Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12.

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.

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.

Development of a method for expression and purification of the regulatory C2-like domain of human 5-lipoxygenase.

5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is built of a catalytic C-terminal domain and a regulatory N-terminal C2-like domain. The C2-like domain is the target of many regulatory factors or proteins including Ca(2+), phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-LO by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. In order to obtain the 5-LO C2-like domain as purified protein in good yields for further mechanistic studies and structure elucidation, a novel expression and purification approach has been applied. A plasmid was constructed expressing a fusion protein of maltose-binding protein (MBP) and the regulatory C2-like domain of 5-LO (AS 1-128), separated by a tobacco etch virus (TEV) protease-cleavage site. The fusion protein MBP-5LO1-128 could be essentially expressed as a soluble protein in Escherichia coli and was efficiently purified by amylose affinity chromatography. By means of this procedure, approximately 80mg purified fusion protein out of 1L E. coli culture were obtained. Digestion with TEV protease yielded the C2-like domain that was further purified using hydrophobic interaction chromatography. Alternatively, the uncleaved fusion protein MBP-5LO1-128 may be suitable to immobilize the C2-like domain on an amylose resin for co-factor interaction studies. Together, we present a convenient expression and purification strategy of the 5-LO C2-like domain that opens many possibilities for structural determination and mechanistic studies, aiming to reveal the precise role and function of this regulatory domain.

5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.

Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.

12-Lipoxygenase from rat basophilic leukemia cells, an oxygenase with leukotriene A4-synthase activity.

Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.

Expression of a cDNA encoding human 5-lipoxygenase under control of the STA1 promoter in Saccharomyces cerevisiae.

A yeast-expression vector utilizing the STA1 promoter was constructed, and shown to be useful for the expression of a heterologous gene. A cDNA, encoding human 5-lipoxygenase (5LO), was inserted into the vector and expressed in Saccharomyces cerevisiae. The enzyme (yh5LO) produced in the transformant was purified to homogeneity from the cellular soluble fraction. The purified enzyme showed both 5LO and leukotriene A4 synthase activities, which were stimulated by Ca2+ and ATP. The N-terminal end of yh5LO contained five extra amino acids not present in 5LO purified from human leukocytes. A human 5LO-secretion vector containing the STA1 signal sequence was also constructed. When this hybrid gene was expressed in S. cerevisiae, its product was glycosylated and accumulated in the fractions related to the secretory pathway.

Criteria for the identification of non-redox inhibitors of 5-lipoxygenase.

Methoxyalkyl thiazoles have been identified as a novel series of selective 5-lipoxygenase inhibitors with anti-inflammatory properties (Bird et al., J Med Chem 34: 2176-2186, 1991). Based on their structure, it was proposed that the potency of these compounds is not due to redox or iron-chelating properties. In the studies reported here, it was found that the model compounds 1-[3-(naphth-2-ylmethoxy)phenyl]-1-(thiazol-2-yl)propy l methyl ether (ICI 211965) and 3-[1-(4-chlorobenzyl)-4-methyl-6-(5- phenylpyridin-2-ylmethoxy)-4,5-dihydro-1H-thiopyrano[2 ,3,4-c,d]indol-2- yl]-2,2-dimethylpropanoic acid (L-689,065) (1) are inactive as reducing substrates in the 5-lipoxygenase-catalyzed decomposition of lipid hydroperoxides, (2) inhibit the 5-lipoxygenase-catalyzed reaction of reducing agents with lipid hydroperoxides, and (3) strongly inhibit the turnover-dependent inactivation of 5-lipoxygenase. These three observations with ICI 211965 and L-689,065 are in contrast to the behavior of other potent 5-lipoxygenase inhibitors from other structural classes, such as L-670,630, BW A4C, and zileuton, which all function as reducing substrates for 5-lipoxygenase. The data indicate that methoxyalkyl thiazoles and thiopyranoindoles are reversible dead-end inhibitors of 5-lipoxygenase and that the effects of inhibitors on the pseudoperoxidase activity and rate of enzyme inactivation provide simple tests to distinguish between redox and non-redox inhibitors of 5-lipoxygenase.

Sensitivity of immunoaffinity-purified porcine 5-lipoxygenase to inhibitors and activating lipid hydroperoxides.

The requirement for hydroperoxide activation and the effect of inhibitors from different structural classes on 5-lipoxygenase activity were determined on the immunoaffinity-purified enzyme from porcine leukocytes. The 5-lipoxygenase activity was measured using a continuous spectrophotometric assay monitoring the increase in conjugated diene formation (A235) upon incubation of the enzyme with arachidonic acid. Under standard assay conditions, the reaction progress curves showed little or no lag phase, with a rapid first-order decay in enzyme activity (T1/2 = 0.7 to 1.1 min). Both the initial rate of the reaction and total product formation were stimulated by the addition of ATP, Ca2+ and phosphatidylcholine (PC). PC (24 micrograms/ml) was also found to increase the recovery of radiolabeled arachidonic acid from the assay mixture and thus part of the stimulation may be due to an increase in substrate availability and reduction of surface adsorption effects. The requirement of hydroperoxides for the initiation of the reaction was shown by the induction of 0.1 to 1-min lag phases using NaBH4 or glutathione peroxidase and by the reduction in lag times by 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 13-hydroperoxyoctadecadienoic acid (13-HPOD). The following compounds were evaluated as inhibitors of the 5-lipoxygenase reaction and caused a 50% decrease in product accumulation (IC50) at the indicated concentrations: quercetin, L-651,896, L-656,224, MTPPH and L-651,392 (0.3-0.5 microM); diphenyldisulfide (2-5 microM); phenidone (5-10 microM); AA861 (4-10 microM) and BW755C (4-15 microM). In addition, the presence of inhibitors extended the initial lag phase of the reaction and increased the dependence of the initiation of the reaction on exogenous lipid hydroperoxides. The inhibition by phenidone was accompanied by a 2-fold increase in the rate of enzyme inactivation, whereas other compounds such as AA861 and L-656,224 did not show this effect. The results indicate that the presence of inhibitors can modify the kinetics of 5-lipoxygenase at the levels of the initiation of the reaction and the rate of enzyme inactivation, with variations depending on the structural class of the inhibitor and the concentration of lipid hydroperoxides.

WY-50,295 tromethamine, a novel, orally active 5-lipoxygenase inhibitor: biochemical characterization and antiallergic activity.

WY-50,295 tromethamine demonstrates significant 5-lipoxygenase inhibitory activity with IC50 values ranging from 0.055 microM in rat peritoneal exudate cells, to 0.16 microM in mouse macrophages, 1.2 microM in human peripheral neutrophils and 8.1 microM in rat blood leukocytes. This activity appeared selective for 5-lipoxygenase as concentrations up to 10 microM in rat peritoneal exudate cells, and 1 microM in mouse macrophages did not effect prostaglandin generation. In non-cellular enzyme assays, WY-50,295 tromethamine displayed inhibitory activity against a soluble 5-lipoxygenase from guinea pig peritoneal exudate cells (IC50 = 5.7 microM), while it was essentially inactive against 12-lipoxygenase, 15-lipoxygenase, or prostaglandin H synthetase at concentrations up to 500 microM, or against human phospholipase A2 at concentrations up to 50 microM. In purified human blood neutrophils the inhibitory activity was reversible but did not appear dependent upon substrate concentration. IN contrast, in the guinea pig cell-free 5-lipoxygenase assay changing the arachidonic acid substrate concentration from 5 to 500 microM produced a concentration-dependent reduction in inhibitory activity. WY-50,295 tromethamine inhibited the release of peptidoleukotrienes from fragmented guinea pig lung with an IC50 of 0.63 microM. When administered p.o. with a 4 h pretreatment time, WY-50,295 tromethamine inhibited ex vivo leukotriene B4 production in rat blood leukocytes with an ED50 of 19.6 mg/kg. Against an ovalbumin-induced leukotriene dependent bronchoconstriction in anesthetized sensitized guinea pigs, WY-50,295 tromethamine inhibited the ovalbumin-induced bronchoconstriction with an i.v. ED50 of 2.5 mg/kg (5 min pretreatment) and a p.o. ED50 of 7.3 mg/kg (4 h pretreatment). Significant activity was also evident with an 18 h pretreatment. Thus WY-50,295 tromethamine is an potent and selective 5-lipoxygenase inhibitor in a number of in vitro systems. Additionally the compound is orally efficacious and has a long duration of action in an allergic bronchoconstriction model. This data suggests that WY-50,295 tromethamine may have utility in the treatment of asthma and other leukotriene-dependent pathologies.

Demonstration of multiple regulatory factors for purified human leukocyte 5-lipoxygenase.

The human leukocyte 5-lipoxygenase is a unique enzyme in its class because of its involvement in the synthesis of the biologically active leukotrienes. Furthermore, unlike most other lipoxygenases, this enzyme requires multiple stimulatory factors for maximal activity. These include Ca2+, ATP, and three non-dialyzable cellular components, two cytosolic, and one membrane-associated. The mechanism of action of these factors is not yet well understood; however, a Ca2+-dependent association of the enzyme and one of the cytosolic factors with the membrane has been demonstrated. These findings suggest that stimulation of the leukocyte, resulting in an increased intracellular Ca2+ concentration, may result in the translocation of the enzyme and the factor to a membrane site, thereby facilitating the interaction of these two proteins with other enzymes involved in the 20:4 metabolic cascade. The development of a better understanding of these processes should not only help to define the biochemical basis for the regulation of leukotriene formation, but should also yield valuable information concerning the more general aspects of stimulus-response coupling in the leukocyte.

Physiochemical characterization of ATP binding to human 5-lipoxygenase.

Human 5-lipoxygenase requires ATP as a stimulatory factor. At the two preferred concentrations of the free Ca2+, 0.02 microM with a resting cell and 20 microM with a stimulated cell, Scatchard analysis revealed that 5-lipoxygenase has one affinity ATP binding site with a Kd of 4.6 microM at the low Ca2+ concentration but has two affinity ATP binding sites with a higher Kd of 4.4 microM and a lower Kd of 14.5 microM at the high Ca2+ concentration. In contrast, in a Tween 20 reaction system, 5-lipoxygenase had similar activation coefficients for ATP at both Ca2+ concentrations; these were 12.7 microM at the low Ca2+ concentration and 12.0 microM at the high Ca2+ concentration. These results showed that 5-lipoxygenase has an ATP binding site and suggest that self-association of 5-lipoxygenase in 20 microM Ca2+ may affect ATP binding affinity as measured by Scatchard analysis.