Anders Wetterholm

Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase.

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 Å resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the ω-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

Leukotriene A4 hydrolase: a zinc metalloenzyme.

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.

Advances in eicosanoid research, novel therapeutic implications.

Eicosanoids are a family of oxygenated metabolites of arachidonic acid, including the prostaglandins, thromboxanes, leukotrienes and lipoxins. These lipid mediators play essential roles in normal cellular homeostasis as well as in a number of disease states. This review will focus on recent advances in the field of eicosanoids and highlight specific discoveries and achievements. Emphasis will be placed on structure and receptor biology, which are of significant pharmacological and clinical relevance.

Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses

In humans, the antimicrobial peptide LL‐37 and the potent chemotactic lipid leukotriene B4 (LTB4) are important mediators of innate immunity and host defense. Here we show that LTB4, at very low (1 nM) concentrations, strongly promotes release of LL‐37 peptides from human neutrophils (PMNs) in a time‐ and dose‐dependent manner, as determined by Western blot, enzyme‐linked immunoassay (ELISA), and antibacterial activity. The LTB4‐induced LL‐37 release is mediated by the BLT1 receptor, and protein phosphatase‐1 (PP‐1) inhibits the release by suppressing the BLTl‐mediated exocytosis of PMN granules. Conversely, LL‐37 elicits translocation of 5‐lipoxygen‐ase (5‐LO) from the cytosol to the perinuclear membrane in PMNs and promotes the synthesis and release of LTB4, particularly from cells primed with LPS or GM‐CSF. Furthermore, LL‐37 stimulates PMN phagocytosis of Escherichia coli particles, a functional response that is enhanced by LTB4, especially in GM‐CSF pretreated cells. In these cells, LL‐37 also enhances LTB4‐induced phagocytosis. Hence, in human PMNs, positive feedback circuits exist between LL‐37 and LTB4 that reciprocally stimulate the release of these mediators with the potential for synergistic bioactions and enhanced immune responses. Moreover, these novel lipid‐peptide signaling pathways may offer new opportunities for pharmacological intervention and treatment of chronic inflammatory diseases.—Wan, M., Sabirsh, A., Wetterholm, A., Agerberth, B., Haegg‐ström, J. Z. Leukotriene B4 triggers release of the cathelicidin LL‐37 from human neutrophils: novel lipid‐peptide interactions in innate immune responses. FASEB J. 21, 2897–2905 (2007)

Leukotriene A4 hydrolase: an anion activated peptidase

The peptidase activity of leukotriene A4 hydrolase purified from human leukocytes has been characterized, utilizing synthetic amides as substrates. The enzyme was stimulated by several monovalent anions. Thiocyanate ions were most effective followed by chloride and bromide ions. In phosphate buffer alone the peptidase activity towards alanine-4-nitroanilide was barely detectable and addition of 100 mM NaCl increased the specific activity more than 20-fold. Increasing the concentration of NaCl (or NaSCN) did not significantly affect the apparent Km for the substrate alanine-4-nitroanilide, but resulted in a dose dependent increase of Vmax. The stimulatory effect of these anions on the reaction velocities appeared to obey saturation kinetics and thus indicated the presence of an anion binding site. Apparent affinity constants for chloride and thiocyanate ions were calculated to 100 and 50 mM, respectively. In contrast to the effect on the peptidase activity, no chloride-stimulation could be detected of the epoxide hydrolase activity of this enzyme, i.e., the conversion of leukotriene A4 into leukotriene B4. In conclusion, the results indicate that under physiological conditions, chloride ions may selectively stimulate the peptidase activity of LTA4 hydrolase. Also, the differences in chloride concentrations between cellular compartments suggest that a possible proteolytic function of the enzyme may be limited to the extracellular space.

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzymes prostaglandin E synthase activity (85–95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H2 into prostaglandin F2α. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites.

Recombinant mouse leukotriene A4 hydrolase : a zinc metalloenzyme with dual enzymatic activities

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Amino hydroxamic acids as potent inhibitors of leukotriene A4 hydrolase.

Leukotriene A4 hydrolase is a zinc-containing enzyme which catalyzes the hydrolysis of LTA4 to LTB4, a proinflammatory mediator. The enzyme also exhibits an aminopeptidase activity. Due to its biological importance, it is of considerable interest to develop selective inhibitors of this enzyme. The design and synthesis of a number of potent beta-amino hydroxylamine and amino hydroxamic acid inhibitors are described here. It was found that having a free amine was essential for high activity. Hydroxylamines were found to be about an order of magnitude less potent than their analogous hydroxamic acids. Our investigation of amino hydroxamic acids as inhibitors of leukotriene A4 hydrolase has led to the development of hydroxamates 16 and 17, which are among the most potent inhibitors found to date. These, compounds were found to be competitive inhibitors with Ki values of 1.6 nM and 3.4 nM respectively, against the peptidase activity. Inhibitor 16 has an IC50 value of < or = 0.15 microM against the epoxide hydrolase activity and is also potent against the production of LTB4 by isolated polymorphonuclear leukocytes (PMNL) activated with ionophore A23187 (IC50 approximately 0.3 microM).

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

Human leukotriene C4 synthase (hLTC4S) is an integral membrane enzyme that conjugates leukotriene (LT) A4 with glutathione to form LTC4, a precursor to the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC4S, 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 LTA4. Steady-state kinetics of R104A and R104S revealed that the Km 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 λmax at 239 nm, could be detected using R104A or R104S hLTC4S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pKa to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA4. On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC4S by 88 and 70%, respectively, without significantly affecting the kcat/Km values for GSH, and a crystal structure of R31Q hLTC4S (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 hLTC4S, 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.

Cysteinyl Leukotriene Signaling Aggravates Myocardial Hypoxia in Experimental Atherosclerotic Heart Disease

Background Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe−/− mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia. Methods and principal findings Myocardial biopsies from Apoe−/− mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C4 synthase (LTC4S) and CysLT1 expression in the myocardium of Apoe−/− mice. Acute bouts of hypoxia further induced LTC4S expression, increased LTC4S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe−/− mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC4S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling. Conclusion Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.