Identification and Characterization of a Novel ... - ScienceDirect.com

Biochemical and Biophysical Research Communications 291, 884 – 889 (2002) doi:10.1006/bbrc.2002.6531, available online at http://www.idealibrary.com on

Identification and Characterization of a Novel Type of Membrane-Associated Prostaglandin E Synthase Naomi Tanikawa,* Yoshihiro Ohmiya,* Hiroaki Ohkubo,† Katsuyuki Hashimoto,‡ Kenji Kangawa,§ Masami Kojima,* Seiji Ito, ¶ and Kikuko Watanabe储 ,1 *Special Division for Human Life Technology, Cell Dynamics Research Group, National Institute of AIST, Midorigaoka, Ikeda, Osaka 563-8577, Japan; †Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto, Japan; ‡Division of Genetic Resources, National Institute of Infectious Diseases, 23-1, Toyama 1-chome, Shinjuku-ku, Tokyo 162-8640, Japan; §Department of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan; ¶Department of Medical Chemistry, Kansai Medical University, 10-15 Fumizono, Moriguchi 570-8506, Japan; and 储Division of Life Science, Graduate School of Integrated Science and Art, University of East Asia, 2-1 Ichinomiya-gakuencho, Shimonoseki, Yamaguchi 751-8503, Japan

Received January 30, 2002

Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim. Biophys. Acta 1439, 406 – 414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M r of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V max and K m values for PGH 2 of the purified recombinant mPGE synthase were about 3.3 ␮mol/min 䡠 mg of protein and 28 ␮M, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and ␤-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine Abbreviations used: PG, prostaglandin; mPGE synthase, membrane-associated prostaglandin E synthase; GSH, glutathione; KPB, potassium phosphate buffer; DTT, dithiothreitol; Mer, 2-mercaptoethanol; COX, cyclooxygenase. 1 To whom correspondence should be addressed at Division of Life Science, Graduate School of Integrated Science and Art, University of East Asia, 2-1 Ichinomiya-gakuencho, Shimonoseki, Yamaguchi 751-8503, Japan. Fax: 0832-57-5152. E-mail: [email protected]. ac.jp. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution. © 2002 Elsevier Science (USA)

Key Words: nonspecific for glutathione; membraneassociated prostaglandin E synthase; expression; enzymatic property.

E series of prostaglandin (PG)s were first discovered in sheep seminal vesicles [1]. PGE 2 is widely distributed in various organs, and exhibits various biologically important activities such as smooth muscle dilatation/contraction [cf. ref. 2], Na ⫹ excretion [3], body temperature regulation [4], induction of pain [2], stimulation of bone resorption [5], and inhibition of immune responses [2]. PGE synthase (EC 5.3.99.3.) catalyzes the conversion of PGH 2 to PGE 2. The membrane-associated PGE synthase (mPGE synthase) was partially purified from microsomal fractions of bovine [6] and sheep [7] vesicular glands, and shown to require glutathione (GSH). Tanaka et al. [8] characterized PGE synthase in sheep vesicular gland microsomes by use of a monoclonal antibody. Recently, using a clone of microsomal glutathione S-transferase 1-like 1, Jakobsson et al. [9] expressed human GSH-specific, membrane-associated PGE synthase (mPGE synthase-1) in E. coli. The mPGE synthase-1 had high GSH-dependent PGE synthase activity, and the protein expression was induced by the proinflammatory cytokine IL-1beta. We reported that PGE synthase activity was widely distributed in the microsomal fractions of rat [10] and sheep organs [11]. Although most of the PGE synthase activities in many organs absolutely required GSH

884

Vol. 291, No. 4, 2002

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[10], the enzyme activity in the heart, spleen, and uterine microsomes did not specifically require GSH for its catalytic activity, as GSH could be replaced by other SH-reducing reagents [10]. Previously, we purified PGE synthase from the microsomal fraction of bovine heart to apparent homogeneity, and examined its enzymatic properties [11]. In this present study, we defined its primary structure, and expressed it in E. coli. Moreover, we purified the expressed protein to homogeneity, and identified that it was the PGE synthase, which has the same enzymatic properties as the enzyme purified from bovine heart microsomes. This membrane-associated PGE synthase was different from mPGE synthase-1 reported by Jakobsson et al. [9], and we named this enzyme as mPGE synthase-2. MATERIALS AND METHODS Materials. [1- 14C]PGH 2 (2.07 GBq/mmol) was obtained from Daiichi Pure Chemicals Co, Ltd. (Tokyo, Japan). Authentic PGs were kindly donated by Ono Pharmaceutical Company (Osaka, Japan). Other materials and commercial sources were as follow: nickelchelating resin (ProBond Purification System) and pTrc-HisA, from Invitrogen Corp. (CA, USA), [␣- 32P]dCTP and Thermo sequenase Cy5.5 Dye Terminator Cycle Sequencing kit, from Amersham Pharmacia Biotech (Buckinghamshire, UK); molecular mass markers for polyacrylamide gel electrophoresis (PAGE), from Bio-Rad (CA); and Penta-His antibody, from Qiagen (Hilden, Germany). Other chemicals were at least of reagent grade. Construction of expression vector. The Macaca fascicularis brain cDNA clone (QccE-16688) was used as template. The full amino acid sequence deduced from the cDNA clone was named fPGE synthase. The fPGE synthase fragment was amplified by PCR using primers PB1 (5⬘-CGG ATC CAT GGC CCC GGC TAC GCG GGT-3⬘) and PE1 (5⬘-GGA ATT CTT CAG TGC GCT GGG GAG G-3⬘). The amplified fPGE synthase fragment was digested with BamHI/ EcoRI, and inserted into the BamHI/ EcoRI site of pTrc-HisA as pTH-fPGE synthase. Then, fPGE synthase with the N-terminal 87 amino acids deleted was prepared and designated as mPGE synthase-2. The fragment of mPGE synthase-2 was amplified by PCR using a pair of primers; PB2 (5⬘-CTG GGA TCC GAG CGC TCA GCA GTG CAG CTC-3⬘) and PE1; digested with BamHI/ EcoRI; and inserted into the BamHI/ EcoRI site of pTrc-HisA as pTH-mPGE synthase-2. The nucleotide sequences of both amplified DNA fragments were verified by the dideoxy chain termination method using a Thermo sequenase Cy5.5 Dye Terminator Cycle Sequencing kit specifically developed for the Gene rapid Amersham Pharmacia Biotech sequencer. The resultant plasmids were used to transform E. coli BL21. Purification of the expressed enzyme. Cells (BL21) were cultured in 150 ml of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) containing 50 ␮g/ml ampicillin at 37°C for 14 h, following the addition of 1 mM IPTG at 0.1 of OD 600 after the preculture. The cells were harvested by centrifugation at 3,000 g for 20 min and stored at ⫺80°C until the preparation. All procedures were carried out at 0 – 4°C. The cells were suspended in 10 ml of Buffer A (30 mM potassium phosphate buffer (KPB), 1 M NaCl, and 0.5 mM dithiothreitol (DTT), pH 7.0) containing 1 mM pepstatin A, 1 ␮g/ml PMSF, 50 ␮g/ml DNase; and 50 ␮g/ml RNase, and were disrupted by sonication followed by the centrifugation at 10,000 g for 30 min. After the addition of 10 mM imidazole, the supernatant was applied to an affinity column (0.5 ⫻ 1.3 cm) containing nickel-chelating resin equilibrated with Buffer A following the manufacturer’s directions with a minor modifications. The column was washed with Buffer A, followed by Buffer A containing 10 –140 mM imidazole. The enzyme

was then eluted with Buffer A containing 150 mM imidazole. The fractions with the PGE synthase activity were pooled. Two polyclonal antibodies against mPGE synthase-2 were raised in rabbits, with mPGE synthase-2 purified from E. coli, and the C-terminal 14 amino-acid oligopeptide of mPGE synthase-2 used as the immunogens. For Western-blot analysis, the purified enzyme was subjected to SDS–PAGE (12%) and electrophoretically transferred to a polyvinylidene disulfide membrane (Amersham Pharmacia Biotech, UK). Protein bands were immunostained with the antirecombinant mPGE synthase-2, anti-C-terminus of mPGE synthase-2, or Penta-His antibody and reagents from a Vectastain ABC kit (Vector Laboratories, U.S.A.), and visualized with an Enhanced Chemiluminesence Kit (ECL, Amersham Pharmacia Biotech, UK). Enzyme assay of PGE synthase. The mPGE synthase-2 activity was measured as described previously [10]. The reaction mixture contained 0.1 M KPB (pH 6.5), 0.5 mM DTT, and enzyme. One unit of enzyme activity was defined as the amount that produced 1 ␮mol of PGE 2 per min at 24°C. Specific activity was expressed as the number of units/mg of protein. Protein concentrations were determined according to the method of Bradford [13], with bovine serum albumin used as the standard. Dot blot and Northern blot analyses. As a probe for hybridization, human cDNA (371–1179 bp) for the coding region of mPGE synthase-2 was isolated from a human Multiple-Choice First-Strand cDNA Set 1 (OriGene Technologies, Inc., MD, Cat. No. CH-1101) by the PCR method, and the sequence was confirmed. Dot blot analysis of human multiple tissue expression array (Clontech, Cat. No. 7776-1) and Northern blot analyses of human multiple tissue blot (Clontech, Cat. No. 7780-1 and 7755-1) were performed according to the instructions of the manufacturer. Three membranes were hybridized with the random-primed cDNA probe described above. The probe was labeled by using a Prime-it Random Primer Labeling Kit (Strategene, CA) and [␣- 32P]dCTP, according to the instructions of the manufacturer. After hybridization and washing, the dot blot/blot were exposed overnight and visualized by use of a FUJIX BAS2000 II system (Fujifilm, Tokyo, Japan).

RESULTS Identification and Expression of mPGE Synthase-2 The N-terminal 22-amino acid sequence of the mPGE synthase-2 purified from the microsomal fraction of bovine heart was determined with automatic amino acid sequencer based on the Edman degradation method, and was ERSATQLSLSSRLQLTLYQYKT. This amino acid sequence was identical to the corresponding region (88 –109) deduced from human (Accession No. AK024199) and monkey (Accession No. AB046026) cDNAs, except that Thr at position 5 of the mPGE synthase-2 purified from bovine heart was replaced by Ala and Val at position 92 in human and monkey sequences, respectively. The amino acid sequences derived from the human and monkey cDNAs showed 97% identity with each other, and had 87 amino-acid residues N-terminal to the sequence of PGE synthase purified from the microsomal fraction of bovine heart. The amino acid sequences derived from human and monkey cDNAs did not belong to any GSH S-transferase family, but the amino acid sequence from 104 Leu to 120Leu was the consensus region of glutaredoxin [14, 15] and of thioredoxin [15]. We constructed 2 expression plasmids using polyhistidine-tagged pep-

885

Vol. 291, No. 4, 2002


FIG. 1. SDS–PAGE (A) and Western blot analysis (B) of mPGE synthase-2. PGE synthase were analyzed by (A) SDS–PAGE Coomassie brilliant blue stain (3 ␮g for each lane) and (B) Western blot analysis (0.3 ␮g for each lane) using the antiserum against the purified recombinant PGE synthase: the crude extract after sonication of E. coli (lane 1), 10,000 g supernatant after sonication of E. coli (lane 2), and the purified recombinant enzyme (lane 3) were loaded into the indicated lanes. The positions of the molecular mass standards are shown: MBP-␤-galactosidase (175,000), MBP-paramyosin (83,000), glutamic dehydrogenase (62,000), aldolase (47,500), triosephosphate isomerase (32,500), ␤-lactoglobulin A (25,000), lysozyme (16,500).

tide fused vector (pTrc-HisA) and the monkey cDNA (AB046026) coding a 377-amino acid polypeptide (fPGE synthase) with M r of 41,914 or that coding a 290-amino acid polypeptide (mPGE synthase-2) with M r of 33,107, and expressed the 2 proteins in E. coli (BL21). The crude extracts of the 2 expressed proteins had PGE synthase activity (390 nmol/min 䡠 mg of protein for fPGE synthase and 430 nmol/min 䡠 mg of protein for mPGE synthase-2). The expressed pTH-mPGE synthase-2 was released from the membrane to the cytosol fraction by 1 M NaCl, but pTH-fPGE synthase could not be released from the membrane fraction. First, we purified the expressed protein of pTH-mPGE synthase-2 to apparent homogeneity by nickel-chelating column chromatography. In a typical purification, 20% of the initial PGE synthase activity was recovered with a specific activity of 3.3 ␮mol/min 䡠 mg of protein. The enzyme was broadly eluted from the immobilized nickel absorption chromatography. Although it leaked out of the column with other proteins by elution with 10–140 mM imidazole and the yield was decreased, the enzyme eluted by 150 mM imidazole had high specific activity. A sample from each of the purification steps was subjected to SDS–PAGE. Coomassie blue staining of the gel indicated that an approximately 33-kDa protein was produced in the cells harboring pTH-mPGE synthase-2, and this protein was purified to apparent homogeneity (Fig. 1A). Western blot analysis of each sample revealed that the 33-kDa protein was recognized by anti-recombinant

mPGE synthase-2 antibody (Fig. 1B). The same results were obtained with antibodies against the C-terminal sequence of mPGE synthase-2 and Penta-His (data not shown). The M r of the expressed enzyme was a little larger than that of the enzyme purified from bovine heart because of 35-amino-acid residues of the His-tag. No protein from the control E. coli. bearing pTrc-HisA without the insert DNA interacted with any of the 3 antibodies (data not shown). The N-terminal sequence of the purified recombinant enzyme (mPGE synthase-2) was GGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSERSAV. The first 35 amino acids, from –34Gly to 0Ser were derived from pTrc-HisA, and the next five, from 1Glu to 5Val, were identical to the N-terminal sequence of the enzyme purified from bovine heart. These results suggest that the clone pTHmPGE synthase-2 was mPGE synthase-2 identical to the enzyme purified from bovine heart, and that its protein expressed in E. coli had been purified to apparent homogeneity. Enzymatic Properties of PGE Synthase The conversion of PGH 2 to PGE 2 was proportional to the enzyme amount up to 0.25 ng (Fig. 2A), and proceeded linearly at least 2 min (Fig. 2B). The specific activity and the K m value for PGH 2 were about 3.3 ␮mol/min 䡠 mg of protein and about 28 ␮M, respectively (Fig. 2C), indicating that the k cat/K m value was 65 ⫻ 10 3 M ⫺1 s ⫺1. When the enzyme activity was measured in the presence of an SH reagent, i.e., GSH, DTT, or 2-mercaptoethanol (Mer), it was most stimulated, about 4.3-fold, by DTT, compared with that in the absence of the reagent (Fig. 2D). Moreover, the enzyme was the most stable in 30 mM KPB (pH 7.0) containing 0.5 mM DTT (data not shown). The optimal pH of the enzyme was pH 6.0 –7.0 (data not shown). These enzymatic properties are similar to those of the enzyme purified from bovine heart [11]. Tissue Distribution of mPGE Synthase-2 mRNA in Humans The expression of mPGE synthase-2 mRNA was investigated in various human tissues by dot blot analysis (Fig. 3A) using the human cDNA as a probe. The mPGE synthase-2 mRNA was detected in the heart, including apex, inter-ventricular septum, both atria and ventricles, but not in the aorta. The mRNA was also detected in fetal heart. Moreover, the mPGE synthase-2 mRNA was detected in various regions of the brain: cerebellum; occipital, frontal, and pariental lobes; and so forth. In the lymph nodes, skeletal muscle, kidney, and trachea, the mRNA was also detected, but not in the thymus or lung. However, the mRNA was found in the fetal thymus and lung, suggesting that the mPGE synthase-2 may be related to development in these tissues. Northern blot analysis (Fig. 3B)

886

Vol. 291, No. 4, 2002


FIG. 2. Enzymatic properties. (A) Dependency of PGE 2 synthesis from PGH 2 by the purified mPGE synthase-2 on enzyme amount and (B) time course of PGE 2 synthesis by the purified mPGE synthase-2. (C) Substrate dependency of PGE synthase-2 by the purified mPGE synthase-2. (D) Effects of SH reagents on the PGE synthase-2 activity. DTT of Buffer A, in which the enzyme was suspended, was removed by NAP10 equilibrated with 30 mM KPB (pH 7.0). The PGE synthase-2 activity was measured in the presence of DTT (E), GSH (F), or Mer (}) at various concentrations.

revealed that the probe was hybridized to a 2-kilobase mRNA in skeletal muscle, heart, kidney, and brain. In the brain, the probe hybridized to a 2-kilobase mRNA present in the cerebral cortex, occipital pole, frontal lobe, temporal lobe, putamen, and cerebellum. These results support those from the dot blot analysis. The result that the mPGE synthase-2 was detected in heart, but not in vesicular gland, coincided with that for the distribution of PGE synthase activity in the absence of GSH in rats [10] and sheep [11]. DISCUSSION In this paper, we identified and characterized a mPGE synthase that had a broad specificity of thiol requirement for its enzyme activity. The N-terminal sequence of the mPGE synthase-2 purified from microsomes of bovine heart started from 88Glu of the protein derived from human and monkey cDNAs, which encode hypothetical proteins with unknown function. We constructed the pTH-mPGE synthase-2 vector, using the vector (pTrc-HisA) and the monkey cDNA for the 290amino-acid polypeptide, and expressed the recombinant mPGE synthase-2 in E. coli. Moreover, we purified the mPGE synthase-2 to apparent homogeneity,

and examined the enzymatic properties. The purified enzyme with a M r of 33-kDa required SH reagent, especially DTT, and the V max and K m values for PGH 2 were about 3.3 ␮mol/min 䡠 mg of protein and 28 ␮M, respectively. The k cat/K m value was about 65 ⫻ 10 3 M ⫺1 s ⫺1, which was the highest among those for other types of PGE synthases [6 –9, 16 –18]. These enzymatic properties of the recombinant protein were the same as those of the enzyme purified from bovine heart microsomes. Furthermore, the amino acid sequence derived from monkey cDNA (AB046026) shows 97% identity with that of human cDNA (AK024100). Therefore, we considered that the structure and function of mPGE synthase-2 seems to be highly conserved among the various species, such as bovine, monkey, and human. We also constructed the pTH-fPGE synthase vector, and expressed the recombinant enzyme (fPGE synthase) with a M r of about 42 kDa. This enzyme had 87 amino-acid residues N-terminal to the sequence of mPGE synthase purified from bovine heart, and these 87-amino acids contained hydrophobic residues. The crude extract of the recombinant fPGE synthase also exhibited PGE synthase activity. However, the recombinant fPGE synthase was not released from membrane fraction with 1 M NaCl. On the other hand, the

887

Vol. 291, No. 4, 2002


FIG. 3. Dot blot analysis (A), and Northern blot analyses of various human tissues (B) and of various regions of the human brain (C). (A) The mRNA amount on each dot of the array was normalized by the manufacturer to yield similar hybridization signals for 8 housekeeping genes. The various dots represent the following: A1, whole brain; B1, cerebral cortex; C1, frontal lobe; D1, parietal lobe; E1, occipital lobe; F1, temporal lobe; G1, paracentral gyrus of cerebral cortex; H1, pons; A2, cerebellum, left; B2, cerebellum, right; C2, corpus callosum; D2, amygdala; E2, caudate nucleus; F2, hippocampus; G2, medulla oblongata; H2, putamen; A3, empty; B3, accumbens nucleus; C3, thalamus; D-H3, empty; A4, heart; B4, aorta; C4, atrium, left; D4, atrium, right; E4, ventricle, left; F4, ventricle, right; G4, interventricular septum; H4, apex of the heart; A5, esophagus; B5, stomach; C5, duodenum; D5, jejunum; E5, ileum; F5, ilocecum; G5, appendix; H5, colon, ascending; A6, colon, transverse; B6, colon, desending; C6, rectum; D-H6, empty; A7, kidney; B7, skeletal muscle; C7, spleen; D7, thymus; E7, peripheral blood leukocytes; F7, lymph node; G7, bone marrow; H7, trachea; A8, lung; B8, placenta; C8, bladder; D8, uterus; E8, prostate; F8, testis; G8, ovary; H8, empty; A9, liver; B9, pancreas; C9, adrenal gland; D9, thyroid gland; E9, salivary gland; F-H9, empty; A10, HL-60; B10, HeLa S3; C10, K-562; D10, Molt-4; E10, Burkitt’s lymphoma, Raji; F10, Burkitt’s lymphoma, Daudi; G10, colorectal adenocarcinoma, SW480; H10, lung carcinoma, A549; A11, fetal brain; B11, fetal heart; C11, fetal kidney; D11, fetal liver; E11, fetal spleen; F11, fetal thymus; G11, fetal lung; H11, empty; A12, yeast, total RNA; B12, yeast tRNA; C12, E. coli rRNA; D12, E. coli DNA; E12, Poly r(A); F12, human CoT-1 DNA; G12, human DNA 100 ng; H12, human DNA 500 ng. (B and C) The human multiple tissue blot contained 2 ␮g of tissue-specific mRNA/lane. PBL is peripheral blood leukocyte.

mPGE synthase-2 was bound to the membrane fraction, but was released with 1 M NaCl. These results suggest that mPGE synthase-2 is attached to membranes and that fPGE synthase is tightly bound to them by its N-terminal 87 amino acid residues. Thus, these N-terminal residues are probably involved in the association of the enzyme with membranes. The reason that the N-terminus of the enzyme purified from bovine heart started from 88Glu of fPGE synthase has not yet been clarified. Perhaps the explanation is artifact formation during the purification or some physiological reason(s) such as post translation. PGE synthase was partially purified from the microsomal fraction of bovine [6] and sheep [7] seminal vesicular glands. Ogino et al. [6] reported that GSH was required for the PGE synthase activity, and laid the groundwork for the study of mPGE synthase. Recently, mPGE synthase-1 was expressed in E. coli, by use of the microsomal glutathione S-transferase 1-like 1 clone, and shown to be an inducible enzyme [9]. However, it has not yet been clarified whether the mPGE synthase partially purified from microsomes of bovine [6] and sheep [7] seminal vesicular glands is the same as the mPGE synthase-1 [9]. Moreover, the PGE synthase was purified from the cytosol fraction of human brain [16] and Ascaridia galli [17], and was identified to be a GSH S-transferase. Recently, the cytosolic

GSH-dependent PGE synthase was purified from rat brain [18], and its partial amino acid sequence was identical to that of p23 [19]. The recombinant p23 expressed in E. coli exhibited GSH-requiring PGE synthase activity [18]. Many PGE synthases require GSH for PGE synthase activity [6 –9, 16 –18], and belong to the GSH S-transferase family, having a M r of about 20-kDa [8, 9, 16 –18]. The amino acid sequence of mPGE synthase-2 did not exhibit homology with that of any GSH S-transferase, indicating that mPGE synthase-2 does not belong to GSH S-transferase family. In 1986, Ogorochi et al. [16] predicted the existence of various types of PGE synthase. It now appears that there exist at least 3 distinct types of PGE synthase, i.e., a cytosolic type and GSH-specific and -nonspecific membrane-associated types. The results of dot blot and Northern blot analyses revealed that the mPGE synthase-2 mRNA was mainly present in various regions of the heart and brain, but not in genital organs. The localization of the mPGE synthase-2 mRNA was different from that of mPGE synthase-1 [9], suggesting that the different PGE synthase may serve the synthesis of PGE 2 in different tissues. Based on broad specificity for thiol requirement, primary structure, and tissue distribution, the mPGE synthase-2 is a novel enzyme, and plays physiological and pathological roles in vivo.

888

Vol. 291, No. 4, 2002


Moreover, the gene of mPGE synthase-2 was localized to chromosome 9q33-q34, and those genes of cyclooxygenase (Cox) 1 [20], mPGE synthase-1 [21], and lipocalin-type PGD synthase [22] were located close together on chromosome 9, at 9q32-q33.3, 9q34.3, and 9q34.2-q34.3, respectively. Cox 1 converts arachidonic acid to PGH 2, which is the substrate for the synthesis of PGE 2 and PGD 2, and its action occurs upstream of that of PGE synthases and PGD synthase. These results suggest that the region of q32-34 in chromosome 9 may be related to the metabolism of PGs.

10.

11.

12.

ACKNOWLEDGMENTS

13.

We are grateful to Dr. Emiko Ashitaka-Okuda of Kansai Medical University for kind support. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

14.

15.

REFERENCES 16. 1. Bergstro¨ m, S., and Sjo¨ vall, J. (1960) J. Acta Chem. Scand. 14, 1693–1700. 2. Narumiya, S., Sugimoto, Y., and Ushikubi, F. (1999) Prostanoid receptors: Structures, properties, and functions. Physiol. Rev. 79, 1193–1226. 3. Vander, A. J. (1968) Direct effects of prostaglandin on renal function and renin release in anesthetized dog. Am. J. Physiol. 214, 218 –221. 4. Milton, A. S., and Wendlandt, S. (1971) Effects on body temperature of prostaglandins of the A, E, and F series on injection into the third ventricle of unanaesthetized cats and rabbits. J. Physiol. 218, 325–336. 5. Kawaguchi, H., Pilbeam, C. C., Harrison, J. R., and Raisz, L. G. (1995) The role of prostaglandins in the regulation of bone metabolism. Clin. Orthop. 313, 36 – 46. 6. Ogino, N., Miyamoto, T., Yamamoto, S., and Hayaishi, O. (1977) Prostaglandin endoperoxide E isomerase from bovine vesicular gland microsomes, a glutathione-requiring enzyme. J. Biol. Chem. 252, 890 – 895. 7. Moonen, P., Buytenhek, M., and Nugteren, D. H. (1982) Purification of PGH-PGE isomerase from sheep vesicular gland. Methods Enzymol. 86, 84 –91. 8. Tanaka, Y., Ward, S. L., and Smith, W. L. (1987) Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes. J. Biol. Chem. 262, 1374 –1381. 9. Jakobsson, P. J., Thoren, S, Morgenstern, R., and Samuelsson,

17.

18.

19.

20.

21.

22.

889

B. (1999) Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc. Natl. Acad. Sci. (USA) 96, 7220 –7225. Watanabe, K., Kurihara, K., and Hayaishi, O. (1997) Two types of microsomal prostaglandin E synthase: Glutathione-dependent and -independent prostaglandin E synthases. Biochem. Biophys. Res. Commun. 235, 148 –152. Watanabe, K., Kurihara, K., and Suzuki, T. (1999) Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart. Biochim Biophys Acta 1439, 406 – 414. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) Glutathione S-transferases: The first enzymatic step in mercaturic acid formation. J. Biol. Chem. 249, 7130 –7139. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. Johnson, G..P., Goebel. S. J., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. (1991) Vaccinia virus encodes a protein with similarity to glutaredoxins. Virology 181, 378 –381. Holmgren, A. (1989) Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264, 13963–13966. Ogorochi, T., Ujihara, M., and Narumiya, S. (1987) Purification and properties of prostaglandin H-E isomerase from the cytosol of human brain: Identification as anionic forms of glutathione S-transferase. J. Neurochem. 48, 900 –909. Meyer, J., Muimo, R., Thomas, M., Coates, D., and Isaac, R. E. (1996) Purification and characterization of prostaglandin H-E isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli. Biochem. J. 313, 223–227. Tanioka, T., Nakatani, Y., Semmyo, N., Murakami, M., and Kudo, I. (2000) Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J. Biol. Chem. 275, 32775–32782. Johnson, J. L., Beito, T. G., Krco, C. J., and Toft, D. O. (1994) Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes. Mol. Cell. Biol. 14, 1956 –1963. Kosaka, T., Miyata, A., Ihara, H., Hara, S., Sugimoto, T., Takeda, O., Takahashi, E., and Tanabe, T. (1994) Characterization of the human gene (PTGS2) encoding prostaglandinendoperoxide synthase 2. Eur. J. Biochem. 221, 889 – 897. Forsberg, L., Leeb, L., Thoren, S., Morgenstern, R., and Jakobsson, P. J. (2000) Human glutathione dependent prostaglandin E synthase: Gene structure and regulation. FEBS Lett. 471, 78 – 82. White, D. M., Mikol, D. D., Espinosa, R., Weimer, B., Le Beau, M. M., and Stefansson, K. (1992) Structure and chromosomal localization of the human gene for a brain form of prostaglandin D 2 synthase J. Biol. Chem. 267, 23202–23208.