Molecular cloning and expression of human leukotriene-C 4 synthase

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. NatI. 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. Inosinecontaining 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, = 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. Leukotrienes (LTs) are potent biological mediators derived from arachidonic acid and are formed in response to a variety ofimmunologic and inflammatory stimuli (1-3). It is now well established that LTs affect leukocyte chemotaxis, pulmonary smooth muscle contraction, vascular tone and permeability, and mucous secretion (4-8). As such, the LTs have been implicated as potential mediators of immediate hypersensitivity and inflammatory conditions (9, 10). Initially, the production of LTs results from the oxygenation and subsequent dehydration of arachidonic acid to yield the unstable epoxide intermediate LTA4; both of these enzymatic reactions are catalyzed by 5-lipoxygenase (5-LO) (11). Cellular 5-LO activity requires an additional cofactor, 5-LO activating protein (FLAP) (12). It has been suggested that FLAP activates 5-LO by specifically binding arachidonic acid and transferring this substrate to the 5-LO enzyme (13). The LTA4 formed by 5-LO-FLAP can then be stereoselectively hydrolyzed to biologically active LTB4 by the cytosolic enzyme LTA4 hydrolase or conjugated with reduced glutathione by the membrane-bound enzyme LTC4 synthase (LTC4S; EC 2.5.1.37) to form the peptidoleukotriene LTC4. LTC4 is then converted to LTD4 by y-glutamyltranspeptidase and subsequently metabolized to LTE4 by cysteinylglycine dipeptidase (14). Collectively, the peptidoleukotrienes (LTC4, LTD4, and LTE4) have been demonstrated to be involved in inflammatory and anaphylactic reactions. They were first isolated and identified as the active mediators of the slow-reacting substance of anaphylaxis and are released from the lung tissue of asthmatics upon exposure to specific allergens (15). Additionally, exogenous application of peptidoleukotrienes results in many phenomena characteristic of human bronchial asthma including bronchoconstriction and cellular infiltration (16, 17). Because of the important role that these mediators may play in the pathophysiology of asthma, compounds that inhibit the production (5-LO inhibitors and FLAP antagonists) or action (LTD4 receptor antagonists) of these mediators are currently being evaluated in clinical trials for amelioration of asthma (18, 19). LTC4S is a unique membrane-bound enzyme that catalyzes the committed step in the biosynthesis of all of the peptidoleukotrienes. LTC4S conjugates reduced glutathione with the unstable epoxide LTA4 to form LTC4 and, as such, is a glutathione S-transferase activity. It has been reported that LTC4S, like other known glutathione S-transferases, is enzymatically active as a multimer composed of low molecular mass subunits (20). However, unlike other members of the glutathione S-transferase multigene family, LTC4S does not appear to be involved in cellular detoxification but rather appears to be exclusively committed to the biosynthesis of LTC4 (21). Due to the lack of an abundant source and the extreme lability of LTC4S, it is the only known human glutathione S-transferase whose complete sequence has not been elucidated. Nicholson et al. (20) reported the purification to homogeneity and N-terminal sequence of human LTC4S from THP-1 cells. They found LTC4S to be a homodimer consisting of 18-kDa subunits. In this report, we describe the molecular cloning and expression of human LTC4SAt MATERIALS AND METHODS Cell Culture. The human monocytic leukemia cell line THP-1 (American Type Culture Collection TIB 202) and ASFTL-3 cells (from J. Pierce, National Institutes of Health) were cultured in sterile RPMI 1640 medium (supplemented with 0.2% NaHCO3 and 0.03% L-glutamine; Sigma) containing 10% (vol/vol) fetal bovine serum (Bioproducts for Science, Indianapolis). All cultures were grown at 370C in a humidified atmosphere containing 6% C02/94% air in either 175-cm2 culture flasks or spinner flasks (25 rpm). Stocks were maintained by subculturing cells every third or fourth day in Abbreviations: LT, leukotriene; LTC4S, LTC4 synthase; 5-LO, 5-lipoxygenase; FLAP, 5-LO activating protein; RT-PCR, reverse transcriptase-PCR. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U11552). 9745 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 91 (1994) fresh medium at a seed density of 1 x 105 cells per ml (subculturing was performed earlier if the cell density exceeded 1.5 x 106 cells per ml). LTC4S Activity Assay. Unless otherwise indicated, LTC4S activity was measured in 0.1 M potassium phosphate (pH 7.4) (200 ILI, final volume) containing 10mM reduced glutathione, 10 mM magnesium chloride, L-a-phosphatidylcholine [0.2 mg/ml; prepared as described (22)], and 1 juM LTA4 (free acid). Incubation mixtures were maintained at 250C for 30 min, the reactions were terminated, and the LTC4 produced was quantitated by EIA essentially as described by the manufacturer (Cayman Chemicals, Ann Arbor, MI). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad). Partial Purification of Human LTC4S from THP-1 Cells. Cells were harvested after -5 days in culture, washed with and resuspended in phosphate-buffered saline (GIBCO), and lysed by sonication using an Ultrasonics model W-375 ultrasonic processor. Complete cell lysis was accomplished using five 30-sec bursts of sonication with 30-sec cooling periods between each burst. All purification procedures were performed either at 4TC or on ice. The microsomal membrane fraction was isolated from the lysed cells by differential centrifugation; after removal of large cellular debris by centrifugation at 5000 x g, the 100,000 X g microsomal fraction was collected. Membrane-bound LTC4S activity was solubilized with 2% (wt/vol) taurocholate (Sigma) essentially as described (22). The LTC4S-containing taurocholate extract of THP-1 cell microsomal membranes was applied to a HiLoad Q-Sepharose HP 26/10 anion-exchange column (Pharmacia, 2.6 x 10 cm) that had been equilibrated in buffer A (20 mM Tris HCl, pH 7.4/1 mM EDTA/2 mM reduced glutathione/l mM dithiothreitol/0.1% taurocholate) at a flow rate of 2 ml/min. After exhaustive column washing with bufferA (500 ml), protein was eluted with a linear NaCl gradient (0-1.0 M, 500-ml gradient volume) in buffer A. Fractions were assayed for LTC4S activity and those having activity were pooled, concentrated -10-fold using a Centriprep-10 (Amicon), and subsequently, dialyzed into buffer B [20 mM Tris HCl, pH 7.4/1 mM EDTA/2 mM reduced glutathione/1 mM dithiothreitol/0.03% taurocholate (similar to buffer A except containing 0.03% taurocholate)]. The final preparation was enriched '100-fold relative to lysed cells and activity was recovered with a yield of =10%. Antisera and Immunoprecipitation. Antibodies directed against human LTC4S were prepared by immunizing rabbits with bovine thyroglobulin-conjugated synthetic peptide corresponding to amino acids 1-13 of the enzyme (MKDEVALLAAVTL). The IgG fractions of preimmune (control) and peptide antisera were prepared by adsorption to protein A-Sepharose (Pierce). The insoluble protein A-Sepharoseantibody complex was incubated with a partially purified preparation of LTC4S from THP-1 cells at 40C for 12 h with gentle mixing. The soluble fraction was assayed for depletion of LTC4S activity. The insoluble immune complexes were washed with buffer B and assayed for LTC4S bioactivity. Molecular Cloning of Human LTC4S. Total RNA was isolated from THP-1 cells using the acid guanidinium thiocyanate/phenol/chloroform procedure (23), and polyadenylylated RNA was isolated from this preparation using an oligo(dT)-cellulose column (Collaborative Research) as described (24). Reverse transcriptase-PCR (RT-PCR) was carried out using this THP-1 poly(A) RNA. First strand was primed with random primers by the manufacturer's instructions (Invitrogen). Inosine-containing primers (5'-ATGAAIGATGAIGTIGCICTICTIGC-3' and 5'-ACICGGAAIGCIATICGIGC-3') were designed based on the reported (20) partial protein sequence for human LTC4S and were use