),
with different Km values for PGD2 (120 and 10 µM, respectively). The regulation by metals, the
sensitivity to chloride ions, the inhibition by CuSO4 and
HgCl2, and the profile of immuno-precipitation with anti-bovine lung PGF synthase antibody are different between the two
isozymes (6). Although Kuchinke et al. (7) isolated a clone
(PGFS II) of PGF synthase from bovine liver and determined its amino
acid sequence, the 99% similarity with the amino acid sequence of lung
PGF synthase and the high Km value for PGD2 of this recombinant PGFS II indicated that its
cDNA was that of the lung-type enzyme even though it had been
isolated from liver. Until now, the primary structure of the liver-type
PGF synthase and the amino acids related to the affinity for
PGD2 have not yet been defined.
Dihydrodiol dehydrogenase (DD, EC 1.3.1.20) catalyzes the NADP-linked
oxidation of trans-dihydrodiols of aromatic hydrocarbons to
the corresponding catechols and is distributed in various mammalian tissues. DD, also belonging to the aldo-keto reductase superfamily, has
been purified from various animal tissues, i.e. human liver (8), rat liver (9, 10), rabbit liver (11), mouse liver (12), bovine
liver (13), guinea pig testis (14), and so forth. Human liver DD exists
in at least four multiple forms (DD1-DD4) with similar mass of about
36 kDa (8). Human DD3 was identified as an aldehyde reductase, and the
other three forms exhibited 3
-hydroxysteroid dehydrogenase (HSD, EC
1.1.1.213) activity. Bovine liver DD also has three multiple forms
(DD1-DD3), namely DD1 (3
-HSD), DD2 (high Km
aldehyde reductase), and DD3 (dihydrodiol-specific enzyme) (13). Human
liver DD1, DD2, and DD3 are not identical with bovine liver DD1, DD2,
and DD3, respectively, on the basis of their enzymatic properties
including the substrate specificity. The amino acid sequence of the
bovine lung-type PGF synthase (3, 7) showed an identity of 81, 79, 78, and 87% with that of human liver DD1, DD2, and DD4 (15, 16) and bovine liver DD3 (17). Among human liver DDs, DD1 and DD2 exhibited PGF
synthase activity with Km values of 12 and 79 µM, respectively, for PGD2, but this activity
of DD4 was not detected (16). Moreover, PGF synthase activity of bovine
liver DD3 has not yet been reported.
In the present study, we describe the primary structure of the bovine
liver-type PGF synthase and the enzymatic properties of the recombinant
enzyme in COS-7 cells. Based on the comparison of the amino acid
sequences among the liver-type and the lung-type PGF synthases and
human liver DDs, several mutants were constructed, and their enzymatic
properties were examined. The results of mutagenesis indicated the
amino acid residues related to the binding sites of PGD2,
PGH2, and PQ.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[5,6,8,9,12,14,15-3H]PGD2
(3.7 TBq/mmol) was obtained from NEN Life Science Products.
[1-14C]PGH2 was prepared as described
previously (18), with acetone powder of sheep vesicular gland
microsomes (Ran Biochemicals, Tel Aviv, Israel) used as a source of PG
endoperoxide synthase. Authentic PGs were kindly donated by Ono
Pharmaceutical Co. pEF-BOS mammalian expression vector was a generous
gift from Dr. S. Nagata. Other materials and commercial sources were as
follows: NADP, NADPH, glucose 6-phosphate, and glucose-6-phosphate
dehydrogenase from bakers' yeast (type IX), from Sigma (Japan); Red
Sepharose and NAP10, from Amersham Pharmacia Biotech (UK); precoated
silica gel glass plates (F254), from Merck (Germany). Other chemicals were at least of reagent grade.
Internal Amino Acid Sequences of the Liver-type PGF
Synthase--
The bovine liver PGF synthase was purified to apparent
homogeneity as described previously (6).
S-Carboxymethylation and lysyl-endopeptidase digestion of
the purified enzyme and the purification of fragments digested by
lysyl-endopeptidase were done as described previously (5). The purified
peptide fragments were sequenced by automated Edman degradation
with an Applied Biosystems Inc. sequencer (Perkin-Elmer).
cDNA Cloning of Bovine Liver PGF Synthase--
Total RNA was
prepared from bovine liver by the acid guanidinium
thiocyanate/phenol/chloroform extraction method (19).
Poly(A)+ RNA was prepared by use of an mRNA
Purification Kit (Amersham Pharmacia Biotech, UK) according to the
manufacturer's manual. A (dT)12-18 (Life Technologies,
Inc.) primed cDNA was synthesized from 2 µg of
poly(A)+ RNA by SuperScript II reverse transcriptase (Life
Technologies, Inc.).
Degenerative reverse transcriptase-polymerase chain reaction (PCR)
using mixed oligonucleotide primers was performed to obtain a partial
cDNA fragment for screening of a bovine liver cDNA library. Mixed oligonucleotide primers were designed according to the amino acid
sequences of peptides I, II, III, and IV shown in Fig. 1 and to the
nucleotide sequence of the lung-type PGF synthase (5). Each primer was
synthesized with an Applied Biosystems Inc. DNA synthesizer
(Perkin-Elmer), and two sets of the sequences of sense and antisense
primers were used as follows: (i) 5'-TTCCGCCATAT(CTA)GACAGTGCT-3' (corresponding to peptide I, 21-mers) named P1 and
5'-GTCAAACACCTG(TGA)ATATTCTC-3' (corresponding to peptide III, 21-mers)
named P2; (ii) 5'-GTGTCCAACTTCAACCACAAG-3' (corresponding to
peptide II, 21-mers) named P3 and 5'-ATATTCTTC(AGCT)ACAAATGGGTA-3' (corresponding to peptide IV, 21-mers) named P4. The reverse
transcriptase-PCR was conducted with mRNA used as a template and
primers P1/P2 for one PCR and P3/P4 for the other one under the
conditions of denaturation at 98 °C for 15 s, annealing at
65 °C for 30 s, and elongation at 74 °C for 30 s by KOD
polymerase (Toyobo, Japan). After 20 cycles of PCR, the products were
ethanol-precipitated and separated on 1% agarose gel. Two bands were
recovered from the gel by use of a QIAEX II Gel Extraction Kit (Qiagen,
Netherlands). Each band was ligated to a BlueScript SK II (+) vector
(Toyobo, Japan) by use of a ligation kit (Takara, Japan), and the
resulting constructs were transfected into Escherichia coli
DH10B competent cells (Life Technologies, Inc.). Plasmids were purified
with a Qiagen plasmid purification kit and sequenced with an Applied
Biosystems Inc. automated DNA sequencer 373A (Perkin-Elmer). Two bands,
one of 720 base pairs (P1/P2) and one of 477 base pairs (P3/P4),
encoded the internal cDNA and were used as two different probes for
screening of the library.
The bovine liver cDNA library was constructed from 5 µg of
poly(A)+ RNA with SuperScriptTM Plasmid System (Life
Technologies, Inc.) for cDNA synthesis and a Plasmid Cloning Kit
(Life Technologies, Inc.) according to the manufacturer's manual. The
resulting constructs were transformed into ElectroMax, DH12S competent
cells (Life Technologies, Inc.) by the electroporation method using a
Gene-Pulser (Bio-Rad). The library yielded 2.0 × 105
independent clones. Full-length cDNA clones were obtained by the
colony hybridization method. All clones were spread on 20 sheets of
nylon filters for the master filter, and then two filters were
replicated from each master filter. The replicate filters were
alkaline-denatured and fixed by baking at 80 °C for 2 h. Two
reverse transcriptase-PCR products, 720 and 477 base pairs described
above, were randomly labeled by [
-32P]dCTP (111 TBq/mmol, Amersham Pharmacia Biotech, UK) with a Megaprime random
primer labeling kit (Amersham Pharmacia Biotech, UK) and used as two
probes for hybridization. After hybridization in 5× SSCP, 0.1% SDS,
100 µg salmon sperm DNA, and 10× Denhardt's solution at 65 °C
for overnight, each filter was washed extensively twice in 2× SSC,
0.1% SDS at room temperature for 5 min and twice in 0.5× SSC, 0.1%
SDS at 60 °C for 30 min. Thirty two double-positive clones against
the two different probes were obtained from 2.0 × 105
independent clones. Six clones were picked up and sequenced with an
Applied Biosystems Inc. automated DNA sequencer 373A. All clones coded
for full-length cDNAs of bovine liver PGF synthase. One of these
six clones was named pSPORT-BLiFS27.
Northern Blot Analysis--
For Northern blot analysis, total
RNA (20 µg), which was isolated from bovine liver with a total RNA
purification kit (Amersham Pharmacia Biotech, UK) according to the
manufacturer's manual, was separated on 1.0% agarose gel and
transferred to nylon membranes. The 477-base pair fragment (P3/P4
described above) was labeled with a BcaBEST Labeling Kit (Takara,
Japan) according to the manufacturer's manual using
[
-32P]dCTP and was used as a probe. The membranes were
prehybridized in Rapid Hybridization buffer (Amersham Pharmacia
Biotech, UK) at 65 °C for 1 h and then hybridized for 2.5 h after addition of the radiolabeled probe. The membranes were washed
for 5 min once at room temperature in 2× SSC, 0.1% SDS, twice for 30 min at 65 °C in 0.5× SSC, 0.1% SDS, and twice for 30 min at
65 °C in 0.2× SSC, 0.1% SDS. Autoradiograms were examined with a
BAS-2000 system analyzer (Fuji Film, Japan).
Expression of Bovine Liver PGF Synthase in COS-7 Cells and
Purification of Its Expressed Protein--
The bovine liver cDNA
insert was removed from pSPORT-BLiFS27 by digestion with
EcoRI and AflII, and its ends were blunted. The
blunt-ended insert containing the complete coding region was subcloned
into the blunt-ended BstXI sites of the pEF-BOS mammalian expression vector (20). Monkey COS-7 cells were cultured in Dulbecco's
modified Eagle's medium (Nissui Co., Tokyo) containing 10% fetal calf
serum. COS-7 cells (5 × 106 cells) were transfected
with 20 µg of plasmid DNA by the electroporation method using a Gene
Pulser (Bio-Rad). These cells were incubated in a 5% CO2
incubator at 37 °C for 72 h. The transfected COS-7 cells were
sonicated in 3 volumes of 10 mM potassium phosphate buffer
(KPB) (pH 7.0). The recombinant enzyme was purified by the method of
Chen et al. (6) with a minor modification. The cytosol
fraction of the homogenated cells, which was centrifuged at
100,000 × g, was subjected to ammonium sulfate
fractionation between 40 and 75% saturation. The precipitate formed
was suspended in 500 µl of 10 mM KPB (pH 7.0) and
desalted by passage through a NAP-10 column. The desalted sample was
applied to a Red Sepharose column, and the enzyme was eluted with 10 mM KPB (pH 7.0) containing 1 M KCl and 1 mM NADP. About 2.8-fold purification of the recombinant protein was achieved, and the apparent homogeneity was concluded following SDS-polyacrylamide gel electrophoresis (PAGE) and staining with Two-dimensional Silver Stain·II "Dai-ichi" (Dai-ichi Pure Chemicals Co., Ltd., Japan). A polyclonal antibody against PGF synthase
was raised in a rabbit by the same procedure as described previously
(3), with the enzyme purified from bovine liver used as the immunogen
(6). For Western blot analysis, the purified enzyme was subjected to
SDS-PAGE and electrophoretically transferred to a polyvinylidene
difluoride membrane (Amersham Pharmacia Biotech, UK). Protein bands
were immunostained with the anti-bovine liver PGF synthase antibody and
reagents from a Vectastain ABC kit (Vector Laboratories) and visualized
with an Enhanced Chemiluminescence Kit (ECLTM, Amersham Pharmacia
Biotech, UK).
Enzyme Assay--
The PGD2 11-ketoreductase,
PGH2 9,11-endoperoxide reductase, and PQ reductase
activities of the recombinant protein were measured as described
previously (3). The standard assay mixture for PGD2
11-ketoreductase contained 0.1 M KPB (pH 6.5), 0.5 mM NADP, 5 mM glucose 6-phosphate,
glucose-6-phosphate dehydrogenase (1 unit), 1.5 mM
[3H]PGD2 (3.7 KBq), and enzyme in a total
volume of 50 µl. Incubation was carried out at 37 °C for 30 min.
The PGH2 9,11-endoperoxide reductase activity was assayed
under the same conditions as those of the PGD2
11-ketoreductase activity except that 40 µM
[1-14C]PGH2 (4 MBq) was used as a substrate
in place of 1.5 mM [3H]PGD2 and
that the incubation time was 2 min. The PQ reductase activity was
measured spectrophotometrically at 37 °C by following a decrease in
absorbance at 340 nm in the assay mixture consisting of 0.1 M KPB (pH6.5), 80 µM NADPH, 10 µM PQ, and enzyme in a total volume of 0.5 ml. One unit
of enzyme activity was defined as the amount that produced 1 µmol of
PGF2 per min at 37 °C. Specific activity was expressed
as the number of units/mg of protein. Protein was determined according
to the method of Lowry et al. (21).
Site-directed Mutagenesis--
A mutagenesis study was performed
by the method of Jones et al. (22). The mutagenesis primers
for the mutant R27E were designed as follows: LiFSPN,
5'-CAAACAATGGATCC-3'; LuFSP1, 5'-TTGCACCTGAGGAGGTTCC-3'; LuFSP2, 5'-CCTCCTCAGGTGCAAAGGTT-3'; LiFSPC,
5'-GAATGCACGTGTACAGCT-3'. The bold letters of LuFSP1 and LuFSP2 were
the sites of mutagenesis. As shown in Fig. 1, one PCR between LiFSPN
and LuFSP2 and the other one between LuFSP1 and LiFSPC were conducted
with pSPORT-BLiFS27 harboring the full-length cDNA for bovine liver
PGF synthase as a template. The conditions of PCR were described above.
After 20 cycles of PCR, the products were purified by 1% agarose gel electrophoresis, and the purified products were treated with Klenow fragment of DNA polymerase I after recovery. These products were mixed
at the ratio of 1 to 1, and the second PCR was conducted using LiFSPN
and LiFSPC as described above. The product of the second PCR was
blunt-ended and then was ligated to the blunted BstXI sites
of the pEF-BOS expression vector. Consequently, the mutant of R27E was
formed. The other mutant enzymes were formed by the same procedure as
used for the R27E mutant.
 |
RESULTS |
cDNA Cloning of Bovine Liver PGF Synthase--
Screening of
2.0 × 105 clones with the two probes (P1/P2 and P3/P4
products described under "Experimental Procedures") gave 32 double-positive clones, and six independent clones of these 32 clones
were picked up. DNA sequencing confirmed that these clones coded for
full-length cDNAs of bovine liver PGF synthase. The deduced amino
acid sequences of bovine liver PGF synthase contained all the amino
acid sequences of the nine peptide fragments obtained from the native
bovine liver enzyme (Fig. 1). The bovine liver PGF synthase cDNA clone BLiFS27 contained a polyadenylation signal after the stop codon (Fig. 1), showing that it coded for a
full-length PGF synthase. BLiFS27 contained an open reading frame of
969 base pairs coding for 323 amino acids. The calculated Mr of the bovine liver enzyme was 36,742, a
value similar to that of the native enzyme, which is about 36 kDa (6).
The identity between the liver and lung enzymes was 87% at the amino
acid level and 90% at the nucleotide level. As shown in Fig.
2, this enzyme showed a high identity in
amino acid sequence with not only bovine lung PGF synthase (87%) but
also human liver DD1 (79%), DD2 (77%), and DD4 (76%). Moreover, its
sequence was identical with that of bovine liver DD3.

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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of bovine liver PGF synthase. The underlined
letters indicate the peptide sequences from the purified native
enzyme. The arrows indicate the primers used for PCR or
mutagenesis as described under "Experimental Procedures." The
bold letters in the nucleotide sequence show the
polyadenylation signal. The dashed lines show the amino acid
sequences of peptides I, II, III, and IV for design of the mixed
oligonucleotide primers.
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Fig. 2.
Amino acid alignment of bovine PGF synthase
isozymes and human liver DD isozymes. The amino acid sequences of
the bovine liver-type PGF synthase (LiPGFS), the bovine
lung-type ones (LuPGFSI (5) and LuPGFSII (7)),
and human liver DD isozymes (hDD1 (16), hDD2
(16), and hDD4 (15)) are aligned. The arrowheads
show the positions of site-directed mutagenesis. Arg27,
Arg91, His170, Arg223,
Lys225, Ser301, and Asn306 were
converted to Glu, Gln, Cys, Leu, Ser, Arg, and Tyr (R27E, R91Q, H170C,
R223L, K225S, S301R, and N306Y), respectively. Dots indicate
the same amino acid residues as LiPGFS, and asterisks
indicate the same amino acid residues among the six proteins.
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Identification and Size Determination of Bovine Liver PGF Synthase
mRNA by Blot Hybridization Analysis--
Fig.
3A shows the result of the
Northern blot analysis of bovine liver mRNA with the P3/P4 PCR
product (477 base pairs) used as a probe. From its migration in a
denaturing gel system, the sequence of PGF synthase mRNA from
bovine liver was estimated to be 1400 nucleotides. Therefore, assuming
a length for the poly(A) tail of 100-150 nucleotides, the insert
cDNA sequence of 1139 nucleotides extended nearly the full length
of the mRNA.

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Fig. 3.
Northern blot analysis of bovine liver
(A), SDS-PAGE (B), and Western blot analysis
(C) of the cytosol and the purified recombinant bovine
liver PGF synthase. A, total RNA (10 µg) obtained
from bovine liver was hybridized with a
[ -32P]dCTP-labeled P3/P4 probe. The positions at 28 S
and 18 S of ribosomal RNA are shown. B, SDS-PAGE silver
stain and (C) Western blot analysis using the antiserum
against the purified native bovine liver PGF synthase: the native
enzyme purified from bovine liver (lane 1, 0.1 µg for
B and 0.01 µg for C), and the cytosol
(lane 2, 0.5 µg for B and 0.05 µg for
C), the ammonium sulfate fraction (lane 3, 0.5 µg for B and 0.05 µg for C), and the Red
Sepharose fraction (lane 4, 0.1 µg for B and
0.01 µg for C) of expressed protein in COS-7 cells were
loaded into the indicated lanes. The positions of the molecular mass
standards are shown: phosphorylase b (97,400), bovine serum
albumin (66,300), ovalbumin (42,400), carbonic anhydrase (30,000),
soybean trypsin inhibitor (20,100).
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Expression of Bovine Liver PGF Synthase in COS-7 Cells and
Purification of Its Expressed Protein--
pBOS-BLiFS27 carrying the
full-length bovine liver PGF synthase in the mammalian expression
vector pEF-BOS was prepared by use of the strategy described under
"Experimental Procedures." COS-7 cells were transfected with
pBOS-BLiFS27. The recombinant protein was expressed transiently in
COS-7 cells and was located in the cytosol fraction. The recombinant
protein was purified to an apparent homogeneity by the following
purification steps: ammonium sulfate fractionation between 40 and 75%
saturation and Red Sepharose column chromatography, as described under
"Experimental Procedures." About 2.8-fold purification of the
PGD2 11-ketoreductase activity was achieved from the
cytosol of COS-7 cells with a yield of 44% (Table
I). A sample of each of the purification
steps was subjected to SDS-PAGE. Silver staining of the gel indicated that an approximately 36.7-kDa protein was produced in the cells harboring pBOS-BLiFS27, and this protein was purified to an apparent homogeneity (Fig. 3B). Western blot analysis of each sample
revealed that the 36.7-kDa protein was recognized by anti-bovine liver PGF synthase antibody (Fig. 3C). The molecular weight of the
expressed enzyme was the same as that of the native enzyme, as shown in Fig. 3, B and C. No protein from the control
COS-7 cells bearing pEF-BOS without the insert DNA interacted with this
antibody (data not shown).
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Table I
Purification of recombinant wild-type PGF synthase and R27E, R91Q,
H170C, R223L, K225S, S301R, and N306Y mutants
All activity measurements were performed under standard assay condition
for the PGD2 11-ketoreductase activity as described under
"Experimental Procedures." One milliunit of enzyme activity was
determined as the amount that produced 1 nmol of PGF2/min at
37 °C.
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The purified recombinant protein exhibited enzymatic properties similar
to those of the native enzyme. The Km values for
PGD2, PGH2, and PQ were 15, 25, and 1.1 µM, respectively (Table II). The low Km value
for PGD2 was essentially identical to that of the native
liver-type enzyme and was different from the high Km
value (120 µM) of the lung-type one. The specific
activities of the recombinant protein for PGD2,
PGH2, and PQ were 23, 9, and 186 milliunits/mg of protein,
respectively. However, PGE2 was not reduced. Moreover, the
IC50 value for inhibition of PGD2
11-ketoreductase activity of the expressed enzyme by CuSO4 was 0.5 mM (data not shown), which was almost the same as
that for inhibition of the native liver-type enzyme (0.4 mM) but unlike that for the lung-type enzyme (0.003 mM) (6). These results indicate that the cloned cDNA
coded for the liver-type PGF synthase and not for the lung-type
synthase.
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Table II
Comparison of kinetic constants for PGD2, PGH2, and
phenanthrenequinone among expressed liver PGF synthase, native liver
PGF synthase, and native lung PGF synthase
Enzyme assays of expressed liver PGF synthase with PGD2,
PGH2, and phenanthrenequinone were conducted under standard
assay conditions including the enzyme (3.4 µg for PGD2, 17 µg for PGH2, and 3.4 µg for PQ), and measured by the
radioisotope method for PGD2 and PGH2 and by the
spectrophotometric method for phenantherenequinone at 37 °C as
described under "Experimental Procedures." One milliunit of enzyme
activity was determined as the amount that produced 1 nmol of
PGF2/min at 37 °C. Enzymes purified to apparent homogeneity
were used as enzyme sources.
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Site-directed Mutagenesis--
The comparison of amino acid
sequences among PGF synthases of bovine liver and lung, and human DD1,
DD2, and DD4 is shown in Fig. 2. The Km values of
the lung-type (3) and the liver-type (6) PGF synthases and the human
liver DD1 and DD2 for PGD2 (16) were 120, 10, 12, and 79 µM, respectively. DD4 does not catalyze the reduction of
PGD2 (16). To determine which amino acid residues were
related to PGF synthase activity, especially to PGD2
11-ketoreductase activity, we conducted a site-directed mutagenesis
study. Arg27, Arg91, Arg223,
Lys225, Ser301, or Asn306 of the
liver-type PGF synthase with a low Km value for PGD2 was changed to Glu, Gln, Leu, Ser, Arg, or Tyr,
respectively, the latter of which are the residues of the lung-type PGF
synthase with a high Km value for PGD2.
In addition to these mutations, His170 was changed to the
Cys of DD4 (Fig. 2), which has no PGD2 11-ketoreductase activity. The mutant enzyme, R27E, R91Q, H170C, R223L, K225S, S301R, or
N306Y, was expressed in COS-7 cells and was purified to an apparent
homogeneity (Fig. 4) as described under
"Experimental Procedures." The results of their final purification
step are shown in Table I. The kcat,
Km, and
kcat/Km values for three
representative substrates, i.e. PGD2,
PGH2, and PQ, of the purified mutant enzymes are shown in
Table III.

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Fig. 4.
SDS-PAGE (A) and Western blot
analysis (B) of PGF synthase mutants. Wild-type and
the mutant PGF synthases were purified in an identical manner and
analyzed by SDS-PAGE silver stain (0.1 µg for each lane)
(A) and Western blot analysis (0.01 µg for each lane)
(B) using the antiserum against the purified native bovine
liver PGF synthase: the native enzyme purified from bovine liver
(lane 1), the purified wild-type recombinant enzyme
(lane 2), and the purified R27E (lane 3), R91Q
(lane 4), H170C (lane 5), R223L (lane
6), K225S (lane 7), S301R (lane 8), and N306
(lane 9) mutants cells were loaded into the indicated lanes.
The positions of the molecular mass standards are shown in Fig.
3.
|
|
View this table:
[in this window]
[in a new window]
|
Table III
Comparison of kinetics for PGD2, PGH2, and
phenanthrenequinone among expressed liver PGF synthase and mutants
The enzyme activities for PGD2, PGH2, and
phenanthrenequinone were measured in the presence of wild-type PGF
synthase and seven mutants (0.06-15 µg for PGD2, 0.3-19
µg for PGH2, and 0.3-10 µg for PQ) by the methods shown in
Table II.
|
|
Although the kcat/Km values
of almost all mutants for PGH2 and PQ were retained above
50% of the wild-type value, these values of all mutants for
PGD2 decreased. The
kcat/Km values of R27E, R91Q,
H170C, and N306Y for PGD2 were only about 10% that of the
wild type, and the Km values of R27E, R91Q, H170C,
R223L, and N306Y for PGD2 were 110, 145, 75, 180, and 100 µM, respectively. These Km values were
5-10-fold higher than the wild-type value and were almost the same as
that of the lung-type PGF synthase. Considering the amino acid residues of these mutants were changed from the liver-type PGF synthase to the
lung-type synthase for R27E, R91Q, R223L, and N306Y or to DD4 for
H170C, these results suggest that Arg27, Arg91,
His170, Arg223, and Asn306 are
essential to give a low Km value for
PGD2 and that these amino acid residues play an important
role on the binding for PGD2 to PGF synthase. In addition,
the R223L mutant increased kcat for
PGD2 5-fold, indicating that the amino acid residue at 223 has a profound effect on kcat for
PGD2 reduction. The
kcat/Km values of R27E, R91Q,
and N306Y mutants for PQ were 50-80% of that value of the wild type,
indicating that Arg27, Arg91, and
Asn306 have little effect on the catalytic efficiency for
PQ, less than that of these residues on the catalytic efficiency for
PGD2. Moreover, the Km values of R223L,
K225S, and S301R for PQ were about 2-10-fold lower than the wild-type
value, and the kcat/Km values
of these mutants for PQ were 3-15-fold higher than the value of the
wild type. These results suggest that the amino acid residues at 223, 225, and 301 are related to the binding for PQ to the enzyme and that
the binding to the carbonyl group is different between PG and quinone
compounds. On the other hand, the Km value of H170C
for PGH2 was 8-fold lower than that of the wild type, and
the kcat/Km values of H170C
and N306Y for PGH2 were 5-6-fold higher than that of the
wild type. These results indicate that the amino acid residue at 170 is
related to the binding for PGH2 and that Cys170
seems to confer greater affinity for PGH2 than His.
Moreover, the amino acid residue at 306 plays an important role in
catalytic efficiency for PGH2.
 |
DISCUSSION |
We isolated a clone of the liver-type PGF synthase with a low
Km value for PGD2 from the cDNA
library of bovine liver, expressed the enzyme in COS-7 cells, and
constructed seven mutants. Moreover, we examined the enzymatic
properties of the wild-type enzyme and of the mutant enzymes, and we
investigated the amino acid residue(s) related to the affinity of PGF
synthase for the substrates.
The amino acid sequence of the liver-type PGF synthase consisted of 323 amino acid residues with a Mr of 36,742 (Fig. 1)
and showed 87% identity with that of the lung-type synthase (Fig. 2).
When the liver-type enzyme was expressed in COS-7 cells (Fig. 3), the
recombinant purified protein (Fig. 3, B and C,
and Table I) was essentially identical to the native liver-type enzyme and not to the lung-type enzyme, based on the enzymatic properties (Table II) including the low Km value (15 µM) for PGD2 and the results of Western blot
analysis (Fig. 3). These results suggest that this liver-type PGF
synthase is distinct from the lung-type PGF synthase isolated from the
cDNA library of bovine lung (5) or liver (7).
Recently, Jez et al. (23) reported on a structure/function
analysis of the aldo-keto reductase superfamily. They reported that
five amino acid residues, i.e. Asp50,
Asn167, Gln190, Ser271, and
Arg276, and three residues, i.e.
Asp50, Tyr55, and Lys84, function
in cofactor binding and in the active site, respectively. Based on the
locations of the cofactor-binding pocket and the active site, a
putative substrate-binding site was also proposed. However, the binding
site for PGs has not yet been reported. The amino acid sequence of the
liver-type PGF synthase was highly homologous with those sequences of
DD1, DD2, and DD4 of human liver (Fig. 2), and DD1 and DD2 exhibited
PGD2 11-ketoreductase activity (16). Based on the
comparison among the amino acid sequences of human liver DDs (15, 16)
and the lung-type and the liver-type PGF synthases, we studied the
site-directed mutagenesis to change seven amino acid residues as
follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. All mutants
expressed the proteins in COS-7 cells to almost the same extent as the
wild-type protein, suggesting that the tertiary structures of these
mutants were not drastically changed. Moreover, all mutants retained
the activity with above 50% of
kcat/Km values of the wild
type for PGH2 and PQ, indicating that the structures of all
mutants were conserved. Therefore, the change in the enzymatic
properties of the mutants reflected the mutation of the amino acid
residue and not a change in the structure. Judging from the results of
this study, Arg27, Arg91, His170,
Arg223, and Asn306 are essential to give a low
Km value for PGD2 and play an important
role in the binding of PGD2; and the amino acid residue at
223 has a significant effect on kcat for
PGD2 reduction. Moreover, the results of
Km of the R223L, K225S, and S301R mutants for PQ and
PGD2 show that R223L, K225S, and S301R mutations acquired high binding for PQ and low for PGD2. Leu223,
Ser225, and Arg301 of the lung-type PGF
synthase have a positive effect on the binding of PQ. In terms of the
binding site for PGH2, the amino acid residue at 170 seems
to be related to the binding of this PG and that at 306 plays an
important role in kcat/Km of
this PG.
Twelve amino acid residues among the 42 amino acid residues of the
liver-type enzyme that differed from those of the lung-type one were
located in the
-helix or
-sheet, and the other 30 amino acid
residues were located in loop structures of the tertiary structure of
the liver-type PGF synthase, as inferred from data on human aldose
reductase (24, 25) and rat 3
-HSD (26), which showed 46 and 70%
identity, respectively, in terms of amino acid sequence with bovine
liver-type PGF synthase. As a general rule, an amino acid(s) located in
an
-helix or a
-sheet is involved in supporting the tertiary
structure and that in loop structures of aldo-keto reductases is
related to define substrate specificity (23, 27). Although
His170 is located in an
-helix of the inferred tertiary
structure of the liver-type PGF synthase, the amino acid residue at
this position in the aldo-keto reductase family shows variation and is
located near Asn167, which makes hydrogen bonds with the
carboxyamide moiety of the cofactor (23). His170 was
changed to Cys of DD4, which has no PGD2 11-ketoreductase activity. The kcat/Km values
of this mutant for PGD2 was 16% that of the wild type, and
its 1/Km value decreased to about 20% that of the
wild type. On the other hand, the
kcat/Km values of this mutant
for PGH2 were about 6-fold that of the wild type, and its
1/Km value of this mutant increased to 8-fold that
of the wild type. These results indicate that the amino acid residue at
170 seems to be related to the binding for PGD2 and to that
for PGH2. The reverse effects of the H170C mutant on
PGD2 and PGH2 reductase activities indicate
that His and Cys at 170 play an important role in the binding of
PGD2 and PGH2.
Arg27, Arg91, Arg223,
Lys225, Ser301, and Asn306 located
in a loop structure are related to the binding site of
PGD2, PQ, or PGH2. Jez et al. (27)
reported that Trp86 and Trp227 of the 3
-HSD
near the active site may have roles in substrate binding. They proposed
that Trp86 is important in binding to a steroid ligand,
whose A-ring lies between this Trp and the cofactor, and that
Trp227 interacts with the C- and/or D-rings of steroid
ligands. The effect of R91Q on PGD2 taken together with the
report on Trp86 of the 3
-HSD suggests that
Arg91 near Trp86 is a part of the
substrate-binding pocket for PGD2. Moreover, although the
effects of W227Y on the
kcat/Km values for steroids
are dramatic, smaller effects of this mutant on those for one-, two-,
and three-ring substrates are also shown in this order (27).
PGD2 has one cyclopentane ring and two long tails (
- and
-chains), and Arg223 and Lys225 are also
located near Trp227. The results of the R223L mutant for
PGD2 suggest that Arg223 is an essential amino
acid residue to give a low Km value for
PGD2 of the liver-type PGF synthase and that the amino acid
residue at 223 plays an important role in kcat
of PGD2 reduction. The results of the R223L and K225S
mutants for PQ suggest that Leu223 and Ser225
have a significant effect on the binding of PQ. PGD2 and PQ
seem to bind to the same apolar pocket of PGF synthase differently, like steroids, non-steroidal anti-inflammatory drugs, and aldose reductase inhibitors of 3
-HSD (27). Moreover, the effect of S301R on
PQ suggests that Ser301 also has a role in the binding of
PQ to the enzyme. Asn306, located in the C-terminal region,
may have structural importance for the binding of the substrate.
Members of the NADPH-dependent aldo-keto reductase
superfamily are in part distinguished by unique C-terminal loops
(28-30). C-terminal loops of aldo-keto reductase are unique for each
member and differ drastically in length and amino acid composition; and
the C-terminal loop is critical for catalytic efficiency and for
substrate and inhibitor specificity (28-30). In the case of PGF
synthases, the C-terminal region may be also critical for the affinity
and specificity for the
substrate.2 The effect on the
binding of the N306Y mutant for PGD2 and that on
kcat/Km of this mutant for
PGH2 also suggest that Asn306 is a critical
determinant of PGs.
Considering the one binding site for NADPH in the inferred tertiary
structure of the liver-type PGF synthase, the catalytic site for the
various substrates of aldo-keto reductase including PGF synthase seems
to be the same. Tyr55 of aldo-keto reductase is favored as
the catalytic acid in the reaction mechanism (23). Tyr55 of
PGF synthase also plays the same role as that of the corresponding position in the other aldo-keto reductases.2 The Y55F
mutation eliminated PGD2, PGH2, and PQ
reductase activities of PGF synthase completely.2 However,
although PGD2 and PQ reductase activities of Y55Q were eliminated completely, only PGH2 reductase activity of this
mutant was stimulated, 27-fold.2 These results suggest that
Tyr55 is favored as the catalytic residue, but the
reduction mechanism, which involves a hydride transfer from NADPH to
the substrate and protonation of the oxygen by a residue of the enzyme
acting as a general acid, may be different between PGH2 and
PGD2/PQ. The reduction site of PGD2 is the keto
group like other carbonyl compounds but that of PGH2 is the
endoperoxide group. Moreover, the results of the reverse effects of
H170C and N306Y on PGD2 and PGH2 reductase
activities shown in this paper, taken together with the results on the
competitive inhibition between PGD2 and PQ (3) and on the
lack of inhibition between PGH2 and PGD2/PQ (3,
6), indicate that the reduction mechanism for PGD2 may be
the same as that for PQ but not the same as that for
PGH2.
3
-HSD, which catalyzes the NADP-dependent reversible
oxidation of the 3
-hydroxy group of various steroids, interacts
extensively with bile acids (31); and it and human liver high affinity
bile acid-binding protein with minimal 3
-HSD activity are
multifunctional proteins in bile acid transport and xenobiotic
metabolism (32). The amino acid sequences of HSD and high affinity bile
acid-binding protein are similar to the sequence of bovine liver PGF
synthase, and DD2 and DD4 exhibited binding activity for bile acids
(33). Therefore, bovine liver PGF synthase may also be expected to
exhibit the ability to bind bile acids. In the liver,
PGF2
, PGE2, and PGD2 reduce bile
flow and bile acid secretion, and especially the effect of
PGF2
is more potent than that of PGE2 or
PGD2 (34). PGF synthase may be multifunctionally involved
in the biosynthesis of PGF and in the binding of bile acids, and PGF synthase in the liver may reduce bile flow. Moreover,
PGF2
stimulates hepatocyte DNA synthesis and may have a
role in promoting hepatocyte proliferation (35, 36). Furthermore,
PGF2
and PGD2 are released from primary Ito
cell cultures after stimulation by noradrenaline and ATP (37).
PGF2
plays an important physiological role in the liver,
and the liver-type PGF synthase mainly contributes to the biosynthesis
of PGF2
there.
Recently, the sequence of the bovine liver DD3 (17) was reported. The
sequence of the liver-type PGF synthase reported in this paper was
found to be identical to that of the bovine liver DD3. The liver-type
PGF synthase showed the enzyme activity for (S)-(+)-indanol
(6.3 µmol/min/mg),3 which
is a typical substrate of DD3 (13, 17). However, PG(s) is the naturally
occurring substrate(s) for this enzyme, as indanole is a xenobiotic compound.
 |
ACKNOWLEDGEMENTS |
We are grateful to Professor S. Nagata of the
Department of Genetics, Medical School of Osaka University, for a
generous gift of the pEF-BOS mammalian expression vector. We are
indebted to Professor S. Kuramitsu of the Department of Biology,
Graduate School of Science, Osaka University, for the guidance of
kinetics of enzyme and for critical reading of this manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science, and
Culture of Japan and by a grant from Sankyo Foundation of Life Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D88749.
§
Present address: Dept. of Anatomy and Cell Biology, School of
Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima
770-8503, Japan.
§§
To whom correspondence should be addressed: Osaka Bioscience
Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: 81-6-872-4812; Fax: 81-6-872-4818; E-mail:
watanaki{at}obisun1.obi.or.jp.
2
M. Sato, M. Tanaka, K. Ikehara, T. Suzuki, and
K. Watanabe, manuscript in preparation.
3
T. Suzuki, and K. Watanabe, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
PG, prostaglandin;
PQ, 9, 10-phenanthrene quinone;
DD, dihydrodiol dehydrogenase;
HSD, 3
-hydroxysteroid dehydrogenase;
KPB, potassium phosphate
buffer;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction..
 |
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