Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

doi:10.1016/S0006-291X(05)80080-4

Biochemical and Biophysical Research Communications

Volume 173, Issue 2, 14 December 1990, Pages 620-626

https://doi.org/10.1016/S0006-291X(05)80080-4 Get rights and content

Summary

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.

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Recent advances in function and structure of two leukotriene B<inf>4</inf> receptors: BLT1 and BLT2
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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.
Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice
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This formulation is novel and found safe and non-toxic following intravitreal injection of vehicle alone and shows no alteration in retinal deformity and angiograms. In the sensory nervous system (Minami et al., 1990; Haeggström et al., 1990) in vitro and in the in vivo studies with mice retina (Husain et al., 2009) aminopeptidase activity of LTA4H is reported to include hydrolysis of short peptides including degradation of locally produced opioid peptide enkephalin (Griffin et al., 1992) and dynorphin (Nissen et al., 1995). In the retina, opioids and their receptor activation is important for cell survival following hypoxic or ischemic injury (Husain et al., 2009; Riazi-Esfahani et al., 2009).
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.
Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling
2015, Progress in Lipid Research
Citation Excerpt :
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.
Product formation controlled by substrate dynamics in leukotriene A4 hydrolase
2014, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
As an epoxide hydrolase, the enzyme catalyzes the hydrolysis of the allylic epoxide LTA4 (5S-trans-5,6-oxido-7,9-trans-11,14-cis eicosatetra-enoic acid) to form LTB4 (5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a potent chemotactic agent and a mediator of inflammation [3] (Fig. 1). In addition to the epoxide hydrolase activity, LTA4H also possesses an anion-dependent aminopeptidase activity [4] that, in recent studies have shown to have a role in resolution of inflammation. LTA4H has been reported to be a soluble cytosolic monomeric enzyme with a molecular mass of 69 kDa.
Leukotriene A₄ hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA₄ to two enzymatic metabolites, LTB₄ and another biologically active product Δ⁶-trans-Δ⁸-cis-LTB₄ (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3 Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB₄ isomeric product Δ⁶-trans-Δ⁸-cis-LTB₄. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA₄. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA₄ conformers.
Leukotriene A<inf>4</inf> Hydrolase
2013, Handbook of Proteolytic Enzymes
A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity
2013, Analytical Biochemistry
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.

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^*: This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

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Leukotriene A4 hydrolase is a zinc-containing aminopeptidase*

Summary

Int. J. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Leukotriene A₄ hydrolase is a zinc-containing aminopeptidase*