Nigulas Samel

The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: Molecular cloning and expression of a novel cyclooxygenase

The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase–PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and ≈50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as by-products. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

The basis of prostaglandin synthesis in coral: molecular cloning and expression of a cyclooxygenase from the Arctic soft coral Gersemia fruticosa.

In vertebrates, the synthesis of prostaglandin hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively expressed enzyme with physiological functions, and COX-2, induced in inflammation and cancer. Prostaglandins have been detected in high concentrations in certain corals, and previous evidence suggested their biosynthesis through a lipoxygenase-allene oxide pathway. Here we describe the discovery of an ancestor of cyclooxygenases that is responsible for prostaglandin biosynthesis in coral. Using a homology-based polymerase chain reaction cloning strategy, the cDNA encoding a polypeptide with ∼50% amino acid identity to both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic soft coral Gersemia fruticosa. Nearly all the amino acids essential for substrate binding and catalysis as determined in the mammalian enzymes are represented in coral COX: the arachidonate-binding Arg120 and Tyr355 are present, as are the heme-coordinating His207 and His388; the catalytic Tyr385; and the target of aspirin attack, Ser530. A key amino acid that determines the sensitivity to selective COX-2 inhibitors (Ile523 in COX-1 and Val523 in COX-2) is present in coral COX as isoleucine. The conserved Glu524, implicated in the binding of certain COX inhibitors, is represented as alanine. Expression of the G. fruticosa cDNA afforded a functional cyclooxygenase that converted exogenous arachidonic acid to prostaglandins. The biosynthesis was inhibited by indomethacin, whereas the selective COX-2 inhibitor nimesulide was ineffective. We conclude that the cyclooxygenase occurs widely in the animal kingdom and that vertebrate COX-1 and COX-2 are evolutionary derivatives of the invertebrate precursor.

Structure of a Calcium-dependent 11R-Lipoxygenase Suggests a Mechanism for Ca2+ Regulation

Background: Lipoxygenases vary in their catalytic specificity and regulation. Results: 11R-LOX, strictly Ca2+-dependent, displays novel structural features in the membrane-binding domain. Conclusion: A model for how access to an enclosed active site is linked to Ca2+-dependent membrane binding is proposed. Significance: The 11R-LOX model provides structural insights into the allosteric regulation of lipoxygenases. 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.

Evidence of a Cyclooxygenase-related Prostaglandin Synthesis in Coral THE ALLENE OXIDE PATHWAY IS NOT INVOLVED IN PROSTAGLANDIN BIOSYNTHESIS

Certain corals are rich natural sources of prostaglandins, the metabolic origin of which has remained undefined. By analogy with the lipoxygenase/allene oxide synthase pathway to jasmonic acid in plants, the presence of (8R)-lipoxygenase and allene oxide synthase in the coral Plexaura homomallasuggested a potential metabolic route to prostaglandins (Brash, A. R., Baertshi, S. W., Ingram, C.D., and Harris, T. M. (1987)J. Biol. Chem. 262, 15829–15839). Other evidence, from the Arctic coral Gersemia fruticosa, has indicated a cyclooxygenase intermediate in the biosynthesis (Varvas, K., Koljak, R., Järving, I., Pehk, T., and Samel, N. (1994) Tetrahedron Lett. 35, 8267–8270). In the present study, active preparations of G. fruticosa have been used to identify both types of arachidonic acid metabolism and specific inhibitors were used to establish the enzyme type involved in the prostaglandin biosynthesis. The synthesis of prostaglandins and (11R)-hydroxyeicosatetraenoic acid was inhibited by mammalian cyclooxygenase inhibitors (indomethacin, aspirin, and tolfenamic acid), while the formation of the products of the 8-lipoxygenase/allene oxide pathway was not affected or was increased. The specific cyclooxygenase-2 inhibitor, nimesulide, did not inhibit the synthesis of prostaglandins in coral. We conclude that coral uses two parallel routes for the initial oxidation of polyenoic acids: the cyclooxygenase route, which leads to optically active prostaglandins, and the lipoxygenase/allene oxide synthase metabolism, the role of which remains to be established. An enzyme related to mammalian cyclooxygenases is the key to prostaglandin synthesis in coral. Based on our inhibitor data, the catalytic site of this evolutionary early cyclooxygenase appears to differ significantly from both known mammalian cyclooxygenases.

Direct evidence of the cyclooxygenase pathway of prostaglandin synthesis in arthropods: genetic and biochemical characterization of two crustacean cyclooxygenases.

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world.

New antiproliferative 9,11-secosterol from soft coral gersemia fruticosa

A new highly antiproliferative 9,11-secosterol 1 was isolated from the White Sea soft coral Gersemia fruticosta. The structure was established

New cytotoxic sterols from the soft coral Gersemia fruticosa

Abstract Six new polyoxygenated sterols 1–6 were isolated from the soft coral Gersemia fruticosa. The structures of these compounds were determined by MS. 1H- and 13C- 1D and 2D FT NMR spectroscopy. Compounds 1–6 showed a moderate cytotoxic activity against human erythroleukemia K-562 cells and other leukemia cell lines.

Comparison of Anti-Aggregatory Effects of PGI2, PGI3 and Iloprost on Human and Rabbit Platelets

In human, prostaglandin I3 (PGI3) is as potent inhibitor of platelet aggregation as prostacyclin (PGI2). However the data on the anti-aggregatiory effect of this prostaglandin is scanty on human and is absent on platelets of other species. The potency of PGI3 on other species may be different if there are differences in the structure of receptors. Comparison of the rank orders of the potency of the selective agonists in different species may provide evidence for the existence of such differences. The aim of this work was to study the anti-aggregatory effect of PGI3 on the platelets of human and rabbit and compare the rank orders of the potency of PGI2, PGI3, and iloprost, a synthetic analogue of PGI2, on the platelets of the two species. Experiments were performed in the suspensions of washed platelets prepared from the blood anticoagulated with trisodium citrate solution. A prostaglandin concentration causing 50% inhibition of ADP-induced platelet aggregation (IC50) was obtained from concentration-effect curves. On human platelets, PGI3 was as effective as PGI2, while on rabbit platelets, the value of IC50 for PGI3 (10.2 ± 1.6 nM) was twofold higher than that of PGI2. The rank orders of agonist potency are different in rabbit compared to those of human. This indicates that the prostacyclin receptors of rabbit platelets are pharmacologically different from those of human.

Platelet activity before and after coronary artery bypass grafting

The normal response of the platelet can be altered either by increased pro-aggregatory stimuli or by diminished anti-aggregatory substances. Increased platelet activation occurs in the cardiovascular disease states of stable angina pectoris and acute coronary syndromes. Also, cardiac surgery involving cardiopulmonary bypass is associated with extensive contact between blood and synthetic surfaces and leads to a strong activation of platelets. Using light transmission aggregometry method, we tested platelet sensitivity to ADP and spontaneous activity in the platelet-rich plasma of patients before and after coronary artery bypass grafting (CABG). The median values of EC50 for ADP were significantly lower at days 3 and 5 after surgery (0.33 and 0.37 µM, respectively) than before surgery (0.65 µM). The medians percentages of spontaneous platelet aggregation were significantly higher after than before surgery (16 and 15% at days 3 and 5 after surgery, respectively, vs. 5% before surgery). Thus, there is a significant increase of spontaneous platelet aggregation and platelet hyperreactivity to ADP in patients after CABG compared with those before surgery.

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.