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Structure and inhibition of mouse leukotriene C4 synthase.

ABSTRACT: Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes' steady-state kinetics. Although structural differences near the active site and along the C-terminal ?-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development.

SUBMITTER: Niegowski D

PROVIDER: S-EPMC4014545 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Structure and inhibition of mouse leukotriene C4 synthase.

Niegowski Damian D Kleinschmidt Thea T Ahmad Shabbir S Qureshi Abdul Aziz AA Mårback Michaela M Rinaldo-Matthis Agnes A Haeggström Jesper Z JZ

PloS one 20140508 5

Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhi ...[more]

PMID: 24810165

Similar Datasets

Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity.

Project description:Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery.

| S-EPMC5000086 | biostudies-literature

Arginine 104 is a key catalytic residue in leukotriene C4 synthase.

Project description:Human leukotriene C(4) synthase (hLTC(4)S) is an integral membrane enzyme that conjugates leukotriene (LT) A(4) with glutathione to form LTC(4), a precursor to the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC(4)S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K(m) for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ(max) at 239 nm, could be detected using R104A or R104S hLTC(4)S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK(a) to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA(4). On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC(4)S by 88 and 70%, respectively, without significantly affecting the k(cat)/K(m) values for GSH, and a crystal structure of R31Q hLTC(4)S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC(4)S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.

| S-EPMC3003377 | biostudies-literature

The catalytic architecture of leukotriene C4 synthase with two arginine residues.

Project description:Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).

| S-EPMC3091245 | biostudies-literature

Molecular cloning and expression of human leukotriene-C4 synthase.

Project description:Leukotriene-C4 synthase (LTC4S; EC 2.5.1.37) catalyzes the committed step in the biosynthesis of the peptidoleukotrienes, which are important in the pathogenesis of asthma. Antibodies were generated to a synthetic peptide based on the partial amino acid sequence previously reported for human LTC4S [Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019] and specifically bound detergent-solubilized LTC4S obtained from THP-1 cells, confirming that the published sequence is associated with enzyme activity. Inosine-containing oligonucleotides based on the partial protein sequence were used to isolate a 679-bp cDNA for LTC4S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M(r) = 16,568) that has a calculated pI value of 11.1. The deduced protein sequence is composed predominantly of hydrophobic amino acids; hydropathy analysis predicts three transmembrane domains connected by two hydrophilic loops. Analysis of the deduced sequence identified two potential protein kinase C phosphorylation sites and a potential N-linked glycosylation site. The amino acid sequence for human LTC4S is unique and shows no homology to other glutathione S-transferases. LTC4S was found to be most similar to 5-lipoxygenase activating protein (31% identity, 53% similarity), another protein involved in leukotriene biosynthesis. Active enzyme was expressed in bacterial, insect, and mammalian cells as shown by the biosynthesis of LTC4 in incubation mixtures containing LTA4 and reduced glutathione. The cloning and expression of human LTC4S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis.

| S-EPMC44893 | biostudies-other

Elevated levels of leukotriene C4 synthase mRNA distinguish a subpopulation of eosinophilic oesophagitis patients.

Project description:Cysteinyl leukotrienes contribute to Th2-type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls.We asked whether mRNA levels of leukotriene C4 synthase (LTC4 S), a key regulator of leukotriene production, could serve as a marker for EoE.Digital mRNA expression profiling (nCounter(®) Technology) was performed on proximal and distal oesophageal biopsies of 30 paediatric EoE patients and 40 non-EoE controls. Expression data were confirmed with RT-qPCR. LTC4 S mRNA levels were quantified in whole blood samples. Leukotriene E4 was measured in urine.LTC4 S mRNA levels were elevated in proximal (2.6-fold, P < 0.001) and distal (2.9-fold, P < 0.001) oesophageal biopsies from EoE patients. Importantly, increased LTC4 S mRNA transcripts identified a subpopulation of EoE patients (28%). This patient subgroup had higher serum IgE levels (669 U/mL vs. 106 U/mL, P = 0.01), higher mRNA transcript numbers of thymic stromal lymphopoietin (TSLP) (1.6-fold, P = 0.009) and CD4 (1.4-fold, P = 0.04) but lower IL-23 mRNA levels (0.5-fold, P = 0.04). In contrast, elevated levels of IL-23 mRNA were found in oesophageal biopsies of patients with reflux oesophagitis. LTC4 S mRNA transcripts in whole blood and urinary excretion of leukotriene E4 were similar in EoE patient subgroups and non-EoE patients.Elevated oesophageal expression of LTC4 S mRNA is found in a subgroup of EoE patients, concomitant with higher serum IgE levels and an oesophageal transcriptome indicative of a more-pronounced allergic phenotype. Together with TSLP and IL-23 mRNA levels, oesophageal LTC4 S mRNA may facilitate diagnosis of an EoE subpopulation for personalized therapy.

| S-EPMC3728707 | biostudies-literature

Seleno-detergent MAD phasing of leukotriene C4 synthase in complex with dodecyl-?-D-selenomaltoside.

Project description:Dodecyl-?-D-selenomaltoside (SeDDM) is a seleno-detergent with a ?-glycosidic seleno-ether in place of the ether moiety in dodecyl-?-D-maltoside. Seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. Crystals of a nuclear membrane-embedded enzyme, leukotriene C(4) synthase (LTC(4)S), in complex with SeDDM were prepared and a multiwavelength anomalous diffraction (MAD) experiment was performed. The SeDDM in the LTC(4)S crystal exhibited sufficient anomalous diffraction for determination of the structure using MAD phasing.

| S-EPMC3232166 | biostudies-literature

Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

Project description:Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

| S-EPMC3869987 | biostudies-literature

14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase.

Project description:We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.

| S-EPMC1218910 | biostudies-other

Association of Leukotriene C4 Synthase A-444C Polymorphism with Asthma and Asthma Phenotypes in Romanian Population.

Project description:IntroductionLeukotriene C4 synthase (LTC4S) gene -444A/C polymorphism has been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. The aim of this study was to investigate the association between the -444A/C polymorphism of LTC4S gene and asthma, asthma phenotypes (aspirin intolerant/tolerant asthma) and different characteristics of the patients.Material and methodsWe included 106 patients with asthma (60 with aspirin tolerant asthma - ATA, 46 with aspirin intolerant asthma - AIA) and 103 controls. All the subjects were genotyped for LTC4S-444 A/C by Real-Time PCR. We assessed the association of LTC4S promoter polymorphism with asthma and its phenotypes and with clinical and biological characteristics of asthmatic patients.ResultsWe did not find a significant association between the studied polymorphism and asthma, but the minor allele tended to be more frequent in AIA patients. We found a significant association between the minor allele C and lower levels of serum total immunoglobulin E and eosinophils, suggesting a possible role of -444A/C LTC4S polymorphism as modulating factor of allergic inflammation in asthma.ConclusionThe results show that LTC4S -444A/C SNP is not associated with susceptibility to asthma in Romanian patients, but could influence asthma phenotype, namely aspirin intolerant asthma.

| S-EPMC5327799 | biostudies-literature

Crystal structures of leukotriene C4 synthase in complex with product analogs: implications for the enzyme mechanism.

Project description:Leukotriene (LT) C4 synthase (LTC4S) catalyzes the conjugation of the fatty acid LTA4 with the tripeptide GSH to produce LTC4, the parent compound of the cysteinyl leukotrienes, important mediators of asthma. Here we mutated Trp-116 in human LTC4S, a residue proposed to play a key role in substrate binding, into an Ala or Phe. Biochemical and structural characterization of these mutants along with crystal structures of the wild type and mutated enzymes in complex with three product analogs, viz. S-hexyl-, 4-phenyl-butyl-, and 2-hydroxy-4-phenyl-butyl-glutathione, provide new insights to binding of substrates and product, identify a new conformation of the GSH moiety at the active site, and suggest a route for product release, aided by Trp-116.

| S-EPMC3931076 | biostudies-literature

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