Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.     

Oxygenation of phosphatidylcholine by human polymorphonuclear leukocyte 15-lipoxygenase

A cell-free human polymorphonuclear leukocyte preparation containing both 15- and 5-lipoxygenase activities was found to oxygenate phosphatidylcholine at carbon-15 of the arachidonic acid moiety. No oxygenation at carbon-5 was found. Under similar incubation conditions, soybean and rabbit reticulocyte 15-lipoxygenases also oxygenated phosphatidylcholine, whereas rat basophilic leukemia cell 5-lipoxygenase, rabbit platelet 12-lipoxygenase and rat liver cytochrome P-450 preparations did not. Our results suggest that the oxygenation of phospholipids may be a unique property of the 15-lipoxygenases.     

Pharmacotherapy of diseases mediated by 5-lipoxygenase pathway eicosanoids

Inflammatory eicosanoids generated by the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism are now known to have at least 6 receptors: OXE, which recognizes 5-HETE and 5-oxo-ETE; a putative receptor recognizing a potent 5-oxo-ETE metabolite, FOG(7); the LTB(4) receptors, BLT1 and BLT2; the cysteinyl leukotriene receptors, CysLT(1) and CysLT(2), which recognize leukotrienes LTC(4), LTD(4), LTE(4) and LTF(4). The 5-LO pathway is activated in many diseases and invokes inflammatory responses not affected by glucocorticoids, but therapy with selective BLT1 or CysLT(1) antagonists in asthma has met with variable success. Studies show that 5-LO pathway eicosanoids are not primary mediators in all cases of asthma, but may be especially important in severe persistent asthma, aspirin- and exercise-induced asthma, allergic rhinitis, COPD, idiopathic pulmonary fibrosis, atherosclerosis, atopic dermatitis, acne and ischemia-related organ injury. These disorders appear to involve multiple 5-LO pathway eicosanoids and receptor subtypes, suggesting that inhibition of the pathway at the level of 5-LO may be necessary for maximal efficacy.     

Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.     

Endogenous hydroxyeicosatetraenoic acids stimulate the human polymorphonuclear leukocyte 15-lipoxygenase pathway

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.     

吡啶酮类衍生物对5-脂氧合酶的抑制作用

合成了一系列3-羟基-4(1H)-吡啶酮类衍生物,并研究了它们对5-脂氧合酶的抑制作用. 发现6-取代-3-羟基-4(1H)-吡啶酮化合物(2a～2e)对5-脂氧合酶具有显著抑制作用, 特别是6-苯硫基-1-苯基-2-甲基-3-羟基-4(1H)-吡啶酮(2a)的抑制效果最好(IC₅₀=2.52 μmol/L). 6-位没有取代基的羟基吡啶酮类化合物对5-脂氧合酶却没有抑制作用. 讨论了6-取代-3-羟基-4(1H)-吡啶酮类化合物对5-脂氧合酶的抑制作用机制.     

Inhibition of mammalian 5-lipoxygenase by 2-benzylaminophenols

A number of 2-benzylaminophenols, prepared from the corresponding 2-aminophenols by reductive alkylation, have been identified as highly potent inhibitors of 5-lipoxygenase with IC50 values in the nanomolar range. Most compounds were also shown to inhibit the release of the peptidoleukotrienes when administered intraperitoneally in a rat model of peritoneal anaphylaxis. Two compounds evaluated for their effects on anaphylactic contractions in isolated human lung were shown to attenuate the leukotriene-induced component of the response.     

Inhibition of mammalian 5-lipoxygenase by aromatic disulfides

As a primary step in leukotriene biosynthesis, arachidonic acid is converted into 5-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid by 5-lipoxygenase. This enzyme is studied in the supernatant fraction from sonified RBL-1 cells, a preparation that converts [1-14C]arachidonic acid to 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid and several 5,12-dihydroxyeicosatetraenoic acids including LTB4. In order to examine the reversibility of inhibitors, the supernatant fraction can be depleted of low molecular weight constituents by vacuum filtration. The 5-lipoxygenase is irreversibly inhibited by 500 microM N-ethyl-maleimide or 300 microM methyl methanethiolsulfonate, reagents that react covalently with protein sulfhydryl groups. In contrast, diphenyl disulfide reversibly inhibits this enzyme at 1-5 microM, irrespective of the GSH concentration in the supernatant. KCN also inhibits 5-lipoxygenase at 4 mM, suggesting the presence of a metal-containing prosthetic group. These observations imply that diphenyl disulfide and similar molecules with electron-releasing substituents on the aromatic rings could inhibit by binding to an electrophilic metallic center, the binding being stabilized by hydrophobic interactions between the enzyme and the aromatic groups on the flexible disulfide. Even though diphenyl disulfide does not inhibit soybean 15-lipoxygenase or endoperoxide synthase in cell-free systems, this compound does suppress prostaglandin as well as leukotriene synthesis in intact murine peritoneal macrophages and CXBG cells. Since lipoxygenases are susceptible to peroxide activation and peroxidase deactivation, changes in the redox state of the cell may alter arachidonic acid metabolism as effectively as actual enzyme inhibition.     

Inhibition of human sPLA2 and 5-lipoxygenase activities by two neo-clerodane diterpenoids.

The inhibitory effect of two neo-clerodane diterpenoids, E-isolinaridial (EI) and its methylketone derivative (EIM), isolated from Linaria saxatilis var. glutinosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Both compounds inhibited human synovial sPLA2 in a concentration-dependent manner with IC50 values of 0.20 and 0.49 microM, respectively, similar to scalaradial. Besides, these compounds decreased the cell-free 5-lipoxygenase activity and A23187-induced neutrophil LTB4 biosynthesis. Another function of human neutrophils, such as receptor-mediated degranulation, was also significantly reduced. In contrast, none of the compounds affected superoxide generation in leukocytes, or cyclooxygenase-1, cyclooxygenase-2 and inducible nitric oxide synthase activities in cell-free assays.     

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.     

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.     

Oxidation of oxa and thia fatty acids and related compounds catalysed by 5- and 15-lipoxygenase

The modified fatty acids, (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid, 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid, N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine and N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid, all react with soybean 15-lipoxygenase. The products were treated with triphenylphosphine to give alcohols, which were isolated using HPLC. Analysis of the alcohols using negative ion tandem electrospray mass spectrometry, and by comparison with compounds obtained by autoxidation of arachidonic acid, shows that each enzyme-catalysed oxidation occurs at the omega-6 position of the substrate. In a similar fashion, it has been found that (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid each undergoes regioselective oxidation at the carboxyl end of the polyene moiety on treatment with potato 5-lipoxygenase. Neither (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid nor N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid reacts in the presence of this enzyme, while N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine affords the C11' oxidation product. The alcohol derived from (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid using the 15-lipoxygenase reacts at the C6' position with the 5-lipoxygenase.     

Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.     

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.     

Impaired polymorphonuclear leukocyte 15-lipoxygenase activity in juvenile and rapidly progressive periodontitis

15-lipoxygenase activity was investigated in sonicated polymorphonuclear leukocytes (PMNLs) from patients with juvenile and rapidly progressive periodontitis and adult periodontitis. The group with juvenile and rapidly progressive periodontitis had 17 patients (6 male, 11 female, mean age 27.4 years), and the age matched control group had 18 normal individuals (11 male, 7 female, mean age 26.3 years). The group with adult periodontitis had 14 patients with 9 male, 5 female, mean age 45.2 years and the age-matched control group had 6 normal subjects with 5 male, 1 female, mean age 43.7 years. 15-hydroxyeicosa-tetraenoic acid (15-HETE) synthesized in the group with juvenile and rapidly progressive periodontitis was 0.219 +/- 0.102 ng/mg protein (mean +/- S.D.), while it was 0.410 +/- 0.138 ng/mg protein in the age-matched control group. There was a significant difference between the two groups. The group with adult periodontitis produced 0.358 +/- 0.124 ng/mg protein and the age matched control group produced 0.448 +/- 0.176 ng/mg protein (no significant difference). These results are relevant to reports that PMNLs of patients with juvenile and rapidly progressive periodontitis have abnormal functions, while those of patients with adult periodontitis are normal.     

Selective expression of 15-lipoxygenase activity by cultured human keratinocytes

Human keratinocytes isolated from neonatal skin express 15-lipoxygenase activity at a level far greater than that of any of the other pathways for lipoxygenation of arachidonic acid. The 10,000 x g supernatant of sonicates of 10(6) keratinocytes generates 15-hydroxy-eicosatetraenoic acid from 5 micrograms/ml of arachidonic acid at a mean maximum rate of 38 ng/30 min at 37 degrees C, that is similar to the activity of the 15-lipoxygenase of human airway epithelial cells and greater than that of endothelial cells and leukocytes. The unique mediators derived from the 15-lipoxygenation of arachidonic acid, that stimulate secretion and exert hyperalgesic effects, may achieve a concentration in skin sufficient to regulate local cellular and neural functions.     

Dimerization of human 5-lipoxygenase

Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.