Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.
Journal: 1999/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 10377395
Abstract:
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.
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Proc Natl Acad Sci U S A 96(13): 7220-7225

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

Department of Medical Biochemistry and Biophysics and Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden
To whom reprint requests should be addressed. e-mail: es.ik.bbm@nossbokaj.nahoj-rep.
Contributed by Bengt Samuelsson
Contributed by Bengt Samuelsson
Accepted 1999 Apr 28.

Abstract

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 μmol/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-1β. If A549 cells were grown in the presence of IL-1β, 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-1β. PGE synthase is a potential novel target for drug development.

Abstract

Prostaglandin endoperoxide H2 (PGH2) is formed from arachidonic acid by the action of cyclooxygenases (cox) -1 or -2. cox-1 is constitutively expressed in many cells and tissues such as platelets, endothelium, stomach, and kidney whereas the cox-2 protein can be induced by proinflammatory cytokines like IL-1β at sites of inflammation (for recent reviews on cox see refs. 13). Downstream of the cyclooxygenases, the product PGH2 can be further metabolized into the various physiologically important eicosanoids: e.g., PGF, PGE2, PGD2, PGI2 (prostacyclin), and thromboxane A2 (4).

The mechanism for the biosynthesis of PGE1 and PGF (formed by using dihomo-γ-linolenic acid instead of arachidonic acid) (5) by sheep vesicular glands was postulated to proceed via a cyclic endoperoxide (6) later designated PGH2 (79). In short, the reactions catalyzed by cyclooxygenase start by stereospecific abstraction of a hydrogen atom from carbon 13 of arachidonic acid. The resulting carbon radical reacts with molecular oxygen followed by the formation of the 9,11-endoperoxide and the bond between C-8 and C-12. Thereafter, a second molecule of oxygen is incorporated at C-15 followed by reduction to a hydroperoxy group, and PGG2 is formed. This hydroperoxy group can subsequently be reduced by the peroxidase activity of the cyclooxygenase (in the presence of a reducing agent: e.g., glutathione) thus forming PGH2. The enzyme(s) responsible for the isomerization of PGH2 into PGE2 are not well known. Attempts have been made to isolate the microsomal PGE synthase from ovine and bovine seminal vesicles, an organ known to contain high levels of activity (10, 11). These studies have shown that the microsomal PGE synthase can be solubilized and partly purified. However, the enzyme activity, which depended on glutathione, rapidly deteriorated during the purification attempts. Two monoclonal antibodies, designated IGG1(hei-7) and IGG1(hei-26) and raised against partly purified PGE synthase from sheep seminal vesicles, could immunoprecipitate two proteins from sheep seminal vesicles with molecular masses of 17.5 and 180 kDa, respectively (12). Both of these precipitated proteins were found to possess glutathione-dependent PGE synthase activity but no glutathione S-transferase activity. The 17.5-kDa protein showed a Km for PGH2 of 40 μM, similar to what had been described by others investigating the microsomal PGE synthase (11). In contrast, the larger protein demonstrated a Km for PGH2 of 150 μM. Of interest, the IGG1(hei-7) antibody [but not the IGG1(hei-26) antibody], when incubated with intact sheep vesicular gland microsomes, caused coprecipitation of PGE synthase and cyclooxygenase activities, thereby demonstrating that the 17.5-kDa protein and the cyclooxygenase both are associated with the same membrane systems. Microsomal PGE synthase activity also has been measured in various rat organs (13), and high glutathione-dependent activity was found in the deferens duct, genital accessory organs, and kidney. In the same study, glutathione-independent microsomal PGE synthase activity was observed in heart, spleen, and uterus. Additional proteins, belonging to the cytosolic glutathione S-transferase superfamily, also have been described to possess PGE, PGD, and PGF synthase activities (14). Recently, a microsomal 16.5-kDa protein was purified from sheep seminal vesicles possessing glutathione-dependent PGF synthase activity (15). The enzyme (prostaglandin endoperoxide reductase) also could catalyze the reduction of cumene hydroperoxide whereas 1-chloro-2,4-dinitrobenzene (typical substrate for various glutathione S-transferases) was not a substrate. In this paper, we describe the identification and characterization of human PGE synthase.

Acknowledgments

We thank Dr. Mats Hamberg for performing mass spectrometric analysis. Support from the Swedish Medical Research Council (Projects nos. 03X-00217 and 03X-12573), the Swedish Cancer Society and the Swedish Society of Medicine, Magnus Bergvall, and Harald Jeansson’s and Harald and Greta Jeansson’s foundations is gratefully acknowledged.

Acknowledgments

ABBREVIATIONS

PGH2prostaglandin H2
PGE2prostaglandin E2
12-HHT12(S)-hydroxy-8,10-trans-5-cis-heptadecatrienoic acid
coxcyclooxygenase
MGSTmicrosomal glutathione S-transferase
ABBREVIATIONS

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