Leukotrienes, the biologically active metabolites of arachidonic acid, have been implicated in a variety of inflammatory responses, including asthma, arthritis and psoriasis. Recently a compound, MK-886, has been described that blocks the synthesis of leukotrienes in intact activated leukocytes, but has little or no effect on enzymes involved in leukotriene synthesis, including 5-lipoxygenase, in cell-free systems. A membrane protein with a high affinity for MK-886 and possibly representing the cellular target for MK-886 has been isolated from rat and human leukocytes. Here, we report the isolation of a complementary DNA clone encoding the MK-886-binding protein. We also demonstrate that the expression of both the MK-886-binding protein and 5-lipoxygenase is necessary for leukotriene synthesis in intact cells. Because the MK-886-binding protein seems to play a part in activating this enzyme in cells, it is termed the five-lipoxygenase activating protein (FLAP).
SummaryThe intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion ofarachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells . In addition, subcellular fractionation techniques showed >83% of immunoblot-detectable FLAP protein and -64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction . In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment . These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope .eukotrienes are products of arachidonic acid metabolism that are produced by leukocytes and that have a variety of effects on the immune system (1) . The enzyme 5-lipoxygenase (5-LO, 1 EC 1.13 .11.12) catalyzes two key steps in the leukotriene biosynthetic pathway (2, 3). These steps are the oxygenation ofarachidonate to 5-(S)-hydroperoxy-6,8, 11,14-eicosatetraenoic acid (5-HPETE), followed by its dehydrogenation, which results in the formation of the unstable epoxide LTA4. In neutrophils the enzyme LTA4 hydrolase then converts LTA4 to LTB4, which is a potent chemotactic and activating factor for neutrophils and eosinophils (4-6). In eo-1 Abbreviations used in this paper. FLAP, 5-lipoxygenase-activating protein; GAM, goat anti-mouse IgG; GAR, goat anti-rabbit IgG ; 5-HPETE, 5-(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid; 5-LO, 5-lipoxygenase; LSM, lymphocyte separation medium ; PLP, paraformaldehyde-lysineperiodate . sinophils and mast cells, LTA4 can also be converted to LTC4, LTD4, and LTE4, all of which stimulate contraction of vascular and bronchial smooth muscle (7,8), resulting in effects on local circulation and bronchoconstriction . These potent biological effects implicate leukotrienes in a number of hypersensitivity and inflammatory disorders, including asthma and in...
Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.
5-Lipoxygenase-activating protein (FLAP) is an 1 S-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates S-LO has not been determined. We have expressed high levels of human FLAP in Spodopteru frugiperda (39) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds ['2sI]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.5-Lipoxygenase-activating protein; 5-Lipoxygenase; Arachidonic acid; Photoaffinity labeling; Baculovirus
-EJB 93 0154/6 5-Lipoxygenase (5-LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5-LO catalyzing both the synthesis of (SS)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) from arachidonic acid and the subsequent synthesis of LTA, from 5-HPETE. We have previously expressed both human 5-LO and human FLAP to high levels in Spodopteru frugiperdu (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5-L0, we compared cellular 5-LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5-LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5-LO as a substrate, and increases the efficiency with which 5-LO converts 5-HPETE to LTA,. LT synthesis in cells coexpressing FLAP and 5-LO is inhibited by 3-[l-(p-chlorophenyl)-5-isopropyl-3-~e~-buty1t~o-1~-indo1-2-y1]-2,2-dimethy1-prop~oic acid (MK-886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5-LO by acting as an arachidonic acid transfer protein. jects, and these levels are increased during an asthmatic attack (Tagari et al., 1990). Consequently, compounds which inhibit the synthesis of LTs or act as LT receptor antagonists are currently being developed as potential therapeutic agents. The first two steps in the synthesis of LTs from arachidonic acid, the oxygenation of arachidonic acid to (5S)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) followed by the conversion of 5-HPETE to the unstable epoxide LT&, are catalyzed by the enzyme 5-lipoxygenase (Rouzer et al., 1986). In neutrophils, LTA, is subsequently metabolized to the proinflammatory compound LTB, by the enzyme LTA, hydrolase, while in other cell types, including macrophages, eosinophils, and epithelial cells, LTA, is converted to the peptidoleukotrienes LTC4, LTD4, and LTE4 in a series of enzymatic reactions (Maycock et al., 1989). In response to a variety of agents which stimulate cellular LT synthesis, including calcium ionophore A231 87 (Rouzer and Kargman, 1988), thapsigargin (Wong et al., 1991), IgE (Wong et al., 1992), and fMetLeuPhe (Kargman et al., 1991), 5-LO translocates from the soluble to a membrane compartment. This specific membrane association of 5-LO appears to be an early step in the cellular synthesis of LTs, and may be required for the efficient utilization of arachidonic acid, which is released from membrane phospholipids by phospholipases.The direct inhibition of 5-LO or blocking of the specific membrane association of the enzyme represent alternative approaches to the inhibition of LT synthesis. Inhibitors based on indole and quinoline moieties and hybrid compounds containing both indole and quinoline groups have been shown to inhibit and reverse the membrane association of 5-LO Evans et al., 1991;Brideau et al.,
The therapeutic action of nonsteroidal anti-inflammatory drugs (NSAIDs) is exerted through the inhibition of prostaglandin G/H synthase (PGHS), which is expressed as two isoenzymes, termed PGHS-1 and PGHS-2. From the crystal structure of sheep PGHS-1, it has been proposed that the carboxylic acid group of flurbiprofen is located in a favorable position for interacting with the arginine 120 residue of PGHS-1 (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249). Mutation of this Arg120 residue to Glu was performed and expressed in COS-7 cells using a vaccinia virus expression system. Comparison of microsomal enzyme preparations show that the mutation results in a 20-fold reduction in the specific activity of PGHS-1 and in a 100-fold increase in the apparent Km for arachidonic acid. Indomethacin, flurbiprofen, and ketoprofen, inhibitors of PGHS activity containing a free carboxylic acid group, do not exhibit any inhibitory effects against the activity of PGHS-1(Arg120-->Glu). Diclofenac and meclofenamic acid, other NSAIDs containing a free carboxylic acid group, were 50-100-fold less potent inhibitors of the activity of the mutant as compared with the wild type PGHS. In contrast, the nonacid PGHS inhibitors, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene (DuP697) and a desbromo-sulfonamide analogue of DuP697 (L-746,483), were both more potent inhibitors of PGHS-1(Arg120-->Glu) than of the wild tyupe PGHS-1. Inhibition of PGHS-1(Arg120-->Glu) was time-dependent for diclofenac and time-independent for DuP697, as observed for the wild type enzyme, indicating that the mutation does not alter the basic mechanism of inhibition. Aspirin is an acid NSAID that inhibits PGHS-1 through a unique covalent acetylation of the enzyme and also showed a reduced rate of inactivation of the mutated enzyme. These data provide biochemical evidence of the importance of the Arg120 residue in PGHS-1 for interaction with arachidonic acid and NSAIDs containing a free carboxylic acid moiety.
The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher K m , similar V max and increased specificity of oxygenation for arachidonic acid, and a similar K m but decreased V max for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells.In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401±429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete a-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates.Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo. Although the LOs produce specific hydroxyeicosatetraenoic (HETE) enantiomers, the enzymes do not all display absolute positional specificity with respect to oxygenation of AA. For example, while 15-LOb exclusively oxygenates C15 of AA [8], 15-LOa oxygenates AA at both C15 and C12 [9]. In addition, the degree of homology between members of the LO family does not define the product profile. For example, amino acid sequence alignments demonstrate that 15-LOb is more closely related to 12R-LO than 15-LOa, suggesting complex evolutionary paths in the LO family.The LOs are responsible for the synthesis of a number of inflammatory mediators, including leukotrienes and lipoxins, which are implicated in inflammatory disorders such as bronchial asthma [10]. Lipoxygenase metabolites have also been implicated in a number of noninflammatory biological processes. For example, 12-HETE and 15-HETE have been implicated in tumour metastases and the formation of atherosclerotic plaques, respectively [11,12]. The important biological roles of these LO metabolites and the complexity of substrate utilization by members of the LO family emphasize the importance of understanding factors which affect the activity and regulation of these enzymes.Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis...
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