[52] Preparation and proteolytic cleavage of apoprostaglandin endoperoxide synthase

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H 2 glycerol ester (PGH 2 -G), in vitro and in cultured macrophages. PGH 2 -G produced by macrophages is a substrate for cellular PGD synthase, affording PGD 2 -G. Pharmacological studies reveal that macrophage production of PGD 2 -G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG. Cyclooxygenase (COX 1; prostaglandin endoperoxide synthase, EC 1.14.99.1) catalyzes the bis-dioxygenation of arachidonic acid, generating prostaglandin (PG) H 2 , the precursor to a diverse family of lipid mediators including PGs, thromboxane, and prostacyclin (1). The discovery of a second COX isoform, COX-2, has provided important insights into the molecular basis of inflammation, hyperalgesia, and cancer and has established a novel pharmacological target for their treatment (2-4). The major functional differences between COX-1 and COX-2 are believed to be related to their differential regulation and tissue distribution (5). COX-1 is a constitutive enzyme, whereas COX-2 is inducible and highly regulated by a range of agonists (6, 7). COX-1 activity accounts for PG and thromboxane production in gastric mucosa, kidney, and platelets (3). COX-2 activity is primarily responsible for PG biosynthesis in the central nervous system and inflammatory cells (8 -10). These observations suggest that COX-1 and COX-2 serve different physiological and pathophysiological functions.The possibility that COX-2 has distinct biochemical functions has not been explored extensively. There are conserved structural differences between the active sites of COX-1 and COX-2 which have been exploited in the development of selective COX-2 inhibitors. It is possible that these or other structural differences may have evolved to support separate biochemical functions for the two COX isoforms. The first indication of a separate biochemical function for COX-2 was provided by the observation that it selectively oxygenates the neutral ethanolamide derivative of arachidonic acid, anandamide (11). Anandamide and 2-arachidonylglycerol (2-AG) are endogenous ligands for the cannabinoid receptors that bind ⌬ 9 -tetrahydrocannabinol and mediate its pharmacological effects (12). The ca...

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“…Apoenzyme was prepared as outlined (22). Site-directed mutagenesis of murine COX-2 was performed as described (23).…”

Section: Methodsmentioning

confidence: 99%

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2

Kozak

Rowlinson²,

Marnett

2000

Journal of Biological Chemistry

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360

314

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“…apoPGH synthase-1 was purified from ram seminal vesicles as described and stored at Ϫ80ЊC (34,35). Recombinant human PGH synthase-2 was expressed in Sf9 cells and purified as described (36 Guaiacol Peroxidase Activity.…”

Section: Methodsmentioning

confidence: 99%

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Landino

Crews

Timmons

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

387

226

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Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes' peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Znsuperoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid) 2 , and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inf lammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.Prostaglandins and thromboxanes are important mediators of inflammation, hyperalgesia, cell growth, and hemostasis inter alia. The committed step in prostaglandin biosynthesis is the oxygenation of arachidonic acid (AA) by prostaglandin endoperoxide (PGH) synthase, a bifunctional, membrane-bound hemeprotein (1-4). Inhibition of the cyclooxygenase activity of PGH synthase is the basis for the pharmacological action of nonsteroidal antiinflammatory drugs (5, 6). Two different PGH synthases exist in vertebrates-PGH synthase-1, which is expressed constitutively and occurs in many tissues, and PGH synthase-2, which is inducible and expressed transiently (7-10). PGH synthase-2 is present at high levels in monocytes͞ macrophages, where it appears to play a major role in the production of inflammatory prostaglandins (7,11).Several recent reports demonstrate that NO stimulates prostaglandin biosynthesis in vivo, in perfused organs, and in macrophages (12-17). The stimulatory effect of NO is rapid and appears to be the result of direct activation of cyclooxygenase activity (16). However, conflicting reports exist regarding the ability of NO to stimulate purified PGH synthase, and it is possible that a derivative of NO is responsible for the activation in inflammatory cells (16,(18)(19)(20).The principal mechanism described for direct activation of the cyclooxygenase activity of PGH synthase is reaction of fatty acid hydroperoxides with the heme prosthetic group to generate a protein radical. This protein radical (probably Tyr-385) serves as the catalytic oxidant of AA (21-23). The identity of the hydroperoxides that activate PGH synthase in different cell types is uncertain because fatty acid hydroperoxides are excellent substrates for glutathione peroxidase (GSH-Px)-catalyzed reduction by glutathione (GSH) (24). The potential for control of prostaglandin biosynthesis by GSH-Px͞GSH reduction of hydroperoxide activators exhibits s...

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“…PGHS-1 Purification-PGHS-1 was purified from ram seminal vesicles as described previously and stored at Ϫ80°C (39). Apo-PGHS-1 was prepared as described and was 99 -100% apoprotein as judged by cyclooxygenase activities in the presence and absence of Fe-PPIX (40 (42). Hydroperoxide Reduction Assays-The PGHS-2 mutants, wtPGHS-2, and apo-PGHS-1 were reconstituted with 0.5 eq of Fe-PPIX for 2 min in 500 l of 0.1 M Tris-HCl, pH 8.0, containing 500 M guaiacol.…”

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confidence: 99%

Mutational Analysis of the Role of the Distal Histidine and Glutamine Residues of Prostaglandin-Endoperoxide Synthase-2 in Peroxidase Catalysis, Hydroperoxide Reduction, and Cyclooxygenase Activation

Landino¹,

Crews²,

Gierse³

et al. 1997

Journal of Biological Chemistry

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Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His 193 , was mutated to alanine and the distal glutamine, Gln 189 , was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wt-PGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to ϳ60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.

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[52] Preparation and proteolytic cleavage of apoprostaglandin endoperoxide synthase

Cited by 37 publications

References 6 publications

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Mutational Analysis of the Role of the Distal Histidine and Glutamine Residues of Prostaglandin-Endoperoxide Synthase-2 in Peroxidase Catalysis, Hydroperoxide Reduction, and Cyclooxygenase Activation

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