Publications

• Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis.

  Huang X., Yan W., Gao D., Tong M., Tai H.H., Zhan C.G.

  Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure-function relationships. Using an ... >> More

  Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure-function relationships. Using an approach of the combined structure prediction, molecular docking, site-directed mutagenesis, and enzymatic activity assay, we have developed the first three-dimensional (3D) model of the substrate-binding domain (SBD) of mPGES-1 and its binding with substrates prostaglandin H2 (PGH2) and glutathione (GSH). In light of the 3D model, key amino acid residues have been identified for the substrate binding and the obtained experimental activity data have confirmed the computationally determined substrate-enzyme binding mode. Both the computational and experimental results show that Y130 plays a vital role in the binding with PGH2 and, probably, in the catalytic reaction process. R110 and T114 interact intensively with the carboxyl tail of PGH2, whereas Q36 and Q134 only enhance the PGH2-binding affinity. The modeled binding structure indicates that substrate PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the -SH group of GSH. The -SH group of GSH is expected to attack the peroxy group of PGH2, initializing the catalytic reaction transforming PGH2 to prostaglandin E2 (PGE2). The overall agreement between the calculated and experimental results demonstrates that the predicted 3D model could be valuable in future rational design of potent inhibitors of mPGES-1 as the next-generation inflammation-related therapeutic. << Less

  Bioorg. Med. Chem. 14:3553-3562(2006) [PubMed] [EuropePMC]

• Essential 110Cys in active site of membrane-associated prostaglandin E synthase-2.

  Watanabe K., Ohkubo H., Niwa H., Tanikawa N., Koda N., Ito S., Ohmiya Y.

  The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in ... >> More

  The amino acid sequence of membrane-associated prostaglandin (PG) E synthase-2 (mPGE synthase-2), which has a broad specificity in its thiol requirement for a catalytic activity, has the consensus region from 104Leu to 120Leu found in glutaredoxin and of thioredoxin. The sequence of Cys-x-x-Cys in the consensus region is the active site for thioredoxin and mPGE synthase-2 also has this amino acid sequence (110Cys-x-x-113Cys). The mutation from 110Cys to Ser or the double mutation from 110Cys and 113Cys to Ser caused loss of PGE synthase activity, whereas the single mutation from 113Cys to Ser did not affect the enzyme activity. These results indicate that 110Cys, but not 113Cys, is the essential amino acid in the active site of mPGE synthase-2. 110Cys is an important amino acid in PGE synthase activity and plays the critical role as Cys at the same position in redoxin. Moreover, we found that the reduced form of lipoic acid (dihydrolipoic acid) serves as one of the natural activators of mPGE synthase-2 in the cells. << Less

  Biochem. Biophys. Res. Commun. 306:577-581(2003) [PubMed] [EuropePMC]

• Identification and characterization of a novel type of membrane-associated prostaglandin E synthase.

  Tanikawa N., Ohmiya Y., Ohkubo H., Hashimoto K., Kangawa K., Kojima M., Ito S., Watanabe K.

  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 ... >> More

  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 mumol/min center dot mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-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 heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution. << Less

  Biochem. Biophys. Res. Commun. 291:884-889(2002) [PubMed] [EuropePMC]

• Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart.

  Watanabe K., Kurihara K., Suzuki T.

  Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) w ... >> More

  Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) was 24 microM. The molecular weight of the enzyme was about 31000 on SDS-polyacrylamide gel electrophoresis and was about 60000 by gel filtration. The enzyme was separated from glutathione (GSH) S-transferase by DEAE-Toyopearl column chromatography, and did not exhibit any GSH S-transferase activity towards four different substrates. The purified enzyme was active in the absence of GSH, but it was activated by various SH-reducing reagents including dithiothreitol, GSH, or beta-mercaptoethanol. This is the first reported purification of membrane-bound PGE synthase to apparent homogeneity. << Less

  Biochim. Biophys. Acta 1439:406-414(1999) [PubMed] [EuropePMC]

• Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.

  Jakobsson P.-J., Thoren S., Morgenstern R., Samuelsson B.

  Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was ... >> More

  Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was expressed in Escherichia coli, and both cytosolic and membrane fractions were prepared. Western blot analysis specifically detected a 15-to 16-kDa protein in the membrane fraction. Both fractions were incubated with prostaglandin H2 in the presence or absence of reduced glutathione. The membrane but not the cytosolic fraction was found to possess high glutathione-dependent PGE synthase activity (0.25 micromol/min/mg). The human tissue distribution was analyzed by Northern blot analysis. High expression of PGE synthase mRNA was detected in A549 and HeLa cancer cell lines. Intermediate level of expression was demonstrated in placenta, prostate, testis, mammary gland, and bladder whereas low mRNA expression was observed in several other tissues. A549 cells have been used as a model system to study cyclooxygenase-2 induction by IL-1beta. If A549 cells were grown in the presence of IL-1beta, a significant induction of the PGE synthase was observed by Western blot analysis. Also, Western blot analysis specifically detected a 16-kDa protein in sheep seminal vesicles. In summary, we have identified a human membrane bound PGE synthase. The enzyme activity is glutathione-dependent, and the protein expression is induced by the proinflammatory cytokine IL-1beta. PGE synthase is a potential novel target for drug development. << Less

  Proc. Natl. Acad. Sci. U.S.A. 96:7220-7225(1999) [PubMed] [EuropePMC]

• Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis.

  Tanioka T., Nakatani Y., Semmyo N., Murakami M., Kudo I.

  Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, i ... >> More

  Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis. << Less

  J. Biol. Chem. 275:32775-32782(2000) [PubMed] [EuropePMC]

• Purification and characterization of recombinant microsomal prostaglandin E synthase-1.

  Ouellet M., Falgueyret J.-P., Ear P.H., Pen A., Mancini J.A., Riendeau D., Percival M.D.

  Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. Th ... >> More

  Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation. << Less

  Protein Expr. Purif. 26:489-495(2002) [PubMed] [EuropePMC]

• Studies on membrane-associated prostaglandin E synthase-2 with reference to production of 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT).

  Watanabe K., Ito S., Yamamoto S.

  Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH(2) primarily to PGE(2). The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic P ... >> More

  Membrane-associated prostaglandin (PG) E synthase (mPGE synthase)-2 catalyzes the conversion of PGH(2) primarily to PGE(2). The enzyme is activated by various sulfhydryl reagents including dithiothreitol, dihydrolipoic acid, and glutathione, and it is different from mPGE synthase-1 and cytosolic PGE synthase, both of which require specifically glutathione. Recently, other investigators reported that their preparation of mPGE synthase-2 containing heme converted PGH(2) to 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) rather than to PGE(2) [T. Yamada, F. Takusagawa, Biochemistry 46 (2007) 8414-8424]. As we examined presently, the heme-bound enzyme expressed and purified according to their method synthesized HHT from PGH(2), but also PGE(2) in a decreased amount. Whereas the PGE synthase activity was completely lost at 50 degrees C for 5 min, the HHT synthase activity remained even at 100 degrees C for 5 min. In contrast, when the heme-bound enzyme was purified in the presence of dithiothreitol, only PGE(2) was produced, but essentially no HHT was detected. Thus, native mPGE synthase-2 enzymatically catalyzes only the conversion of PGH(2) to PGE(2), but not to HHT, and heme is not involved in this reaction. << Less

  Biochem. Biophys. Res. Commun. 367:782-786(2008) [PubMed] [EuropePMC]

• Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides.

  Kozak K.R., Crews B.C., Morrow J.D., Wang L.H., Ma Y.H., Weinander R., Jakobsson P.J., Marnett L.J.

  Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endo ... >> More

  Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo. << Less

  J. Biol. Chem. 277:44877-44885(2002) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.

  Thoren S., Weinander R., Saha S., Jegerschold C., Pettersson P.L., Samuelsson B., Hebert H., Hamberg M., Morgenstern R., Jakobsson P.-J.

  Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affi ... >> More

  Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 micromol min-1 mg-1) and high kcat/Km (310 mm-1 s-1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 micromol min-1 mg-1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 micromol min-1 mg-1), 5-hydroperoxyeicosatetraenoic acid (0.043 micromol min-1 mg-1), and 15-hydroperoxy-PGE2 (0.04 micromol min-1 mg-1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 micromol min-1 mg-1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1. << Less

  J. Biol. Chem. 278:22199-22209(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.