Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E 2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE 2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE 2 generation. Arg 110 , a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A 2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE 2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

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. GSHdependent 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. Biosynthesis of prostaglandin (PG)1 E 2 , the most common prostanoid with potent bioactivities, is regulated by three sequential steps of the cyclooxygenase (COX) pathway. Phospholipase A 2 (PLA 2 ) initiates this pathway by releasing arachidonic acid (AA) from membrane glycerophospholipids. Of more than 10 members of the PLA 2 family characterized to date, cytosolic PLA 2 (cPLA 2 ) and several secretory PLA 2 s are involved in supplying AA to either of the two COX isozymes, COX-1 and COX-2, depending upon the phases of cell activation (1-3). The constitutive COX-1 is mainly utilized in immediate PGE 2 biosynthesis, which occurs within several minutes after stimulation with Ca 2ϩ mobilizers, whereas the inducible COX-2 mediates the delayed PGE 2 biosynthesis, which lasts for several hours following proinflammatory stimuli. Although COX-1 and COX-2 have been reported to exhibit subtle differences in AA requirements in that COX-2 is favored over COX-1 at low AA concentrations (3-5) and subcellular localizations (6), their functional segregation in the PGE 2 biosynthetic response cannot be fully explained only by these aspects.The activity of PGES, which catalyzes conversion of COXderived PGH 2 to PGE 2 , has been detected in both cytosolic and microsomal fractions of various cells, and in most, if not all, cases it requires glutathione (GSH) for optimal activity (7-9). Although several groups have attempted to purify this critical enzyme to near homogeneity for the last 20 years (7-9), such trials have been unsuccessful. The PGES enzyme purified from human brain cytosol was identified as a GSH S-transferase (GST), which converts PGH 2 to PGE ...

We have recently shown that two distinct prostaglandin (PG) E 2 synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE 2 biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE 2 synthase, the perinuclear enzymes thromboxane synthase and PGI 2 synthase generated their respective products via COX-2 in preference to COX-1 in both the A23187-induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD 2 synthase preferentially used COX-1 and COX-2 in the A23187-induced immediate and interleukin-1-induced delayed PGD 2 -biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A 2 . Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA 2 s at a moment when PG production takes place.Biosynthesis of prostaglandins (PGs) 1 through the cyclooxygenase (COX) pathway involves oxidation and subsequent isomerization of membrane-derived arachidonic acid (AA) via three sequential enzymatic reactions. The initial step of this metabolic pathway is the stimulus-induced liberation of AA from membrane glycerophospholipids by the action of phospholipase A 2 (PLA 2 ) enzymes, including cytosolic PLA 2 ␣ (cPLA 2 ; group IVA) and several secretory PLA 2 (sPLA 2 ) isozymes (groups IIA, IID, V, and X) (1-10). The released AA is sequentially metabolized to PGG 2 and then to PGH 2 by either COX-1 or COX-2. PGH 2 is then converted to various bioactive PGs (thromboxane (TX) A 2 , PGD 2 , PGE 2 , PGF 2␣ , and PGI 2 ) by the respective terminal PG synthases, which have different structures and exhibit cell-and tissue-specific distributions.Segregated utilization of COX-1 and COX-2 in the PG biosynthetic events has been demonstrated by a number of cell biological, pharmacological, and genetic studies (3,(11)(12)(13)(14)(15)(16)(17). Generally, the constitutive COX-1 is mainly utilized in the immediate PG biosynthesis, which occurs within several minutes after stimulation with Ca 2ϩ mobilizers, whereas the inducible COX-2 is an absolute requirement for delayed PG biosynthesis, which lasts for several hours after proinflammatory stimuli. When cells are first treated with proinflammatory stimuli and subsequently exposed to Ca 2ϩ mobilizers, the induced COX-2 can also promote the immediate response (priming response) (3, 18 -22). However, the precise molecular me...

Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectinspecific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 6 5.5 mg/ml (mean 6 SD) in healthy women and 6.2 6 3.6 mg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).-Nakano, Y., S. Tajima, A. Yoshimi, H. Akiyama, M. Tsushima, T. Tanioka, T. Negoro, M. Tomita, and T. Tobe. A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin. Adiponectin is an adipocyte-specific secretory protein that is highly and specifically expressed in adipose tissue (1-3). Adiponectin includes a collagen-like domain, and in this domain, three adiponectin peptides form one stable trimer and the trimers further multimerize to form "bouquet" forms (Fig. 1). In human plasma, adiponectin was found to circulate as a trimer, a hexamer, and a highmolecular-weight (HMW) multimer, and we purified the HMW adiponectin of 420 kDa from human serum using gelatin-Cellulofine and previously reported it as the gelatin binding protein of 28 kDa (GBP28) in 1996 (4).Plasma adiponectin levels are reported to be decreased in obese individuals, to be negatively correlated with visceral fat accumulation, and to be significantly lower in type 2 diabetic patients with coronary artery disease (5-7). Adiponectin mRNA levels are significantly reduced in omental adipose tissue of obese patients with type 2 diabetes compared with lean and obese normoglycemic subjects, and although less pronounced, the levels are also reduced in subcutaneous adipose tissue of type 2 diabetic patients (8). Plasma adiponectin concentrations in patients with acute coronary syndrome, both acute myocardial infarction and unstable angina pectoris, are significantly lower than those in patients with stable angina pectoris and in controls, and a low adiponectin concentration is correlated independently with the development of an acute coronary disease (9). Plasma adiponectin levels are an inverse predictor of the cardiovascular outcome in patients with end-stage renal disease (10). Tietge et al. (11) reported that plasma adipon...

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X) 18 E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-␥. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.