Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase.

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 µM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance.

Distinct characteristics of the substrate binding between highly homologous catalase-related allene oxide synthase and hydroperoxide lyase.

A catalase-related allene oxide synthase (cAOS) or a hydroperoxide lyase (cHPL) fused together with an 8R-lipoxygenase is involved in the stress signaling of corals via an arachidonic acid pathway. cAOS gives rise to α-ketol and cyclopentenone, while cHPL catalyzes the cleavage of 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) to C8-oxo acid and C12 aldehyde. In silico analysis of the substrate entry sites of highly identical coral cAOS and cHPL indicated that two positively charged residues of cAOS, K60 and K107, and the corresponding residues of cHPL, E60 and K107, may be involved in the anchoring of the carboxy group of polyunsaturated fatty acid (PUFA) hydroperoxides. A mutational analysis of cAOS and cHPL revealed that K60 or E60 and K107 were not necessary in the tethering of 8R-HpETE, however, the E60 of cHPL was essential in the productive binding of PUFA hydroperoxides. The substrate preferences of cAOS and cHPL were determined with hydroperoxy derivatives of C18, C20, C22 PUFAs, anandamide (AEA), 1-arachidonoyl glycerol (1-AG) and selected methylated substrates. Although cAOS and cHPL were able to metabolize different free PUFA substrates and arachidonoyl derivatives, only cHPL catalyzed the reaction with methylated PUFA hydroperoxides. The differences in the substrate binding and preferences between cAOS and cHPL can be explained by the distinct properties of their substrate entry sites. The current study demonstrated that homologous PUFA metabolizing enzymes may contribute to the versatile usage of the substrate pool.

Eicosanoid Diversity of Stony Corals.

Oxylipins are well-established lipid mediators in plants and animals. In mammals, arachidonic acid (AA)-derived eicosanoids control inflammation, fever, blood coagulation, pain perception and labor, and, accordingly, are used as drugs, while lipoxygenases (LOX), as well as cyclooxygenases (COX) serve as therapeutic targets for drug development. In soft corals, eicosanoids are synthesized on demand from AA by LOX, COX, and catalase-related allene oxide synthase-lipoxygenase (cAOS-LOX) and hydroperoxide lyase-lipoxygenase (cHPL-LOX) fusion proteins. Reef-building stony corals are used as model organisms for the stress-related genomic studies of corals. Yet, the eicosanoid synthesis capability and AA-derived lipid mediator profiles of stony corals have not been determined. In the current study, the genomic and transcriptomic data about stony coral LOXs, AOS-LOXs, and COXs were analyzed and the eicosanoid profiles and AA metabolites of three stony corals, Acropora millepora, A. cervicornis, and Galaxea fascicularis, were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with MS-MS and a radiometric detector. Our results confirm that the active LOX and AOS-LOX pathways are present in Acropora sp., which correspond to the genomic/sequence data reported earlier. In addition, LOX, AOS-LOX, and COX products were detected in the closely related species G. fascicularis. In conclusion, the functional 8R-LOX and/or AOS-LOX pathways are abundant among corals, while COXs are restricted to certain soft and stony coral lineages.

A PDZ-like domain mediates the dimerization of 11R-lipoxygenase.

Lipoxygenases (LOXs), participating in inflammatory processes and cancer, are a family of enzymes with high potential as drug targets. Various allosteric effects have been observed with different LOX isozymes (e.g. lipid/ATP binding, phosphorylation), yet there is a lot of uncertainty concerning the regulation of these enzymes. It has been recently found that a number of LOXs form dimers, extending the list of possible allosteric mechanisms with oligomerization. Coral 11R-LOX is, unlike several mammalian counterparts, a stable dimer in solution facilitating quaternary structure studies that demand high sample homogeneity. By combining previous crystallographic data of 11R-LOX with small-angle X-ray scattering and chemical cross-linking, we were able to narrow down the possible dimerization interfaces, and subsequently determined the correct assembly by site-directed mutagenesis of potential contacting residues. The region of interest is located in the vicinity of an α+ß formation in the catalytic domain, also coined the PDZ-like domain. Being situated just between the active site and the dimer interface, our results further implicate this putative subdomain in the regulation of LOXs.

Glucose impairs aspirin inhibition in platelets through a NAD(P)H oxidase signaling pathway.

Hyperglycemia has been suggested to play a role in the increased platelet resistance to antiplatelet therapy in patients with diabetes mellitus. Exposure to high glucose impairs platelet inhibition by aspirin. It has been found that antioxidant agents reduce the effect of glucose, confirming the involvement of reactive oxygen species (ROS) in the effect of glucose. The aim of the study was to examine the mechanism of ROS increase by high glucose in aspirin-treated platelets. Platelet aggregation was measured by the optical method, and the production of ROS was detected using luminol-dependent horseradish peroxidase-enhanced chemiluminescence. We found that glucose did not affect ADP-induced platelet aggregation. However, it reduced the effect of aspirin on platelet aggregation, which was accompanied by an increase in ROS generation. The inhibition of NAD(P)H oxidase (NOX) prevented the glucose effect and ROS generation. The same result was recorded after the inhibition of p38 mitogen-activated protein kinases (p38 MAPK), phospholipase A2 (PLA2) or 12-lipoxygenase (12-LOX). The inhibition of TxA2 receptor did not decrease the effect of glucose indicating that the effect was not caused by activation of TxA2 receptors.

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE.

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom.

A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation.

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase.

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG2 and PGH2. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.

Lactate is a possible mediator of the glucose effect on platelet inhibition.

Abstract Glucose has been found to impair the inhibition of platelets with aspirin and alter the basal activity of nitric oxide synthase (NOS) in platelets. The aim of this work was to study the effects of glucose on the inhibitory pathways in activated platelets. A short-term incubation of glucose impaired the inhibition of platelet aggregation induced by agents activating an NOS-dependent pathway, such as l-arginine, adenosine and α-tocopherol. However, glucose had no effect on the inhibition induced by iloprost and BW245C, agents that activate the cyclic adenosine monophosphate (cAMP) signaling pathway. Potassium lactate attenuated the effects of the same inhibitors as glucose did. The inhibitors of glucose transport prevented the effect of glucose. Dichloroacetate, known to prevent the conversion of pyruvate to lactate and to decrease lactate in platelets, significantly attenuated the effect of glucose in platelets. The data support the suggestion that the effect of glucose on the inhibition of platelets by agents activating an NOS-dependent pathway is mediated by glucose metabolite lactate.

Structure of a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2+ regulation.

Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge.

Activation of 11R-lipoxygenase is fully Ca(2+)-dependent and controlled by the phospholipid composition of the target membrane.

Activation of some lipoxygenases (LOX) is found to be related to the selective membrane binding upon cell stimulation. In this study, a systematic analysis of the effect of the lipid composition on the membrane binding efficiency, Ca(2+) affinity, and enzymatic activity of 11R-LOX was performed. The analysis of the membrane targeting by fluorometric and surface plasmon resonance measurements in the absence of Ca(2+) showed an exclusive binding of 11R-LOX to the anionic phospholipids (phosphatidylinositol < phosphatidylglycerol ≈ phosphatidylserine) containing model membranes. The presence of Ca(2+) enhanced the rate of interaction and influenced its mode. The modulation of the activity of 11R-LOX indicated that (i) Ca(2+) binding is a prerequisite for productive membrane association, (ii) the reaction of 11R-LOX with arachidonic acid coincided with and was driven by its Ca(2+)-mediated membrane association, and (iii) phosphatidylethanolamine and anionic phospholipids had a synergistic effect on the Ca(2+) affinity, in line with a target-activated messenger affinity mechanism [Corbin, J. A., et al. (2007) Biochemistry 46, 4322-4336]. According to the mechanism proposed in this report, 11R-LOX can bind to the membranes in two different modes and the efficiency of productive membrane binding is determined by a concerted association of Ca(2+) and lipid headgroups.

Purification and characterization of the recombinant human prostaglandin H synthase-2 expressed in Pichia pastoris.

Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2.

Short-term exposure of platelets to glucose impairs inhibition of platelet aggregation by cyclooxygenase inhibitors.

Aspirin treatment reduces cardiovascular events and deaths in high-risk non-diabetic patients, but not in patients suffering from diabetes. In these patients, hyperglycemia has been found to cause reduced platelet sensitivity to aspirin. It is supposed that long-term exposure of platelets to glucose leads to non-enzymatic glycosylation and impairs aspirin inhibition of platelet aggregation. On the other hand, short-term exposure of platelets to glucose also attenuates the effect of aspirin on platelets. The aim of the present work was to analyse the effect of short-term exposure of glucose on the inhibition of platelet aggregation by aspirin and other cyclooxygenase (COX) inhibitors. Already a 15 min exposure of platelets to glucose impaired aspirin inhibition of the platelet aggregation induced by collagen, thrombin, adenosine diphosphate (ADP), and arachidonic acid (AA). Aspirin inhibition of platelet aggregation in platelet-rich plasma (PRP) was attenuated by 5.6, 11.2, 16.8, and 22.4 mM of glucose in a concentration-dependent way. The same effect was observed with indomethacin and acetaminophen used as cyclooxygenase inhibitors instead of aspirin. N-methyl-L-arginine, an inhibitor of nitric oxide synthase, prevented the effect of glucose on aspirin, indomethacin and acetaminophen inhibition of platelet aggregation. Other monosaccharides, for example fructose and galactose, impaired aspirin inhibition as did glucose. Lactic acid (0.1, 0.2, 0.4, 0.8 mM), the end product of anaerobic glycolysis in platelets, impaired the inhibition of platelet aggregation with aspirin in a concentration-dependent way but did not affect indomethacin. It is suggested that lactic acid might be a mediator of the effect of glucose on aspirin inhibition in platelets.

Calorimetric approach for comparison of Angiopoietin-like protein 4 with other pancreatic lipase inhibitors.

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.

Identification of a functional allene oxide synthase-lipoxygenase fusion protein in the soft coral Gersemia fruticosa suggests the generality of this pathway in octocorals.

The conversion of fatty acid hydroperoxides to allene epoxides is catalysed by a cytochrome P450 in plants. In contrast, in the coral Plexaura homomalla, a catalase-related hemoprotein fused to the lipoxygenase (LOX) was found to function as an allene oxide synthase. This work reports the homology-based RT-PCR cloning and functional expression of a Gersemia fruticosa analogue of the allene oxide synthase-lipoxygenase (AOS-LOX) fusion protein. The G. fruticosa mRNA codes for a protein with 84% sequence identity to the P. homomalla AOS-LOX. Our data indicate that the AOS-LOX fusion protein pathway is used by another coral and P. homomalla represents no exception.

Effect of vitamin B(6) vitamers on platelet aggregation.

The vitamers of vitamin B(6) inhibit platelet aggregation. However, their effect is weak when used separately. After the supplementation of one vitamer, the concentrations of the other ones also increase in plasma due to the interconversion of vitamin B(6) forms. It can be suggested that different vitamers in blood can interact with each other. The aim of this work was to test the effect of different vitamer combinations on platelet aggregation in vitro. Platelet aggregation was induced by ADP, collagen or arachidonic acid and measured photometrically in a Chronolog aggregometer. The inhibition of platelet aggregation by the pairwise combinations of the vitamers was significantly stronger than that of each vitamer separately. The combinations of three and four vitamers were yet more effective, inhibiting platelet aggregation at the concentration of 4 microM. Possible involvement of inhibitory prostaglandins in the effect of vitamin B(6) was studied. The inhibition of platelet aggregation by the vitamers could be prevented by the antagonist of prostacyclin receptors, CAY10441 while the antagonist of prostaglandin D(2) receptors, MK 0524 was ineffective. The results suggest that vitamin B(6) vitamers cause a synergistic inhibitory effect on platelet aggregation at concentrations that can be mediated by the activation of prostacyclin receptors with prostaglandin E(1).

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL-R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD-with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

Enhanced expression of human prostaglandin H synthase-2 in the yeast Pichia pastoris and removal of the C-terminal tag with bovine carboxypeptidase A.

Vertebrate prostaglandin H synthases (PGHSs) are membrane-bound disulphide-containing hemoglycoproteins. Therefore, eukaryotic expression systems are required for the production of recombinant PGHSs. Recently we announced the expression of human PGHS-2 (hPGHS-2) in the yeast Pichia pastoris. Here we report improved production of hPGHS-2 in P. pastoris and a convenient method for the purification and de-tagging of the protein. An affinity tag comprised of a proline, a glycine and eight histidines was introduced into the C-terminal end of hPGHS-2. The tagged hPGHS-2 was expressed intracellularly in P. pastoris under the control of a constitutive or methanol-inducible promoter. Compared to constitutive expression, methanol-induced expression yielded approximately four times more protein. The analysis of high and low gene copy number recombinants revealed a positive correlation between the gene copy number and the expression level of hPGHS-2. The recombinant hPGHS-2 was purified using immobilised metal ion affinity chromatography. A novel elution method, treatment of the affinity resin with bovine carboxypeptidase A, was employed. The yield of pure de-tagged hPGHS-2 from 1l of yeast culture was approximately 3mg. The protein purification process with simultaneous removal of the C-terminal polyhistidine tag could be easily applied for the affinity purification of other proteins.

N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris.

Prostaglandin H synthases (PGHSs) are N-glycosylated membrane proteins that catalyse the committed step in prostaglandin synthesis. Unlike PGHS-2, the production of recombinant PGHS-1 in non-mammalian expression systems is complicated. The majority of the heterologous enzyme is inactive due to misfolding. Correct N-glycosylation is proposed to be obligatory for proper folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Accumulation of non-glycosylated hPGHSs was not observed in the crude lysate of the yeast cells. The N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. The isoforms exhibited similar N-glycosylation site occupancy. The results indicate that there are more complex grounds for the inactivity of the recombinant hPGHS-1 produced in yeast.

Novel membrane-associated prostaglandin E synthase-2 from crustacean arthropods.

Prostaglandins (PG) have been shown to play important physiological roles in insects and marine invertebrates, yet the knowledge of their biosynthetic pathways is often lacking. Recently, we described cyclooxygenases in two amphipod crustaceans, Gammarus sp. and Caprella sp. In the present study, we report the cloning and characterization of prostaglandin E synthases (PGES) from the same organisms. The amphipod membrane-bound PGES-2-type enzymes share about 40% of the amino acid sequence identity with human mPGES-2, contain a conserved Cys110-x-x-Cys113 motif and have very low heme-binding affinity. The recombinant enzymes purified in the absence of dithiothreitol specifically catalyze the isomerization of PGH2 into PGE2. The PGES activity is increased in the presence of reduced glutathione and inhibited with a sulfhydryl group inhibitor. We assume that the amphipod mPGES-2, unlike in their mammalian counterparts, is responsible for PGE2 synthesis, not only in vitro but also in vivo.