Bing K. Lam

Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 Å resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane α-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

Cysteinyl Leukotrienes Regulate Th2 Cell-Dependent Pulmonary Inflammation

The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C4 synthase (LTC4S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC4Snull mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC4Snull mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC4Snull mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC4Snull mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.

MOLECULAR CLONING OF THE GENE FOR HUMAN LEUKOTRIENE C4 SYNTHASE : ORGANIZATION, NUCLEOTIDE SEQUENCE, AND CHROMOSOMAL LOCALIZATION TO 5Q35

Leukotriene C (LTC) synthase catalyzes the conjugation of LTA with reduced GSH to form LTC, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase superfamily. Genomic cloning from a P1 library now reveals that the gene for LTC synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC synthase align identically with those of FLAP; however, the small size of the LTC synthase gene contrasts with the >31-kilobase pair size reported for FLAP. Confirmation of the LTC synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A) RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5′ of the ATG translation start site. The 5′-flanking region revealed a GC-rich promotor sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5′ from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5′ and 3′ oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

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

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.

The Catalytic Architecture of Leukotriene C4 Synthase with Two Arginine Residues

Leukotriene (LT) C4 and its metabolites, LTD4 and LTE4, are involved in the pathobiology of bronchial asthma. LTC4 synthase is the nuclear membrane-embedded enzyme responsible for LTC4 biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA4 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 LTA4 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 LTA4.

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures

Prostaglandin E2 (PGE2) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone‐marrow cultures. Because cysteinyl leukotrienes (cys‐LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone‐marrow were due to blockade of PGE2 production alone, or involved further promotion of cys‐LTs production/signalling.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL‐13 and eotaxin play important, inter‐related roles in asthma models. In the lungs, CysLT, produced by the 5‐LO‐LTC4S pathway, mediate some local responses to IL‐13 and eotaxin; in bone marrow, CysLT enhance IL‐5‐dependent eosinophil differentiation. We examined the effects of IL‐13 and eotaxin on eosinophil differentiation. Semi‐solid or liquid cultures were established from murine bone marrow with GM‐CSF or IL‐5, respectively, and the effects of IL‐13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5‐LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5‐LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM‐CSF‐dependent eosinophil colony formation and IL‐5‐stimulated eosinophil differentiation. Although IL‐13 did not induce eotaxin production, its effects were abolished by anti‐eotaxin and anti‐CCR3 antibodies, suggesting up‐regulation by IL‐13 of responses to endogenous eotaxin. Anti‐CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5‐LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL‐13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals.

Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters

Human leukotriene C(4) synthase (LTC(4)S) forms highly ordered two-dimensional (2D) crystals under specific reconstitution conditions. It was found that control of a larger number of parameters than is usually observed for 2D crystallization of membrane proteins was necessary to induce crystal formation of LTC(4)S. Here, we describe the parameters that were optimized to yield large and well-ordered 2D crystals of LTC(4)S. Careful fractioning of eluates during the protein purification was essential for obtaining crystals. While the lipid-to-protein ratio was critical in obtaining order, four parameters were decisive in inducing growth of crystals that were up to several microns in size. To obtain a favorable diameter, salt, temperature, glycerol, and initial detergent concentration had to be controlled with great care. Interestingly, several crystal forms could be grown, namely the plane group symmetries of p2, p3, p312, and two different unit cell sizes of plane group symmetry p321.

Leukotriene C4 synthase

Leukotriene C4 synthase polypeptide identified by photoaffinity labeling, and purification of leukotriene C4 synthase to homogeneity is described.