Leukotriene A4 hydrolase: An epoxide hydrolase with peptidase activity

doi:10.1016/S0006-291X(05)81076-9

Biochemical and Biophysical Research Communications

Volume 173, Issue 1, 30 November 1990, Pages 431-437

https://doi.org/10.1016/S0006-291X(05)81076-9 Get rights and content

Purified leukotriene A₄ hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70°C, 30 min). At 37°C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A₄ hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A₄ reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A₄ into leukotriene B₄.

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However, LTA4 hydrolase is ubiquitously distributed, suggesting that this enzyme is constitutively produced and awaits the substrate produced by 5-LO. In addition, LTA4 hydrolase is a dual-function enzyme that acts as a zinc protease with multiple substrates [20,21]. AA and AA-derived intermediates (e.g., LTA4) are able to permeate cell membranes, thereby allowing the transcellular biosynthesis of LTB4 [4].
Leukotriene B₄ (LTB₄) is generated by the enzymatic oxidation of arachidonic acid, which is then released from the cell membrane and acts as a potent activator of leukocytes and other inflammatory cells. Numerous studies have demonstrated the physiological and pathophysiological significance of this lipid in various diseases. LTB₄ exerts its activities by binding to its specific G protein-coupled receptors (GPCRs): BLT1 and BLT2. In mouse disease models, treatment with BLT1 antagonists or BLT1 gene ablation attenuated various diseases, including bronchial asthma, arthritis, and psoriasis, whereas BLT2 deficiency exacerbated several diseases in the skin, cornea, and small intestine. Therefore, BLT1 inhibitors and BLT2 activators could be beneficial for the treatment of several inflammatory and immune disorders. As a result, attractive compounds targeting LTB₄ receptors have been developed by several pharmaceutical companies. This review aims to understand the potential of BLT1 and BLT2 as therapeutic targets for the treatment of various inflammatory diseases. In addition, recent topics are discussed with major focuses on the structure and post-translational modifications of BLT1 and BLT2. Collectively, current evidence on modulating LTB₄ receptor functions provides new strategies for the treatment of various diseases.
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Leukotriene B₄ (LTB₄) is a lipid mediator derived from arachidonic acid (AA) by the sequential action of 5-lipoxygenase (5-LOX), 5-lipoxygenase-activating protein (FLAP) and LTA₄ hydrolase (LTA₄H). It was initially recognized for its involvement in the recruitment of neutrophils and is one of the most potent chemotactic agents known to date. A large body of data has indicated that LTB₄ plays a significant role in many chronic inflammatory diseases, such as arthritis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer and more recently, metabolic disorder. In this review, we focus on the biosynthesis of LTB₄ and its biological effects. In particular, we will describe a basic biochemical understanding integrated with recent developments in the field of structural biology of the three key enzymes (5-LOX, FLAP and LTA₄H) in LTB₄ biosynthesis, and also summarize the most outstanding work on in vivo biological and pathogenic roles of these enzymes and the development of enzyme inhibitors.
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Thus, different catalytic pockets for separate activities share a carboxylate group recognition zone. ( Haeggström et al., 1990; Medina et al., 1991). Recently, (Stsiapanava et al., 2014) provided structural support to selectively inhibit LTA4 hydrolysis.
Leukotriene A₄ hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A₄ to leukotriene B₄ and the hydrolysis of the peptide proline-glycine-proline.
Imbalances in leukotriene B₄ synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B₄ or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A₄ Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10 μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme.
Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A₄ hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells.
Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme.
Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A₄ hydrolase using in vivo models.
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CD13/APN (aminopeptidase N) was first identified as a selective angiogenic marker expressed in tumor vasculature and is considered a target for anti-cancer therapy. CD13 was also reported to express in non-diabetic, hypoxia-induced retinal neovascularization. Whether diabetes induces upregulation of CD13 expression in the retina is unknown. We hypothesize that at an early stage of non-proliferative diabetic retinopathy (NPDR) characterized by disruption of blood-retinal barrier (BRB) permeability is related to upregulated expression of CD13 because of its known role in extracellular matrix (ECM) degradation. The purpose of this study is to evaluate the role of CD13/APN and the therapeutic efficacy of a CD13/APN inhibitor in a mouse model of streptozotocin-induced NPDR. Hyperglycemic C57Bl/6 mice 26 weeks after streptozotocin (STZ) injection were intravitreally injected with a sustained release formulation of CD13/APN inhibitor bestatin. At 15th day of post-bestatin treatment, mouse retinas were evaluated for vascular permeability by Evans blue dye extravasation assay, fluorescent angiography of retinal vascular permeability and leukostasis. Retinal protein extracts were analyzed by Western blot to determine the effects of bestatin treatment on the expression of CD13/APN related inflammatory mediators of ECM degradation and angiogenesis. Intravitreal bestatin treatment significantly inhibited retinal vascular permeability and leukostasis. This treatment also significantly inhibited retinal expression of CD13, ECM degrading proteases (heparanase and MMP9 and angiogenic molecules (HIF-1α and VEGF). Intravitreal CD13 inhibition may relate to furthering our knowledge on the protective effect of bestatin against diabetic retinal vasculature abnormalities through inhibition of retinal permeability, leukostasis, inflammatory molecules of ECM degradation and angiogenesis.
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This study aimed to understand the genomic architecture of Argentinean dairy herds by measuring linkage disequilibrium (LD) and identifying loci associated with parameters calculated from somatic cell count (SCC). Phenotypic data consisted of 3530 SCC records from 544 Holstein and Holstein x Jersey cows owned by a single dairy company located in the Central dairy area of Argentina. SCC was recorded every 40 days. After quality control, genotypic data consisted in 38,872 single nucleotide polymorphisms (SNP). The squared correlation of the alleles at two loci (r²) was computed for all SNP pairs on each chromosome. At marker distances less than 10 kb the average r² was 0.40. Between 40 and 50 kb the average r² was 0.25 and 0.18 for 100 kb apart. Three different variables were calculated from the somatic cell score (SCS): the arithmetic mean (AM), the maximum value (MAX) and the arithmetic mean of the top 3 values (TOP3). Few significant SNP associations were found. As expected, polygenic traits such as SCC are influenced by multiple loci throughout the genome, each with a relatively small effect. AM on one side and TOP3 and MAX on the other, showed different SNP associated showing that they capture different aspects of mastitis response. AM was significantly associated with two SNP: ARS-BFGL-NGS-114608 (BTA1) and Hapmap60306-rs29023088 (BTA5). MAX and TOP3 were significantly associated with four SNP: ARS-BFGL-NGS-107594, ARS-BFGL-NGS-104220 (BTA10), BTA-43543-no-rs (BTA18) and ARS-BFGL-NGS-109705 (BTA26). MAX and TOP3 were equivalent phenotypic variables to be used in a GWAS. These results contribute to gain insight into the genomic regions influencing the SCC in Argentinean herds.
Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling
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LTA4H is a bifunctional enzyme that has an epoxide hydrolase activity for LTA3, LTA4 and LTA5 [320,321] but also harbors a zinc-dependent aminopeptidase activity with a zinc binding motif [322]. Three amino acids (His295, His299 and Glu318) form the Zn-binding cluster and mutagenesis studies indicated that the loss of one of these residues blocks the catalytic activity [323–326]. LTA4H undergoes suicidal inactivation during LTA4 hydrolysis and as molecular reason for the loss in catalytic activity chemical modification of Tyr378 was suggested [327,328].
Leukotrienes are pro-inflammatory lipid mediators, which are biosynthesized via the lipoxygenase pathway of the arachidonic acid cascade. Lipoxygenases form a family of lipid peroxidizing enzymes and human lipoxygenase isoforms have been implicated in the pathogenesis of inflammatory, hyperproliferative (cancer) and neurodegenerative diseases. Lipoxygenases are not restricted to humans but also occur in a large number of pro- and eucaryotic organisms. Lipoxygenase-like sequences have been identified in the three domains of life (bacteria, archaea, eucarya) but because of lacking functional data the occurrence of catalytically active lipoxygenases in archaea still remains an open question. Although the physiological and/or pathophysiological functions of various lipoxygenase isoforms have been studied throughout the last three decades there is no unifying concept for the biological importance of these enzymes. In this review we are summarizing the current knowledge on the distribution of lipoxygenases in living single and multicellular organisms with particular emphasis to higher vertebrates and will also focus on the genetic diversity of enzymes and receptors involved in human leukotriene signaling.

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Leukotriene A4 hydrolase: An epoxide hydrolase with peptidase activity

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Methods Enzymol.

Anal. Biochem.

J. Biol. Chem.

FEBS Lett.

Gene

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Leukotriene A₄ hydrolase: An epoxide hydrolase with peptidase activity