Publications

• Membrane localization and topology of leukotriene C4 synthase.

  Christmas P., Weber B.M., McKee M., Brown D., Soberman R.J.

  Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance ... >> More

  Leukotriene C(4) (LTC(4)) synthase conjugates LTA(4) with GSH to form LTC(4). Determining the site of LTC(4) synthesis and the topology of LTC(4) synthase may uncover unappreciated intracellular roles for LTC(4), as well as how LTC(4) is transferred to its export carrier, the multidrug resistance protein-1. We have determined the membrane localization of LTC(4) synthase by immunoelectron microscopy. In contrast to the closely related five-lipoxygenase-activating protein, LTC(4) synthase is distributed in the outer nuclear membrane and peripheral endoplasmic reticulum but is excluded from the inner nuclear membrane. We have combined immunofluorescence with differential membrane permeabilization to determine the topology of LTC(4) synthase. The active site of LTC(4) synthase is localized in the lumen of the nuclear envelope and endoplasmic reticulum. These results indicate that the synthesis of LTB(4) and LTC(4) occurs in different subcellular locations and suggests that LTC(4) must be returned to the cytoplasmic side of the membrane for export by multidrug resistance protein-1. The differential localization of two very similar integral membrane proteins suggests that mechanisms other than size-dependent exclusion regulate their passage to the inner nuclear membrane. << Less

  J. Biol. Chem. 277:28902-28908(2002) [PubMed] [EuropePMC]

• Leukotriene C4 synthase: a pivotal enzyme in cellular biosynthesis of the cysteinyl leukotrienes.

  Lam B.K., Austen K.F.

  Leukotriene C4 synthase (LTC4S) conjugates LTA4 with glutathione (GSH) to form LTC4, the parent compound of the cysteinyl LTs. LTC4S is an 18 kDa membrane protein and functions as a noncovalent homodimer. The enzyme activity of LTC4S is augmented by Mg2+ and inhibited by Co2+ and the function of 5 ... >> More

  Leukotriene C4 synthase (LTC4S) conjugates LTA4 with glutathione (GSH) to form LTC4, the parent compound of the cysteinyl LTs. LTC4S is an 18 kDa membrane protein and functions as a noncovalent homodimer. The enzyme activity of LTC4S is augmented by Mg2+ and inhibited by Co2+ and the function of 5-lipoxygenase (LO) activating protein (FLAP) inhibitor MK-886. The Km and Vmax values are 3.6 microM and 1.3 micromol/mg/min for LTA4 and 1.6 mM and 2.7 micromol/mg/min for GSH, respectively. The deduced amino acid sequence and the predicted secondary of LTC4S shares significant homology to FLAP, mGST-2 and mGST-3 which are all members of MAPEG protein superfamily. LTC4S and FLAP exhibited identical genomic organization of five exons and four introns. Site-directed mutagenesis suggests that Arg-51 is involved in opening the epoxide ring of LTA4 and Tyr-93 in GSH thiolate anion formation during catalytic conjugation. LTC4S is a TATA-less gene whose transcription assessed in a reporter construct involved both cell-specific and nonspecific regulatory elements. LTC4S-/-mice grow normally, and are attenuated for innate and adaptive immune inflammatory permeability responses. << Less

  Prostaglandins Other Lipid Mediat 68-69:511-520(2002) [PubMed] [EuropePMC]

• Enzymes functional in the syntheses of leukotrienes and related compounds.

  Shimizu T.

  1. Leukotrienes (LT) constitute a family of bioactive compounds mainly involved in inflammatory and immunological responses. 2. LTs are produced via an unstable intermediate, LTA4 which is synthesized by the action of arachidonate 5-lipoxygenase, a calcium-dependent enzyme. 3. LTA4 is converted to ... >> More

  1. Leukotrienes (LT) constitute a family of bioactive compounds mainly involved in inflammatory and immunological responses. 2. LTs are produced via an unstable intermediate, LTA4 which is synthesized by the action of arachidonate 5-lipoxygenase, a calcium-dependent enzyme. 3. LTA4 is converted to either LTB4 by cytosolic LTA4 hydrolase or to LTC4 by LTC4 synthase present in the microsomal fraction. 4. Three enzymes (5-lipoxygenase, LTA4 hydrolase and LTC4 synthase) were purified, and molecular and kinetic properties have been extensively investigated. 5. The cDNA of LTA4 hydrolase was recently isolated, and the primary structure was fully determined in our laboratory. 6. I report herein recent progress in studies on arachidonic acid metabolism, with the focus primarily on enzymes linked to the 5-lipoxygenase pathway. << Less

  Int J Biochem 20:661-666(1988) [PubMed] [EuropePMC]

• Molecular cloning, expression and characterization of mouse leukotriene C4 synthase.

  Lam B.K., Penrose J.F., Rokach J., Xu K., Baldasaro M.H., Austen K.F.

  Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A4 to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C4 synthase shares substantial amino acid identity in its consensus N-terminal two-thir ... >> More

  Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A4 to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C4 synthase shares substantial amino acid identity in its consensus N-terminal two-thirds with 5-lipoxygenase-activating protein and has a region (residues 37-58) that exhibits 46% amino acid identity with a domain of this protein (residues 41 -62) to which an inhibitor binds. We have now cloned mouse leukotriene C4 synthase CDNA using the polymerase chain reaction to screen a mouse pcDNA3 expression library with oligonucleotide primers based on the translated human leukotriene C4 synthase cDNA sequence. Mouse leukotriene C4 synthase cDNA is 667 bp in length, including the poly(A)-rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150-amino-acid sequence of mouse leukotriene C4 synthase (differs from the human enzyme by only 18 amino acids, of which 9 reside at the C terminus. The potential N-glycosylation site, two protein kinase C phosphorylation sites, the two cysteine residues, and the putative inhibitor-binding domain (substitutions Thr4l-->Ser and Tyr50-->Phe) were conserved in mouse leukotriene C4 synthase. Northern blot analysis indicated that the leukotriene C4 synthase RNA transcript is widely distributed. The Km values for leukotriene A4 methyl ester, leukotriene A4 free acid and GSH were 7.6 microM, 3.6 microM and 1.6 mM, respectively, for purified human recombinant enzyme, and 10.3 microM, 2.5 microM and 1.9 microM, respectively, for purified recombinant mouse enzyme; the corresponding Vmax values were 2.5, 1.3 and 2.7 micromol x min(-1) x mg(-1) protein, respectively, for human enzyme, and 2.3, 1.2 and 2.2 micromol x min(-1) x mg(-1) protein, respectively, for mouse enzyme. The 5-lipoxygenase-activating-protein inhibitor, MK-886, was active against both human and mouse recombinant leukotriene C4 synthase with IC50 values of 3.1 microM and 2.7 microM respectively. These findings confirm that the leukotriene C4 synthases belong to a gene family that includes the 5-lypoxygenase-activating protein and suggest that the C-terminal domain of leukotriene C4 synthase may not be critical for its conjugation function. << Less

  Eur. J. Biochem. 238:606-612(1996) [PubMed] [EuropePMC]

• The catalytic formation of leukotriene C4: a critical step in inflammatory processes.

  MacDonald C.A., Bushnell E.A., Gauld J.W., Boyd R.J.

  Leukotrienes (LT) are a family of drug-like molecules involved in the pathobiology of bronchial asthma and are responsible for smooth muscle contraction. Leukotriene C4 synthase (LTC4S) is a nuclear-membrane enzyme responsible for the conjugation of leukotriene A4 (LTA4) to glutathione to form LTC ... >> More

  Leukotrienes (LT) are a family of drug-like molecules involved in the pathobiology of bronchial asthma and are responsible for smooth muscle contraction. Leukotriene C4 synthase (LTC4S) is a nuclear-membrane enzyme responsible for the conjugation of leukotriene A4 (LTA4) to glutathione to form LTC4, a cysteinyl leukotriene. In this study, the mechanism of LTA4 binding by LTC4S has been computationally examined. More specifically, docking and molecular dynamics simulations were used to gain insight into the substrate-bound active site. These studies identified two possible orientations for bound LTA4: 'tail-to-head' and 'head-to-tail'. An ONIOM(QM/MM) approach was then used to elucidate the mechanism by which glutathione may add to LTA4. In particular, the thiolate of glutathione acts as a nucleophile attacking C6 of LTA4 forming a S-C6 bond. Concomitantly, a proton is transferred from the guanidinium of Arg31 to the epoxide ring oxygen. This results in opening of the epoxide ring and stabilization of the LTC4 product complex. Within the present computational methodology the 'tail-to-head' orientation appears to be the most likely substrate orientation. << Less

  Phys Chem Chem Phys 16:16284-16289(2014) [PubMed] [EuropePMC]

• Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

  Dalli J., Vlasakov I., Riley I.R., Rodriguez A.R., Spur B.W., Petasis N.A., Chiang N., Serhan C.N.

  Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined mar ... >> More

  Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC₄S and GSTM4. Incubation of human macrophages with LTC₄S inhibitors blocked LTC₄ and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration. << Less

  Proc. Natl. Acad. Sci. U.S.A. 113:12232-12237(2016) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Molecular cloning and expression of human leukotriene-C4 synthase.

  Welsch D.J., Creely D.P., Hauser S.D., Mathis K.J., Krivi G.G., Isakson P.C.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:9745-9749(1994) [PubMed] [EuropePMC]

• Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase.

  Gupta N., Gresser M.J., Ford-Hutchinson A.W.

  The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid ( ... >> More

  The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-end inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the initial velocities of LTC4 formation in the absence of the inhibitor using non-linear regression fits of various models to the data favoured a random, rapid equilibrium mechanism, with strong substrate inhibition by LTA4, over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be Vmax = 14 +/-4 microM/min, KLTA4 = 40 +/- 18 microM, KGSH = 0.4 +/-0.2 mM, and a KiLTA4 = 2.3 +/-1.7 microM for the rapid equilibrium random model. Inhibition of enzymatic activity by L-699,333 was found to be reversible as assessed by the ability of the enzyme to restore its activity by 95% upon dilution. L-699,333 was found to be a competitive inhibitor against GSH and non-competitive against LTA4. Non-linear least squares regression analysis yielded estimated parameters of Km = 0.7 +/- 0.1 mM, Vmax = 2.5 +/- 0.1 microM/min, and Ki = 0.7 +/-0.1 microM for GSH at a fixed LTA4 concentration of 20 microM, and Km = 45 +/-3 microM, Vmax = 4.9 +/-0.2 microM/min, and a Ki = 5.8+/-0.4 microM for LTA4 at a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed for L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation. << Less

  Biochim Biophys Acta 1391:157-168(1998) [PubMed] [EuropePMC]