Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis

Abstract

The leukotrienes (LTs) 1 are a family of lipid mediators that play important roles in a variety of allergic and inflammatory reactions (1, 2). These molecules are formed by leukocytes and are divided into two classes, the spasmogenic cysteinyl leukotrienes and LTB 4 , which is a classical chemoattractant that triggers adherence and aggregation of leukocytes to the endothelium at nanomolar concentrations. Recent data also indicate that LTB 4 is a chemoattractant for T-cells, creating a functional link between early innate and late adaptive immune responses to inflammation (3-5). In addition, LTB 4 participates in the host defense against infections (6) and is a key mediator of platelet-activating factor-induced lethal shock (7). Because of these powerful biological effects, LTB 4 is regarded as an important chemical mediator in a variety of acute and chronic inflammatory diseases, e.g. nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease (8). Moreover, only recently, several lines of pharmacological, morphological, biochemical, and genetic evidence have been gathered implicating LTs, in particular LTB 4 , as a mediator of vascular inflammation and arteriosclerosis (9). This article gives an overview of the biochemical, structural, and catalytic properties of LTA 4 hydrolase (LTA4H), which catalyzes the final and committed step in LTB 4 biosynthesis. LTA 4 Hydrolase Is a Key Enzyme in the 5-Lipoxygenase PathwayIn cellular biosynthesis of LTs, 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein, converts arachidonic acid into the unstable epoxide LTA 4 , which in turn may be enzymatically conjugated with GSH to form LTC 4 , the parent compound of the cysteinyl leukotrienes, or hydrolyzed into LTB 4 by LTA4H (Fig. 1). Leukotrienes can also be formed via transcellular routes, where LTA 4 is donated from an activated leukocyte to a recipient cell for further metabolism by downstream enzymes, a process that was recently shown to occur in vivo (10). LTB 4 signals via a specific, high affinity, G-protein-coupled receptor (BLT 1 ) (11). In addition, a second receptor for LTB 4 (BLT 2 ) has been discovered, the functional role of which is presently not known (12). Interestingly, LTB 4 is also a natural ligand of the peroxisome proliferator-activated receptor ␣ class of nuclear receptors and has been suggested to play a role in lipid homeostasis (13). Leukotriene A 4 Hydrolase Is a Zinc-dependent Epoxide Hydrolase and AminopeptidaseLTA4H is a monomeric soluble protein that is widely distributed and has been purified from several mammalian sources (14). It resides in the cytosol, although nuclear localization has also been reported recently (15). The cDNAs encoding the human, mouse, rat, and guinea pig enzymes have been cloned and sequenced. The proteins contain 610 amino acids, excluding the first Met, and the calculated molecular mass of the human enzyme is 69,153 Da. The primary, secondary, and tertiary structures of LTA4H bear no resemblance to soluble xenobiotic epoxide hydrolase, alth...

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…This is evident in the 5-LOX pathway, where synthesis of the lipoxins A 4 (LXA 4 ) and B 4 (LXB 4 ) requires 12-LOX activity [55,56]. Further metabolites for which LTA 4 serves as precursor are leukotriene B 4 (LTB 4 ), catalysed by LTA 4 hydrolase [57] and the cysteinyl leukotrienes (LTC 4 , LTD 4 , LTE 4 ), LTC 4 being synthesised with the aid of a specific glutathione-S-transferase [58]. Formation of 5-oxo-7E,9E,11Z,14Z-eicosatetraenoic acid (5-oxo-7,9,11,14-ETE) is the result of non-enzymatic metabolism [59].…”

Circadian proteomics of the mouse retina

“…Generally, LTB 4 is an inflammatory mediator, and it activates immune systems [45]. The amount of LTB 4 is increased by the light stimulation in the retina [46].…”

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

“…Ligand Binding Assay-The membrane preparation (10 or 5 g of protein) was incubated in 100 l of LTB 4 binding buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 10 mM NaCl) containing 0.05% bovine serum albumin (BSA; fatty acid-free) and various concentrations of [ 3 H]LTB 4 . To determine nonspecific binding, concentrations of unlabeled LTB 4 at least 1,000 times higher than labeled were used.…”

“…GTP␥S Binding Assay-The membrane preparation (10 g of protein) was incubated in 100 l of GTP␥S binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 5 M GDP, and 0.1% BSA) containing 0.5 nM [ 35 S]GTP␥S with or without various concentrations of LTB 4 for 30 min at 30°C. To determine nonspecific binding, unlabeled GTP␥S was added to the binding mixture to a final concentration of 10 M. Bound [ 35 S]GTP␥S was separated from free by rapid filtration through GF/C filters and washed with ϳ2 ml of ice-cold TMN buffer (10 mM Tris-HCl, pH 7.5, 25 mM MgCl 2 , and 100 mM NaCl).…”

“…LTB 4 Mutagenesis-Using pcDNA3-HA-tagged human BLT1 (19) as a template, tandem alanine BLT1 mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following primers were used for the mutagenesis: i1-1S (5Ј-ctttgtggtgtggagtgccgcggcagcgatgcagaagcgctc-3Ј), i1-1AS (5Ј-gagcgcttctgcatcgctgccgcggcactccacaccacaaag-3Ј), i1-2S (5Ј-gtatcctgaaaagggcggcggcggcctctgtcactgccctg-3Ј), i1-2AS (5Ј-cagggcagtgacagaggccgccgccgcccttttcaggatac-3Ј), i1-3S (5Ј-ggatgcagaagcgcgctgccgctgccctgatggtgctgaac-3Ј), i1-3AS (5Ј-gttcagcaccatcagggcagcggcagcgcgcttctgcatcc-3Ј), i2-1S (5Ј-ggccatgagtctagccgccg-3Ј), i2-1AS (5Ј-cgggccaccgccgctgcggcggct-3Ј), i2-2S (5Ј-ctcactggcggcggccgccccctttgtgtc-3Ј), i2-2AS (5Ј-gacacaaagggggcggccgccgccagtgag-3Ј), i2-3S (5Ј-gcggtggcccgcgccgctgcggcccagaagctacgc-3Ј), i2-3AS (5Ј-gcgtagcttctgggccgcagcggcgcgggccaccgc-3Ј), i2-4S (5Ј-cgcccctttgtgtccgcggcggcagccaccaaggcgatggcc-3Ј), i2-4AS (5Ј-ggccatcgccttggtggctgccgccgcggacacaaaggggcg-3Ј), i2-5S (5Ј-cagaagctacgcgccgcggcggcggcccggcgggtg-3Ј), i2-5AS (5Ј-cacccgccgggccgccgccgcggcgcgtagcttctg-3Ј), i3-0S (5Ј-gctgtggtggccgccgccgccgacatagggcgt-3Ј), i3-0A (5Ј-acgccctatgtcggcggcggcggccaccacagc-3Ј), i3-1S (5Ј-ggccagctactcggccgcagcggctcggctacaggcc-3Ј), i3-1AS (5Ј-ggcctgtagccgagccgctgcggccgagtagctggcc-3Ј), i3-2S (5Ј-ggacatagggcgtgcggcagcggcccggcgcttccgcc-3Ј), i3-2AS (5Ј-ggcggaagcgccgggccgctgccgcacgccctatgtcc-3Ј), i3-3S (5Ј-cgtcggctacaggccgcggccgccgcccgcagccgccgcacc-3Ј), i3-3AS (5Ј-ggtgcggcggctgcgggcggcggccgcggcctgtagccgacg-3Ј), i3-4S (5Ј-gcccggcgcttccgcgccgccgccgccaccggccgcctggtg-3Ј), i3-4AS (5Ј-caccaggcggccggtggcggcggcggcgcggaagcgccgggc-3Ј), i3-5S (5Ј-cgcagccgccgcgccgccgccctggtggtgctc-3Ј), i3-5AS (5Ј-gagcaccaccagggcggcggcgcggcggctgcg-3Ј).…”