Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.     

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 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.     

Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ

Leukotrienes A4 and D4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD4 was much higher than that of LTA4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD4 was very active whereas LTA4 was inactive. Since the activities of both leukotrienes were blocked by FPL-55712, our results suggested that the transformation of LTA4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA4 was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP-HPLC). Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4. Incubation of LTA4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA4 undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activities of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its biotransformation.     

Modulation of human neutrophil LTA hydrolase activity by phorbol myristate acetate

The phorbol ester, phorbol 12-myristate 13-acetate enhanced leukotriene B4 production stimulated by formyl-methionyl-leucyl-phenylalanine and arachidonic acid and reduced the production of the all-trans isomers of LTB4 by human neutrophils. Production of 5-hydroxyeicosatetraenoic acid was unaffected. These observations are consistent with a stimulatory effect of phorbol ester on LTA hydrolase, the enzyme which catalyses the conversion of LTA4 to LTB4. We demonstrate that a protein of the same molecular weight as LTA hydrolase is phosphorylated upon stimulation of neutrophils with PMA. These data suggest that the activity of LTA hydrolase may be regulated by protein kinase C-dependent phosphorylation.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Estrone sulfatase activity in guinea pig tissues

Estrone sulfatase activity is widespread in guinea pig tissues. Whole homogenates of adult testis. uterus. lung, adrenal, amnion, ovary, chorion, small intestine, placenta, spleen, kidney and liver exhibit approximately descending order of specific activity. Certain properties, including pH requirement, lack of inhibition by inorganic sulfate and magnitude of estimated K_mvalues, are similar to that for arylsulfatase C of rat liver. Of the subcellular fractions prepared from guinea pig tissues, microsomes exhibit the highest specific activity although considerable enzyme activity remains associated with large cellular fragments sedimenting at 750 g. The sulfatase activity is readily inhibited by inorganic phosphate even when substrate concentration satisfies zero order kinetics. Rat liver arylsulfatase C is not inhibited under these conditions. Sensitivity of the guinea pig enzyme activity to inhibition by a variety of steroids and related compounds, is markedly less than for rat liver. Diethylstilbestrol (DES) strongly inhibits the rat liver enzyme but has little effect on the guinea pig liver system. Guinea pig testicular activity is suppressed to a degree intermediate between these extremes by increasing DES concentration. In guinea pig lung. kidney, and possibly liver, elevated fetal enzyme activities decrease from neonatal to adult life. Teslicular activity appears to follow the opposite trend. Uterine enzyme activity is not markedly affected by pregnancy.     

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.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

Immunohistochemical localization of histamine N-methyltransferase in guinea pig tissues.

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.     

Leukotriene A4 hydrolase in the human B-lymphocytic cell line Raji: indications of catalytically divergent forms of the enzyme

Leukotriene A4 hydrolase was purified 1400-fold, with an approximate yield of 25%, to apparent homogeneity from the human B-lymphocytic cell line Raji. The purification included ammonium sulfate precipitations followed by anion exchange, hydrophobic interaction, and molecular exclusion fast protein liquid chromatography. Kinetic properties at 2 degrees C varied between different enzyme preparations. Two patterns were observed, one with a Km of about 12 microM and Vmax of about 1.1 mumol LTB4/mg protein/min which correlated well with the properties of the human leukocytic LTA4 hydrolase. In other enzyme preparations a higher catalytic activity was observed. These enzyme batches did not obey Michaelis-Menten kinetics but were compatible with a mixture of enzymatic species. Heat treatment (60 degrees C) led to a time-dependent decline in catalytic activity. However, certain enzyme preparations contained a subfraction of enzymatic activity which was more resistant to heat treatment, yielding a biphasic inactivation pattern. It is thus suggested, on the basis of the kinetic properties and the heat-inactivation pattern, that these enzyme preparations contained an addition form of LTA4 hydrolase.     

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.     

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.     

Peritoneal macrophages of guinea pig possibly lack LTC4 synthetase

Peritoneal cells and adherent cells of mice and rats synthesized LTC4 and LTB4 when stimulated with A23187 in vitro. On the other hand, neither peritoneal cells nor adherent cells of guinea pigs generated LTC4, D4, and E4, but did the lower amounts of LTB4. Only generation of LTB4 was potentiated by simultaneous addition of 10 microM A.A. in this species. Enzyme solutions which were extracted from peritoneal cells of these three species were capable of converting DNCB to a colored product in the presence of glutathione and then these potencies were in the following order; guinea pig greater than mouse greater than rat. On the other hand, the potencies of converting LTA4 to LTC4 in the presence of glutathione were in the following order; mouse greater than rat much greater than guinea pig approximately equal to 0. These results suggest that macrophages of guinea pigs lack "LTC4 synthetase" and also this enzyme is different from usual GSH S-transferases.     

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.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Immunochemical characterization of rat testicular asparagine synthetase.

We studied immunochemical properties of rat testicular asparagine synthetase. Western blot analysis of testis extract with polyclonal antibody raised against purified asparagine synthetase revealed an immunoreactive band at 62 kDa. The pancreas, brain, thymus, and spleen also showed 62-kDa bands. The intensities of these bands were roughly proportional to the specific activities of the enzyme in these tissues. The antibody showed some degree of cross-reactivity to asparagine synthetases from human, beef, pig, mouse, guinea pig, chicken, and frog, but not carp. But the enzyme from human HL-60 cells and lower vertebrates reacted with the antibody less strongly than enzyme from rats. The N-terminal amino acid sequence of the enzyme, determined by the Edman degradation method, in 10 recovered residues was identical to that of human asparagine synthetase deduced from corresponding cDNA (I.L. Andrulis et al., 1987, Mol. Cell. Biol. 7, 2435-2443). Immunohistochemical staining of the testis showed the presence of asparagine synthetase mainly in Sertoli cells in the seminiferous tubules.