Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

Determination of the contribution of cysteinyl leukotrienes and leukotriene B4 in acute inflammatory responses using 5-lipoxygenase- and leukotriene A4 hydrolase-deficient mice

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.     

Involvement of leukotriene B4 in arthritis models

We investigated the role of leukotriene B4 (LTB4) in murine arthritis models using a leukotriene A4 (LTA4) hydrolase inhibitor, SA6541. SA6541 inhibited the severity of collagen-induced arthritis and muramyl dipeptide (MDP)-induced hyperproliferation of synovial cells in vivo. SA6541 also inhibited LTA4-induced hyperproliferation of synovial stromal cells in vitro. These results suggest that LTB4 may play an important role in arthritis models.     

Increased leukotriene A(4) hydrolase expression in the heart of angiotensin II-induced hypertensive rat

Leukotriene A(4) (LTA(4)) hydrolase is essential for the conversion of LTA(4) to LTB(4), an inflammatory lipid mediator. We investigated whether LTA(4) hydrolase was regulated in the heart by angiotensin II (ang II) infusion. Continuous ang II infusion via an osmotic minipump for up to 7 days upregulated mRNA and protein levels of LTA(4) hydrolase ( approximately 3.5-fold of control) in the heart in a pressor-dependent manner. Immunohistochemistry demonstrated intense LTA(4) hydrolase staining in the myofibroblast as well as migrated monocytes/macrophages. These data suggest that the cardiac LTA(4) hydrolase-LTB(4) system plays a positive role in the promotion of cardiac inflammation in hypertension.     

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.     

Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes.

Bestatin, an inhibitor of aminopeptidases, was also a potent inhibitor of leukotriene (LT) A4 hydrolase. On isolated enzyme its effects were immediate and reversible with a Ki = 201 +/- 95 mM. With erythrocytes it inhibited LTB4 formation greater than 90% within 10 min; with neutrophils it inhibited LTB4 formation by only 10% during the same period, increasing to 40% in 2 h. Bestatin inhibited LTA4 hydrolase selectively; neither 5-lipoxygenase nor 15-lipoxygenase activity in neutrophil lysates was affected. Purified LTA4 hydrolase exhibited an intrinsic aminopeptidase activity, hydrolyzing L-lysine-p-nitroanilide and L-leucine-beta-naphthylamide with apparent Km = 156 microM and 70 microM and Vmax = 50 and 215 nmol/min/mg, respectively. Both LTA4 and bestatin suppressed the intrinsic aminopeptidase activity of LTA4 hydrolase with apparent Ki values of 5.3 microM and 172 nM, respectively. Other metallohydrolase inhibitors tested did not reduce LTA4 hydrolase/aminopeptidase activity, with one exception; captopril, an inhibitor of angiotensin-converting enzyme, was as effective as bestatin. The results demonstrate a functional resemblance between LTA4 hydrolase and certain metallohydrolases, consistent with a molecular resemblance at their putative Zn2(+)-binding sites. The availability of a reversible, chemically stable inhibitor of LTA4 hydrolase may facilitate investigations on the role of LTB4 in inflammation, particularly the process termed transcellular biosynthesis.     

Bestatin inhibits covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase.

The covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase is inhibited in a concentration-dependent fashion by pre-incubation with bestatin. This inhibition correlated with the concentration-dependent inhibition by bestatin of LTB4 and LTB5 synthesis by LTA4 hydrolase. Epibestatin, a diastereomer of bestatin, neither inhibited LTB4 or LTB5 production by LTA4 hydrolase nor prevented the covalent coupling of [3H]LTA4 to the enzyme. In contrast, captopril inhibited both LTB4 synthesis by LTA4 hydrolase and covalent coupling of [3H]LTA4 to the enzyme.     

Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).     

Leukotriene A4 hydrolase, insights into the molecular evolution by homology modeling and mutational analysis of enzyme from Saccharomyces cerevisiae

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.     

Leukotriene A4 hydrolase activity of human airway epithelial cells

Human tracheal epithelial cells were incubated with LTA4 and metabolic products were identified in extracted supernatants by high pressure liquid chromatography, ultraviolet spectroscopy, and gas chromatography-mass spectrometry. In the presence of epithelial cells, LTA4 was converted to LTB4, but not to LTC4 or LTD4. Maximum LTB4 was released at an LTA4 concentration of 3 microM and had occurred by 30 min. LTB4 release was increased in the presence of albumin, but was not affected by extracellular calcium or A23187. This LTA4 hydrolase activity had a slower time course and could not be clearly inactivated by repeated exposure to substrate as is the case for previously described LTA4 hydrolase enzymes. This hydrolase appears to have novel biochemical characteristics.     

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Characterization of leukotriene A4 and B4 biosynthesis

We have studied LTA4 and LTB4 synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB4 from arachidonic acid are localized in the soluble fraction (100,000 x g supernatant) of these cells. The formation of LTA4 and LTB4 is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 microM, the synthesis of LTB4 leveled off at 30 microM and of LTA4 at 100 microM while 5-HETE had not reached a plateau at 300 microM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA4 and only a relatively small amount of LTB4. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA4 to LTB4. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA4 synthesis did not produce any additional LTA4 or LTB4. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase in guinea pig tissues

We prepared a highly specific polyclonal antibody against leukotriene (LT) A4 hydrolase using a recombinant human enzyme. Using this antibody, we quantified LTA4 hydrolase protein content in the cytosols of guinea pig tissues. The enzyme protein content correlated well with the enzyme activity with a correlation coefficient of 0.87. However, the enzyme activity per mg of the enzyme in the cytosols was low, particularly in the liver and adrenal gland, compared with the specific activity of the purified enzyme. These observations suggest the presence of inhibitory substances and/or inactive enzymes in the cytosols of these tissues. To determine the cellular localization of LTA4 hydrolase in tissues other than blood cells, we carried out immunohistochemical examinations of guinea pig tissues. We identified epithelial cells in the tracheobronchial system and gastrointestinal tract, smooth muscle cells in the bronchi and aorta, vascular endothelial cells, and the intestinal plexus as novel cellular sources of the enzyme in the parenchyme of the tissue. Thus, LTA4 hydrolase was widely distributed in various types of parenchymal cells in the tissues, and this observation warrants further investigations on the biological activities of LTB4 in these cells and tissues.     

Relationship between leukotriene A4 metabolism and biological activity

The data on the pharmacology of leukotrienes showed that LTA4, LTC4 and LTD4 were equipotent on the guinea-pig lung parenchyma whereas LTB4 was slightly less active. However, on the trachea, the myotropic activity of LTC4 and LTD4 was equivalent and higher than LTB4 and LTA4. The potency of these compounds was also different on the ileum where LTD4 was more active than LTC4; at the concentration used, LTA4 and LTB4 were inactive on this tissue. These results suggested that the transformation of leukotrienes by the smooth muscle preparations was a prerequisite for its biological activity. To verify this hypothesis, LTA4 (100 ng) was incubated for 10 min. with 20,000 g supernatants of homogenates of guinea-pig lung parenchyma, trachea and ileum; the metabolites were analysed by bioassay using strips of guinea-pig ileum and lung parenchyma in a cascade superfusion system and by RP-HPLC. Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4, which is in agreement with the myotropic potency of these leukotrienes on the lung parenchymal strip. Conversely, incubation of LTA4 with homogenates of guinea-pig ileum showed the formation of LTB4 and its isomers which are inactive on this preparation. Similarly, incubation of homogenates of trachea with LTA4 led to the formation of LTB4; this finding is again in agreement with the potency of these two leukotrienes on the trachea. Our results suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its transformation.     

The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.