Leukotriene C₄ Synthase
A Pivotal Enzyme in the Biosynthesis of the Cysteinyl Leukotrienes

BING K. LAM and K. FRANK AUSTEN

Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts

	CYSTEINYL LEUKOTRIENES AND ASTHMA

TOP CYSTEINYL LEUKOTRIENES AND... BIOSYNTHESIS OF CYSTEINYL... LTC4 SYNTHASE PROTEIN LTC4 SYNTHASE cDNA LTC4 SYNTHASE GENE CATALYTIC MECHANISM OF LTC4... EXPRESSION OF LTC4 SYNTHASE... REFERENCES

Episodic bronchoconstriction, persistent hyperresponsiveness, and inflammation of the conducting airways (1) characterize bronchial asthma. The inflammatory responses in asthma include lumenal plugging with mucus, epithelial cells, and eosinophils (2); denudation of the epithelium (3) with subepithelial cells, and eosinophils (2); denudation of the epithelium (3) with subepithelial collagen deposition (4); and infiltration of the epithelium and submucosa with eosinophils and helper T type 2 (Th2) cells (5, 6). Importantly, there is partial degranulation of resident mast cells and infiltrating eosinophils (5). These cells release both preformed granule proteins such as mast cell tryptase and eosinophil major basic protein, respectively, and the newly generated lipid mediators such as leukotriene C₄ (LTC₄), which by metabolism to receptor-active LTD₄/LTE₄ contribute to the pathologic changes in asthma (1).

The specific involvement of the cysteinyl leukotrienes in asthma is suggested by their potent biologic activities, their presence in the alveolar lavage fluid of patients with asthma, and the clinical efficacy of 5-lipoxygenase (5-LO) inhibitors and LTD₄/LTE₄ receptor antagonist in the management of patients with bronchial asthma. Inhaled LTC₄ or LTD₄ is 1,000-fold more potent than histamine in compromising airway function in normal individuals. LTC₄, LTD₄, and LTE₄ are each even more potent in reducing the lung function of patients with bronchial asthma (7). These lipid mediators stimulate the secretion of mucus from bronchial epithelial cells and increase pulmonary vascular permeability via endothelial cell contraction at the postcapillary venules (8). The cysteinyl leukotrienes have been recovered from the airways of asthmatic individuals at rest (9) and in increased amounts after allergen challenge (10), isocapnic hyperventilation (11), or aspirin challenge of susceptible patients (12). Clinical studies with 5-LO inhibitors and 5-lipoxygenase-activating protein (FLAP) inhibitors, agents that inhibit leukotriene formation, and with cysteinyl leukotriene receptor antagonists have established the role of cysteinyl leukotrienes in bronchial asthma. Administration of these agents, which are devoid of intrinsic bronchodilatory activity, leads to rather immediate bronchodilatation in patients with asthma (13), suggesting that chronic overproduction of the cysteinyl leukotrienes may contribute to abnormal resting airway tone in some patients with asthma. These therapeutic agents inhibit the acute asthmatic responses to challenge by exercise (14), cold-dry air (15), allergen (16), and aspirin (17). They decrease the late asthmatic response to allergen challenge and the accompanying airway hyperresponsiveness to various pharmacologic agonists (16), but most importantly reduce the severity of chronic asthma (18). Thus a range of clinical data highlight the role of cysteinyl leukotrienes as mediators contributing to the pathogenesis of bronchial asthma.

	BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES

TOP CYSTEINYL LEUKOTRIENES AND... BIOSYNTHESIS OF CYSTEINYL... LTC4 SYNTHASE PROTEIN LTC4 SYNTHASE cDNA LTC4 SYNTHASE GENE CATALYTIC MECHANISM OF LTC4... EXPRESSION OF LTC4 SYNTHASE... REFERENCES

Biosynthesis of cysteinyl leukotrienes in mast cells, basophils, and eosiniophils is initiated on agonist stimulation, by an increase in intracellular Ca²⁺, and with activation of cytosolic phospholipase A₂ (cPLA₂) and perhaps other low molecular weight PLA₂s. cPLA₂ hydrolyzes and releases arachidonic acid from membrane phospholipid (19). The released arachidonic acid binds to FLAP and is presented to 5-LO (20). 5-Lipoxygenase, which translocates from either cytoplasm or nucleoplasm (21) to the perinuclear membrane and is modulated in function by phosphorylation by tyrosine kinase (22), converts arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently to LTA₄ (23). LTC₄ synthase conjugates LTA₄ with glutathione (GSH) to form LTC₄ (24). LTC₄ synthase is the only enzyme in hematopoietic cells committed to conjugate GSH with LTA₄ to form LTC₄. After carrier-mediated export (25), sequential cleavage of glutamic acid and glycine from the glutathione moiety of LTC₄ yields the receptor-active derivatives LTD₄ and LTE₄, respectively (26).

	LTC4 SYNTHASE PROTEIN

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LTC₄ synthase differs from conventional glutathione S-transferase (GST) by its selectivity for LTA₄ and its analogs (24), microsomal localization, and failure to conjugate xenobiotics. LTC₄ synthase also exhibits differential susceptibility to inhibitors (27) and lacks immunoreactivity to antibodies for known GSTs (28). The enzyme is expressed in a limited number of cell types such as eosinophils, mast cells, basophils, and monocyte-macrophages and in leukemic cell lines such as KG-1 cells and THP-1 cells; it is also present in platelets, which lack 5-lipoxygenase.

LTC₄ synthase was purified to homogeneity as an 18-kD protein from THP-1 (29) and KG-1 cells. The 18-kD protein was proposed to function as a homodimer on the basis of the size of the active fraction by gel-filtration column chromatography of both the native and the recombinant protein (29, 30). LTC₄ synthase activity is augmented by Mg²⁺ ions, and is inhibited by Co²⁺ ion (28) and by the FLAP inhibitor MK-886 (31). N-Ethylmaleimide, an agent that activates microsomal GST-I (mGST-I), inhibits LTC₄ synthase both from guinea pig lung and from U-937 cells (24, 28).

LTC₄ synthase purified from dimethyl sulfoxide (DMSO)- differentiated U-937 cells displayed K_m values for LTA₄ and GSH of 19.6 µM and 1.83 mM, respectively, and with a Vmax value of 2-4 µmol/min/mg (28). These values are similar to the values obtained with purified recombinant proteins (31).

	LTC4 SYNTHASE cDNA

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We used an expression cloning technique to isolate the cDNA clone for LTC₄ synthase from a human KG-1 expression library. The clone contains a 694-bp cDNA insert with an open reading frame of 450 bp encoding a 150-amino acid polypeptide (32). The nucleotide sequence of the cDNA and the deduced amino acid sequence of human LTC₄ synthase display no significant homology with cytosolic GSTs or mGST-I. The deduced amino acid sequence reveals 31% overall amino acid identity with FLAP. The identity of the N-terminal two-thirds of these proteins, which includes the putative FLAP-like inhibitor binding site, is 44%. Furthermore, the secondary structure analysis of the deduced LTC₄ synthase indicated that it is nearly identical to FLAP, as their three respective hydrophobic domains overlap each other and their two hydrophilic loops are similar in size. The structural homology between LTC₄ synthase and FLAP is consistent with the ability of the FLAP inhibitor, MK-886, to inhibit LTC₄ synthase activity (31, 32). The predicted secondary structure of LTC₄ synthase and FLAP is also observed in mGST-II and mGST-III (33, 34). The deduced amino acid sequence of LTC₄ synthase reveals two potential protein kinase C (PKC) phosphorylation sites and one potential N-glycosylation site. These features of LTC₄ synthase are consistent with reports of downregulation of this enzyme by PKC activation (35)

Using oligonucleotides based on the human LTC₄ synthase sequence for polymerase chain reaction screening of a mouse expression cDNA library, the cDNA for mouse LTC₄ synthase was also isolated (31). Mouse LTC₄ synthase is also a 150-amino acid polypeptide and shows an 88% amino acid identity to human LTC₄ synthase. Of the 18 different amino acid residues in mouse LTC₄ synthase, 9 are located at the carboxy terminus of the protein. The putative FLAP-like inhibitor binding domain, the potential N-glycosylation site, and the two potential PKC phosphorylation sites present in human LTC₄ synthase are all conserved in mouse LTC₄ synthase. The difference in amino acid residues does not appear to alter the enzyme function, as evidenced by nearly identical enzyme kinetics and susceptibility to the FLAP inhibitor MK-886 (31, 32).

	LTC4 SYNTHASE GENE

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The genomic structure of the human LTC₄ synthase gene revealed five exons and four introns, which span 2.5 kbp (36). The exon sequences responsible for encoding the LTC₄ synthase protein are small, ranging from 71 to 257 nucleotides, and are interspersed with small introns. The human gene for FLAP is > 31 kbp in size. Nonetheless, the second, third, and fourth exons of LTC₄ synthase are identical in size to those of the FLAP gene, while the first and fifth exons differ minimally by the number of nucleotides in the 5' and the 3' untranslated regions. Hence, the intron/exon junctions and gene organization are the same for FLAP and LTC₄ synthase. In addition, the exons of LTC₄ synthase and FLAP align identically with regard to the amino acids that they encode. These findings also allow the deduced amino acids within the respective predicted secondary structures of the two molecules to be aligned as previously depicted into two hydrophilic loops of comparable size, which separate three hydrophobic domains (32, 36).

A 5' extension analysis of KG-1 mRNA revealed three putative transcription initiation sites in the human LTC₄ synthase gene, located 66, 69, and 96 nucleotides upstream of the ATG translation start site. The 5' flanking region of human LTC₄ synthase gene contains the typical features of genes that have been identified with multiple transcription initiation sites: a high G/C content and at least one consensus sequence for Sp1 binding 18 nucleotides upstream of the first transcription start site (Figure 1). In addition, consensus sequences for AP-1 and AP-2 binding sites were identified 807 and 877 nucleotides 5' of the distal transcription initiation site, both of which are PKC-responsive elements. The presence of these cis-acting elements in the 5' flanking region of the human LTC₄ synthase gene is consistent with the reports that LTC₄ synthase activity is induced in human erythroleukemia cells after treatment with phorbol 12-myristate 13-actate (PMA) (37).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Schematic representation of the human LTC4 synthase gene with its putative 5' regulator elements. The solid boxes with Roman numerals underneath represent the exons. The three transcription initiation sites and the ATG translation start site are boxed. Numbers indicate the nucleotide position, with the adenine nucleotide of ATG translation as +1. The polymorphism at position 444, with its respective nucleotides, is shaded, The relative positions of the putative binding sites for Sp1, AP-1, and AP-2 are as indicated.

The 2.0-kb gene for mouse LTC₄ synthase is identical in intron/exon organization to the human LTC₄ synthase and FLAP genes. The 5' flanking sequence of the mouse LTC₄ synthase gene, however, contains only one transcription initiation site 64 bp upstream of the ATG start site by primer extension with lung RNA, and consensus sequences for AP-2, CEBP, and PEA-3 sites (38).

Fluorescent in situ hybridization with the P1 plasmid containing the gene for LTC₄ synthase localized this gene to the long arm of human chromosome 5, an area that corresponds to band 5q35 (36). The LTC₄ synthase gene is located distal to the gene cluster for cytokines central to the Th2 cell phenotype and to genes implicated in allergic inflammation (36, 39); this chromosomal organization is mimicked in mouse by a syntenic location of the LTC₄ synthase gene on mouse chromosome 11 (38).

Promoter analysis with luciferase gene reporter constructs revealed a 550-bp 5' flanking genomic fragment of human LTC₄ synthase gene that induces the expression of luciferase when transfected into LTC₄ synthase-expressing THP-1 cell (B. K. Lam, J. Zhao, and K. F. Austen, unpublished results). Further sequence analysis revealed consensus nucleotide sequence for the GT box, and a mammalian transcription initiator element within this 550-bp fragment (Figure 1). Furthermore, an A-C polymorphism within the 550-bp fragment has been identified at nucleotide 444, the frequency of which is higher in aspirin-intolerant patients with asthma (Figure 1) (40).

	CATALYTIC MECHANISM OF LTC4 SYNTHASE

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Site-directed mutagenesis was carried out to examine the GSH binding site and the catalytic mechanisms for conjugation of LTA₄ and GSH by human LTC₄ synthase (30). A point mutation of the first hydrophilic loop (Arg-51 to Thr or Ile) abolished function of the recombinant mutant protein, whereas mutations of Arg-51 to His or Lys provided fully active mutant protein. Mutations of V49F, A52S, N55A, and Y59F of the first hydrophilic loop, and mutations of Y97F and Y93F of the second hydrophilic loop, increased the K_m of the recombinant enzyme for GSH, suggesting that these residues are involved in GSH binding. The mutation of Y93F also reduced the enzyme activity of the mutant protein to less than 1% of that of the wild-type enzyme. In addition, there is a shift in pH optimum of the mutant protein to that of spontaneous conjugation. Direct linkage of two LTC₄ synthase monomers by a 12-amino acid bridge gave rise to an active covalent-linked dimer, and identical bridging of an inactive R51I monomer with a wild-type monomer created an active pseudo-heterodimer. These results suggest that Arg-51 and Tyr-93 are involved in acid and base catalysis, respectively, with Arg-51 opening the epoxide ring of LTA₄ and Tyr-93 promoting the formation of the thiolate anion of glutathione. It also suggests that each native monomer of LTC₄ synthase has independent conjugation activity and that noncovalent dimerization of LTC₄ synthase maintains the proper protein folding. It is interesting that the amino acid residue Tyr-93 is present in mGST-II, mGST-III, and FLAP. In contrast, Arg-51 is present in mGST-II and mGST-III, which have the ability to conjugate both LTA₄ and xenobiotics with GSH, but is not present in FLAP, which has no known GSH-conjugating activity (30).

	EXPRESSION OF LTC4 SYNTHASE DURING EOSINOPHILOPOIESIS IN VITRO

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The expression of 5-LO/LTC₄ synthase pathway proteins was examined during eosinophil development in vitro from human umbilical cord blood progenitor cells cultured in the presence of interleukin 3 (IL-3), IL-5, and a reconstituted basement membrane (Matrigel). At 7 d, the cultures had cells of mixed granulocytic lineages, produced no LTC₄ when stimulated with ionophore, and had minimal subcellular LTC₄ synthase activity and no immunodetectable LTC₄ synthase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot analyses. By 14 d, more than 93% of the cells possessed both eosinophil and basophil granules (hybrid granulocytes) and the complete repertoire of pathway proteins was present, including LTC₄ synthase as an immunodetectable protein and RNA transcript (41). Further culture to 28 d resulted in a loss of the basophilic granules, a persistent mononuclear cell profile (mononuclear eosinophils), and progressive increases in LTC₄ synthase subcellular activity and immunodetectable protein in SDS-PAGE immunoblots. When the cultures were started with purified CD34-bearing cord blood cells to eliminate contaminating differentiated populations, SDS-PAGE immunoblot analyses revealed the presence of the proximal components of the 5-LO/LTC₄ synthase pathway proteins (cPLA₂, 5-LO, and FLAP) by Day 7 of culture, whereas LTC₄ synthase was absent until Day 10, when the majority of the cells in the culture developed eosinophilic granules. These studies suggest that the incremental acquisition of LTC₄ synthase is a maturation-related phenomenon in eosinophils.

One study has reported that the number of activated eosinophils and the number of cells per cubic millimeter that are immunoreactive for LTC₄ synthase were increased in bronchial mucosa biopsies of aspirin-sensitive patients with asthma, as compared with those from aspirin-tolerant patients with asthma (42). This report thus suggests that the increase in LTC₄ synthase and not other enzymes of the 5-LO pathway of the inflammatory cells of human airways may be a hallmark of aspirin-intolerant asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Bing K. Lam, Ph.D., 1 Jimmy Fund Way, Room 628, Boston, MA 02115.

Acknowledgments: Supported in part by National Institutes of Health Grants AI22531, AI31599, AR36308, ES06105, HL03208, and HL36110.

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