Characterization of human glutathione-dependent microsomal prostaglandin E synthase-1
Prostaglandins (PGs) are lipid mediators, which act as local hormones. PGs are formed in most calls and are synthesized de novo from membrane-released arachidonic acid (AA) upon cell activation. Prostaglandin H synthase (PGHS) -1 or 2, also referred to as COX-1 and COX-2, metabolize AA to PGH2, which is subsequently converted in a cell-specific manner by downstream enzymes to biologically active prostanoids, i.e. PGE2, PGD2, PGF2alpha, PGI2 or TXA2. PGHS-1 is constitutively expressed in many calls and is mainly involved in housekeeping functions, such as vascular homeostasis, whereas PGHS-2 can be induced by proinflammatory cytokines at sites of inflammation. Prostaglandin E synthase (PGES) specifically catalyzes the conversion of PGH2 to PGE2, which is a biologically potent prostaglandin involved in several pathological conditions; including pain, favor, inflammation and possibly some forms of cancers and neurodegenerative diseases.
mPGES-1 was initially identified as a homologue to microsomal glutathione transferase-1 (MGST1) with 37% identity on the amino acid sequence level and referred to as MGST1-like 1 (MGST1-L1). Based on the properties of MGST1-L1, regarding size, amino acid sequence, hydropathy and membrane localization, the protein was identified as a member of the MAPEG-superfamily (membrane-associated proteins in eicosanoid and glutathione metabolism). The superfamily consists of 16- 18 kDa, integral membrane proteins with typical hydropathy profiles and diverse functions. The MAPEG family comprises six human members, which in addition to mPGES-1 are; 5-lipoxygenase activating protein (FLAP), leukotriene C4 synthase (LTC4S), MGST1, MGST2 and MGST3. MGST1 -2 and -3 are glutathione transferases as well as glutathione-dependent peroxidases, while FLAP and LTC4S are crucial for leukotriene biosynthesis.
Human mPGES-1 was cloned and characterized as a 16 kDa, inducible GSH-dependent microsomal PGE synthase. Northern dot blot analysis of mPGES-1 mRNA demonstrated a low expression in most tissues, medium expression in reproductive organs and a high expression in two cancer cell lines (A549 and HeLa). A549 cells had been used earlier as a model system to study PGHS-2 induction by the proinflammatory cytokine IL-1beta and mPGES-1 was also induced by IL-1beta in these calls. A protein of similar size was detected in microsomes from sheep vesicular glands, which are known to contain a highly efficient microsomal PGES, indicating that mPGES-1 was the long-sought membrane bound PGES.
Furthermore, a time study of PGHS-2 and mPGES-1 expression revealed a coordinate induction of these enzymes, which was correlated with increased PGES activity in the microsomal fraction. Tumor necrosis factor-alpha (TNF-alpha) also induced mPGES-1 in these cells and dexamethasone was found to counteract the effect of these cytokines on mPGES-1 induction. A method based on RP-HPLC and UV-detection was developed to efficiently quantify PGES activity. A small set of potential mPGES-1 inhibitors were tested and NS-398, Sulindac sulfide and LTC4 were found to inhibit PGES activity with IC50-values of 20 µm, 80 µm and 5 µm, respectively.
The human mPGES-1 gene structure was investigated. The mPGES-1 gene span a region of approximately 15 kb, is divided into three exons, and is localized on chromosome 9q34.3. A 682 bp fragment directly upstream of the translation start site exhibited promoter activity when transfected in A549 calls. The putative promoter is GC-rich, lacks a TATA box at a functional site and contains numerous potential transcription factor binding-sites. Two GC-boxes, two tandem Barble-boxes and an aryl hydrocarbon response element were identified. The putative promoter region of mPGES1 was transcriptionally active and reporter constructs were regulated by IL-1beta and phenobarbital.
The expression of mPGES-1 was investigated in synovial tissues from patients suffering from rheumatoid arthritis (RA). Primary synovial cells obtained from patients with RA were treated with IL-1beta or TNF-alpha. Both cytokines were found to induce mPGES-1 mRNA from low basal levels to maximum levels after 24 hours and the induction by IL-1beta was inhibited by dexamothasone in a dose-dependent manner. The protein expression of mPGES-1 was also induced by IL-1beta with a linear increase up to 72 h. In contrast, the PGHS-2 induction demonstrated an earlier peak expression (4-8 h). Furthermore, the protein expression of mPGES-1 was correlated with increased microsomal PGES activity. In these biochemical experiments any significant contribution of cytosolic PGES or other cytosolic or nonn-inducible membrane bound PGE syntheses was ruled out.
A purification protocol for mPGES-1 was developed. Human mPGES-1 was expressed with a histidine tag in Eschericha coli, solubilized by Triton X-100 and purified by a combination of hydroxyapatite and immobilized metal affinity chromatography. mPGES-1 catalyzed a rapid GSH-dependent conversion of PGH2 to PGE2 (170 µmol/min mg). The enzyme, also displayed a high GSH-dependent activity against PGG2, forming 15hydroperoxy PGE2 (250 µmol/min mg). In addition, mPGES-1 possessed several other activities; glutathione-dependent peroxidase activity towards cumene hydroperoxide, 5-HpETE and 15-hydroperoxy-PGE2, as well as conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) to GSH. These activities likely reflect the relationship with other MAPEG enzymes. Two-dimensional crystals of purified mPGES-1 were obtained and a 10 A projection map was determined by electron crystallography. Hydrodynamic studies were also performed on the mPGES-1-Triton X-100 complex to investigate the oligomeric state of the protein. Electron crystallography and hydrodynamic studies independently demonstrated a trimeric organization of mPGES-1.
Together with other studies published to date, mPGES-1 has been verified biologically as a drug target and the next stop in this validation process requires specific inhibitors to be tested in animal disease models.
List of scientific papers
I. Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B (1999). Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci U S A. 96(13): 7220-5.
https://pubmed.ncbi.nlm.nih.gov/10377395
II. Thoren S, Jakobsson PJ (2000). Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4. Eur J Biochem. 267(21): 6428-34.
https://pubmed.ncbi.nlm.nih.gov/11029586
III. Forsberg L, Leeb L, Thoren S, Morgenstern R, Jakobsson P (2000). Human glutathione dependent prostaglandin E synthase: gene structure and regulation. FEBS Lett. 471(1): 78-82.
https://pubmed.ncbi.nlm.nih.gov/10760517
IV. Stichtenoth DO, Thoren S, Bian H, Peters-Golden M, Jakobsson PJ, Crofford LJ (2001). Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. J Immunol. 167(1): 469-74.
https://pubmed.ncbi.nlm.nih.gov/11418684
V. Thoren S, Weinander R, Saha S, Jegerschold C, Pettersson PL, Samuelsson B, Hebert H, Hamberg M, Morgenstern R, Jakobsson PJ (2003). Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination. J Biol Chem. 278(25): 22199-209. Epub 2003 Apr 02.
https://pubmed.ncbi.nlm.nih.gov/12672824
History
Defence date
2003-09-19Department
- Department of Medical Biochemistry and Biophysics
Publication year
2003Thesis type
- Doctoral thesis
ISBN-10
91-7349-637-5Number of supporting papers
5Language
- eng