Jesper Z. Haeggström

Lipoxygenase and Leukotriene Pathways: Biochemistry, Biology, and Roles in Disease

4. Biosynthesis of Leukotrienes 5869 4.1. Discovery of the Leukotriene Pathway and a Common Unstable Intermediate 5869 4.2. Conversion of Arachidonic Acid into Leukotriene A4 (LTA4) Is a Two-Step Concerted Reaction Catalyzed by a Single Lipoxygenase 5869 4.3. Structural Elucidation of Slow-Reacting Substance of Anaphylaxis, A Mixture of Leukotrienes 5870 5. Enzymes and Proteins in Leukotriene Biosynthesis 5870 5.1. Cytosolic Phospholipase A2α (cPLA2α) 5870 5.1.1. Molecular Properties and Regulation of cPLA2α 5870 5.1.2. Crystal Structure and Catalytic Mechanism of cPLA2α 5871 5.2. 5-Lipoxygenase (5-LO) 5871 5.2.1. Cellular Expression of 5-LO 5871 5.2.2. Regulation of 5-LO Gene (ALOX5) Transcription 5872 5.2.3. Naturally Occurring Mutations in the Gene Promoter of 5-LO 5872 5.2.4. Allosteric and Post-translational Regulation of 5-LO 5872 5.2.5. Structure function relationships in 5-LO 5872 5.2.6. Crystal Structure of 5-LO 5872 5.3. 5-Lipoxygenase-Activating Protein (FLAP) 5873 5.3.1. FLAP Is Critical for Cellular 5-LO Activity 5873 5.3.2. Effects of FLAP on Leukotriene Production 5873 5.3.3. FLAP Gene (ALOX5AP) and Regulation of Expression 5874 5.3.4. Crystal Structure of FLAP 5874 5.4. LTA4 Hydrolase 5874 5.4.1. LTA4 Hydrolase Is a Substrate-Selective and Suicide-Inactivated Epoxide Hydrolase 5874 5.4.2. LTA4 Hydrolase Is Bifunctional and Belongs to the M1 Family of Zinc Metallopeptidases 5874 5.4.3. LTA4 Hydrolase Cleaves the Chemotactic Pro-Gly-Pro, a Role for the Aminopeptidase Activity during Resolution of Inflammation 5875 5.4.4. Crystal Structure of LTA4 Hydrolase 5875 5.4.5. Mechanism of the Epoxide Hydrolase Reaction 5876 5.4.6. Mechanism of the Aminopeptidase Activity 5876 5.4.7. Two Catalytic Activities Exerted via Specific but Overlapping Active Sites 5877 5.5. LTC4 Synthase 5877 5.5.1. Molecular Properties of LTC4 Synthase 5877 5.5.2. LTC4 Synthase Is a Member of the MAPEG Superfamily of IntegralMembraneProteins 5877 5.5.3. Gene Structure and Regulation of LTC4 Synthase Expression 5877 5.5.4. Crystal Structure of LTC4 Synthase 5877 5.5.5. Catalytic Mechanism of LTC4 Synthase 5878 6. Intracellular Protein Trafficking and Compartmentalization of Leukotriene Biosynthesis 5878 6.1. Translocation of cPLA2α 5878 6.2. Translocation of 5-LO and Association with FLAP on the Nuclear Envelope 5879 6.2.1. 5-LO in the Nucleoplasm 5879 6.2.2. Organization of a Leukotriene Biosynthetic Complex in the Nuclear Membrane 5879 6.3. Leukotriene Biosynthesis in Lipid Bodies and Actions on Extracellular Granules 5879

Crystal structure of human leukotriene A 4 hydrolase, a bifunctional enzyme in inflammation

Leukotriene (LT) A4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn2+ site. A bent and narrow pocket, shaped to accommodate the substrate LTA4, constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB4.

Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic activated the kinase in the presence of calcium at 30 fold lower concentration (1 microM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C4, certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.

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/aminopeptidase, the gatekeeper of chemotactic leukotriene B4 biosynthesis

The leukotrienes (LTs) 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 LTB4, which is a classical chemoattractant that triggers adherence and aggregation of leukocytes to the endothelium at nanomolar concentrations. Recent data also indicate that LTB4 is a chemoattractant for T-cells, creating a functional link between early innate and late adaptive immune responses to inflammation (3–5). In addition, LTB4 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, LTB4 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 LTB4, as a mediator of vascular inflammation and arteriosclerosis (9). This article gives an overview of the biochemical, structural, and catalytic properties of LTA4 hydrolase (LTA4H), which catalyzes the final and committed step in LTB4 biosynthesis. LTA4 Hydrolase Is a Key Enzyme in the 5-Lipoxygenase Pathway In cellular biosynthesis of LTs, 5-lipoxygenase, assisted by 5-lipoxygenase-activating protein, converts arachidonic acid into the unstable epoxide LTA4, which in turn may be enzymatically conjugated with GSH to form LTC4, the parent compound of the cysteinyl leukotrienes, or hydrolyzed into LTB4 by LTA4H (Fig. 1). Leukotrienes can also be formed via transcellular routes, where LTA4 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). LTB4 signals via a specific, high affinity, G-protein-coupled receptor (BLT1) (11). In addition, a second receptor for LTB4 (BLT2) has been discovered, the functional role of which is presently not known (12). Interestingly, LTB4 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).

The novel 13S,14S-epoxy-maresin is converted by human macrophages to maresin 1 (MaR1), inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage phenotype

Maresins are produced by macrophages from docosahexaenoic acid (DHA) and exert potent proresolving and tissue homeostatic actions. Maresin 1 (MaR1; 7R,14S‐dihydroxy‐docosa‐4Z,8E,10E,12Z,16Z,19Z‐hexaenoic acid) is the first identified maresin. Here, we investigate formation, stereochemistry, and precursor role of 13,14‐epoxy‐docosahexaenoic acid, an intermediate in MaR1 biosynthesis. The 14‐lipoxygenation of DHA by human macrophage 12‐lipoxygenase (hm12‐LOX) gave 14‐hydro(peroxy)‐docosahexaenoic acid (14‐HpDHA), as well as several dihydroxy‐docosahexaenoic acids, implicating an epoxide intermediate formation by this enzyme. Using a stereo‐controlled synthesis, enantiomerically pure 13S,14S‐epoxy‐docosa‐4Z,7Z,9E,11E,16Z,19Z‐hexaenoic acid (13S,14S‐epoxy‐DHA) was prepared, and its stereochemistry was confirmed by NMR spectroscopy. When this 13S,14S‐epoxide was incubated with human macrophages, it was converted to MaR1. The synthetic 13S,14S‐epoxide inhibited leukotriene B4 (LTB4) formation by human leukotriene A4 hydrolase (LTA4H) ~40% (P<0.05) to a similar extent as LTA4 (~50%, P<0.05) but was not converted to MaR1 by this enzyme. 13S,14S‐epoxy‐DHA also reduced (~60%; P<0.05) arachidonic acid conversion by hm12‐LOX and promoted conversion of M1 macrophages to M2 phenotype, which produced more MaR1 from the epoxide than M1. Together, these findings establish the biosynthesis of the 13 S,14S‐epoxide, its absolute stereochemistry, its precursor role in MaR1 biosynthesis, and its own intrinsic bioactivity. Given its actions and role in MaR1 biosynthesis, this epoxide is now termed 13,14‐epoxy‐maresin (13,14‐eMaR) and exhibits new mechanisms in resolution of inflammation in its ability to inhibit proinflammatory mediator production by LTA4 hydrolase and to block arachidonate conversion by human 12‐LOX rather than merely terminating phagocyte involvement.—Dalli, J., Zhu, M., Vlasenko, N. A., Deng, B., Haeggström, J. Z., Petasis, N. A., Serhan, C. N. The novel 13S,14S‐epoxy‐maresin is converted by human macrophages to maresin 1 (MaR1), inhibits leukotriene A4 hydrolase (LTA4H) and shifts macrophage phenotype. FASEB J. 27, 2573–2583 (2013). www.fasebj.org

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.

Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions

Incubation of mixed human platelet/granulocyte suspensions with ionophore A23187 led to a platelet dependent formation of several lipoxin isomers from endogenous substrate. The major metabolite coeluted with authentic lipoxin A4 (5(S), 6(R), 15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) in several HPLC-systems and showed an identical UV-spectrum. Furthermore, a similar profile of lipoxins was formed in pure platelet suspensions incubated with exogenous leukotriene A4 (5(S) -5, 6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid). The conversion of exogenous leukotriene A4 to lipoxin A4 was markedly increased in the presence of ionophore A23187.

Dominant Expression of the CysLT2 Receptor Accounts for Calcium Signaling by Cysteinyl Leukotrienes in Human Umbilical Vein Endothelial Cells

Objective—The objective of the present study was to identify and characterize the cell-surface receptors on human umbilical vein endothelial cells (HUVECs) that transduce calcium transients elicited by cysteinyl leukotrienes (CysLTs), potent spasmogenic and proinflammatory agents with profound effects on the cardiovascular system. Methods and Results—Using quantitative reverse transcription–polymerase chain reaction, we found that HUVECs abundantly express CysLT2R mRNA in vast excess (>4000-fold) of CysLT1R mRNA. Lipopolysaccharide, tumor necrosis factor-&agr;, or interleukin-1&bgr; caused a rapid (within 30 minutes) and partially reversible suppression of CysLT2R mRNA levels. Challenge of HUVECs with BAY u9773, a specific CysLT2R agonist, triggered diagnostic Ca2+ transients. LTC4 and LTD4 are equipotent agonists, and their actions can be blocked by the dual-receptor antagonist BAY u9773, but not by the CysLT1R-selective antagonist MK571. Conclusions—HUVECs almost exclusively express the CysLT2R. Furthermore, Ca2+ fluxes elicited by CysLT in these cells emanate from perturbation of the CysLT2R, rather than the expected CysLT1R. Hence, signaling events involving CysLT2R might trigger functional responses involved in the critical components of LT-dependant vascular reactions, which in turn have implications for ischemic heart disease and myocardial infarction.