Proc. Nati. Acad. Sci. USA Vol. 91, pp. 9745-9749, October 1994 Biochemistry Molecular cloning and expression of human -C4 synthase DEAN J. WELSCH*, DAVID P. CREELY, SCOTT D. HAUSER, KARL J. MATHIS, GWEN G. KRIvi, AND PETER C. ISAKSON Searle Research and Development, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Philip Needleman, June 30, 1994

ABSTRACT Leukotriene-C4 synthase (LTC4S; EC LTC4 is then converted to LTD4 by y-glutamyltranspeptidase 2.5.1.37) catalyzes the committed step in the biosynthesis ofthe and subsequently metabolized to LTE4 by cysteinylglycine peptidoleukotrienes, which are important in the pathogenesis dipeptidase (14). of . Antibodies were generated to a synthetic peptide Collectively, the peptidoleukotrienes (LTC4, LTD4, and based on the partial amino acid sequence previously reported LTE4) have been demonstrated to be involved in inflamma- for human LTC4S [Nicholson, D. W., Ali, A., Vaillancourt, tory and anaphylactic reactions. They were first isolated and J. P., Calaycay, J. R., Mumford, R. A., Zamboni, R. J. & identified as the active mediators of the slow-reacting sub- Ford-Hutchinson, A. W. (1993) Proc. NatI. Acad. Sci. USA 90, stance ofanaphylaxis and are released from the lung tissue of 2015-2019] and specifically bound detergent-solubilized asthmatics upon exposure to specific allergens (15). Addi- LTC4S obtained from THP-1 cells, confirming that the pub- tionally, exogenous application of peptidoleukotrienes re- lished sequence is associated with activity. Inosine- sults in many phenomena characteristic of human bronchial containing oligonucleotides based on the partial protein se- asthma including bronchoconstriction and cellular infiltration quence were used to isolate a 679-bp cDNA for LTC4S from (16, 17). Because of the important role that these mediators THP-1 cells. The cDNA contains an open reading frame that may play in the pathophysiology of asthma, compounds that encodes a 150-amino acid protein (M, = 16,568) that has a inhibit the production (5-LO inhibitors and FLAP antago- calculated pI value of 11.1. The deduced protein sequence is nists) or action (LTD4 antagonists) of these media- composed predominantly of hydrophobic amino acids; hydro- tors are currently being evaluated in clinical trials for ame- pathy analysis predicts three transmembrane domains con- lioration of asthma (18, 19). nected by two hydrophilic loops. Analysis of the deduced LTC4S is a unique membrane-bound enzyme that catalyzes sequence identified two potential protein kinase C phosphor- the committed step in the biosynthesis of all of the peptido- ylation sites and a potential N-linked glycosylation site. The . LTC4S conjugates reduced with the amino acid sequence for human LTC4S is unique and shows no unstable epoxide LTA4 to form LTC4 and, as such, is a homology to other glutathione S-transferases. LTC4S was glutathione S-transferase activity. It has been reported that found to be most similar to 5-lipoxygenase activating protein LTC4S, like other known glutathione S-transferases, is en- (31% identity, 53% similarity), another protein involved in zymatically active as a multimer composed of low molecular leukotriene biosynthesis. Active enzyme was expressed in bac- mass subunits (20). However, unlike other members of the terial, insect, and mammalian cells as shown by the biosyn- glutathione S-transferase multigene family, LTC4S does not thesis of LTC4 in incubation mixtures containing LTA4 and appear to be involved in cellular detoxification but rather reduced glutathione. The cloning and expression of human appears to be exclusively committed to the biosynthesis of LTC4S provide the basis for a better understanding of this key LTC4 (21). Due to the lack of an abundant source and the enzyme in peptidoleukotriene biosynthesis. extreme lability of LTC4S, it is the only known human glutathione S-transferase whose complete sequence has not Leukotrienes (LTs) are potent biological mediators derived been elucidated. Nicholson et al. (20) reported the purifica- from and are formed in response to a variety tion to homogeneity and N-terminal sequence of human ofimmunologic and inflammatory stimuli (1-3). It is now well LTC4S from THP-1 cells. They found LTC4S to be a ho- established that LTs affect leukocyte chemotaxis, pulmonary modimer consisting of 18-kDa subunits. In this report, we smooth muscle contraction, vascular tone and permeability, describe the molecular cloning and expression of human and mucous secretion (4-8). As such, the LTs have been LTC4SAt implicated as potential mediators of immediate hypersensi- tivity and inflammatory conditions (9, 10). MATERIALS AND METHODS Initially, the production of LTs results from the oxygen- ation and subsequent dehydration ofarachidonic acid to yield Cell Culture. The human monocytic leukemia cell line the unstable epoxide intermediate LTA4; both of these en- THP-1 (American Type Culture Collection TIB 202) and zymatic reactions are catalyzed by 5-lipoxygenase (5-LO) ASFTL-3 cells (from J. Pierce, National Institutes of Health) (11). Cellular 5-LO activity requires an additional cofactor, were cultured in sterile RPMI 1640 medium (supplemented 5-LO activating protein (FLAP) (12). It has been suggested with 0.2% NaHCO3 and 0.03% L-glutamine; Sigma) contain- that FLAP activates 5-LO by specifically binding arachidonic ing 10% (vol/vol) fetal bovine serum (Bioproducts for Sci- acid and transferring this substrate to the 5-LO enzyme (13). ence, Indianapolis). All cultures were grown at 370C in a The LTA4 formed by 5-LO-FLAP can then be stereoselec- humidified atmosphere containing 6% C02/94% air in either tively hydrolyzed to biologically active LTB4 by the cytosolic 175-cm2 culture flasks or spinner flasks (25 rpm). Stocks were enzyme LTA4 hydrolase or conjugated with reduced glu- maintained by subculturing cells every third or fourth day in tathione by the membrane-bound enzyme LTC4 synthase (LTC4S; EC 2.5.1.37) to form the peptidoleukotriene LTC4. Abbreviations: LT, leukotriene; LTC4S, LTC4 synthase; 5-LO, 5-lipoxygenase; FLAP, 5-LO activating protein; RT-PCR, reverse transcriptase-PCR. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U11552).

9745 Downloaded by guest on September 27, 2021 9746 Biochemistry: Welsch et al. Proc. Natl. Acad. Sci. USA 91 (1994) fresh medium at a seed density of 1 x 105 cells per ml using AmpliTaq (Perkin-Elmer/Cetus). The =105-bp cDNA (subculturing was performed earlier if the cell density ex- fragment that was amplified was gel-purified and cloned into ceeded 1.5 x 106 cells per ml). the TA vector (Invitrogen). The resultant clones were se- LTC4S Activity Assay. Unless otherwise indicated, LTC4S quenced with the Cyclist DNA sequencing kit (Stratagene). activity was measured in 0.1 M potassium phosphate (pH 7.4) Three oligonucleotides (5'-CGAGATCACCTGCAGG- (200 ILI, final volume) containing 10 mM reduced glutathione, GAGAAGTAGGCTTGCAGCAGGACTCCCAGGAGGGT- 10 mM magnesium chloride, L-a-phosphatidylcholine [0.2 GACAGCA-3', 5'-TGCAGCAGGACTCCCAGGAGGGT- mg/ml; prepared as described (22)], and 1 juM LTA4 (free GACAGCA-3', and 5'-CGAGATCACCTGCAGGGAGAAG- acid). Incubation mixtures were maintained at 250C for 30 TAGGCTTG-3') were designed based on the cDNA sequence min, the reactions were terminated, and the LTC4 produced that was not derived from the primers used in RT-PCR. These was quantitated by EIA essentially as described by the oligonucleotides were used to screen a cDNA library con- manufacturer (Cayman Chemicals, Ann Arbor, MI). Protein structed from THP-1 poly(A) RNA using a cDNA library concentrations were determined using the Bio-Rad protein synthesis kit (Stratagene). Approximately 2 x 10W recombi- assay reagent (Bio-Rad). nant plaques were screened with all three radiolabeled oligo- Partial Purification of Human LTC4S from THP-1 Cells. nucleotide probes. Three positive clones were plaque- Cells were harvested after -5 days in culture, washed with purified. One clone, pMON23959, was completely sequenced and resuspended in phosphate-buffered saline (GIBCO), and with 7-deaza nucleotide analogues (Pharmacia). lysed by sonication using an Ultrasonics model W-375 ultra- Expression of Human LTC4S. The coding region was am- sonic processor. Complete cell lysis was accomplished using plified using oligonucleotide primers that incorporated either five 30-sec bursts of sonication with 30-sec cooling periods EcoRI or BamHI restriction sites onto the ends of the open between each burst. All purification procedures were per- reading frame of pMON23959. The PCR products were formed either at 4TC or on ice. The microsomal membrane purified and digested with the appropriate endonuclease and fraction was isolated from the lysed cells by differential ligated into the M13mpl9 cloning vector. DNA sequencing centrifugation; after removal of large cellular debris by cen- revealed no differences from pMON23959 except for the trifugation at 5000 x g, the 100,000 X g microsomal fraction appropriate restriction sites flanking the open reading frame. was collected. Membrane-bound LTC4S activity was solu- LTC4S in M13mpl9 as BamHI and EcoRI fragments are bilized with 2% (wt/vol) taurocholate (Sigma) essentially as pMON23960 and pMON23961, respectively. described (22). The LTC4S-containing taurocholate extract of The cDNA encoding human LTC4S was subcloned into THP-1 cell microsomal membranes was applied to a HiLoad expression vectors employing RecA, polyhedrin, and IE175 Q-Sepharose HP 26/10 anion-exchange column (Pharmacia, promoters and, subsequently, expressed in MON102, Sf9, 2.6 x 10 cm) that had been equilibrated in buffer A (20 mM and BHK cells for bacterial, insect, and mammalian expres- Tris HCl, pH 7.4/1 mM EDTA/2 mM reduced glutathione/l sion systems, respectively. Complete descriptions of these mM dithiothreitol/0.1% taurocholate) at a flow rate of 2 expression systems have been reported (25-27). The LTC4S ml/min. After exhaustive column washing with buffer A (500 activity relative to total cellular protein was determined for ml), protein was eluted with a linear NaCl gradient (0-1.0 M, mock- and cDNA-transfected cell lysates by quantitation of 500-ml gradient volume) in buffer A. Fractions were assayed the LTC4 produced in incubation mixtures containing LTA4 for LTC4S activity and those having activity were pooled, and reduced glutathione. concentrated -10-fold using a Centriprep-10 (Amicon), and Sequence Analysis. Nucleotide and amino acid homology subsequently, dialyzed into buffer B [20 mM Tris HCl, pH comparisons were carried out in the GenBank, EMBL, and 7.4/1 mM EDTA/2 mM reduced glutathione/1 mM dithio- Swiss-Prot data bases using the FASTA program of the Wis- threitol/0.03% taurocholate (similar to buffer A except con- consin Package (Genetics Computer Group, Madison, WI). taining 0.03% taurocholate)]. The final preparation was en- riched '100-fold relative to lysed cells and activity was recovered with a yield of =10%. RESULTS AND DISCUSSION Antisera and Immunoprecipitation. Antibodies directed Immunoprecipitation of LTC4S Activity. To immunologi- against human LTC4S were prepared by immunizing rabbits cally characterize the association between LTC4S biological with bovine thyroglobulin-conjugated synthetic peptide cor- activity and protein sequence, antisera was raised to a responding to amino acids 1-13 of the enzyme (MKDEVAL- synthetic peptide based on the partial sequence reported for LAAVTL). The IgG fractions of preimmune (control) and human LTC4S. The IgG fraction ofantisera was immobilized peptide antisera were prepared by adsorption to protein by protein A-Sepharose and used to immunoprecipitate A-Sepharose (Pierce). The insoluble protein A-Sepharose- LTC4S activity from a partially purified enzyme preparation antibody complex was incubated with a partially purified prepared from THP-1 cells, a cell line that expresses high preparation of LTC4S from THP-1 cells at 40C for 12 h with levels of LTC4S biological activity (20). Approximately 85% gentle mixing. The soluble fraction was assayed for depletion of the total LTC4 biosynthetic activity was associated with of LTC4S activity. The insoluble immune complexes were the insoluble complex when peptide antisera was used (Fig. washed with buffer B and assayed for LTC4S bioactivity. 1). Contrastingly, almost all of the activity (.95%) remained Molecular Cloning of Human LTC4S. Total RNA was in the soluble supernatant with control preimmune sera. The isolated from THP-1 cells using the acid guanidinium thio- results of this study provide independent immunological cyanate/phenol/chloroform procedure (23), and polyadenyl- evidence that the reported sequence is associated with ylated RNA was isolated from this preparation using an LTC4S activity. oligo(dT)-cellulose column (Collaborative Research) as de- Molecular Cloning and Properties of Human LTC4S. Ino- scribed (24). Reverse transcriptase-PCR (RT-PCR) was car- sine-containing oligonucleotides were designed based on the ried out using this THP-1 poly(A) RNA. First strand was partial amino acid sequence of LTC4S and a DNA fragment primed with random primers by the manufacturer's instruc- of the appropriate size was amplified from THP-1 poly(A) tions (Invitrogen). Inosine-containing primers (5'-ATGAAI- ftNA by using RT-PCR. The primer-independent DNA se- GATGAIGTIGCICTICTIGC-3' and 5'-ACICGGAAIGCI- quence that was amplified encoded amino acids identical to ATICGIGC-3') were designed based on the reported (20) the published protein sequence (20) except for the amino acid partial protein sequence for human LTC4S and were used for at position 21, identified as glycine by protein sequencing but amplification. Cycles ofamplification consisted of940C for 30 found to be tyrosine based on the amplified DNA sequence. sec, 420C for 60 sec, and 720C for 30 sec and were performed A 679-bp fragment of cDNA was obtained from a human Downloaded by guest on September 27, 2021 Biochemistry: Welsch et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9747

120- acids. The calculated pI value of 11.1 reflects the presence of 16 positively and 6 negatively charged residues in the protein. 100- Analysis of the deduced sequence revealed two Ser-Ala-Arg consensus sequences for protein kinase C phosphorylation X, 80 (28) at positions 28-30 and 111-113, suggesting that human LTC4S may be a phosphoregulated enzyme. Additionally, the sequence contains a potential N-linked glycosylation site t 60 (Asn-Cys-Ser at positions 55-57). The sequence also contains cysteine residues at positions 56 and 82; whether these 40- residues are involved in forming enzymatically active LTC4S homodimer has yet to be determined. 20- Homology Between Human LTC4S and FLAP. The pro- posed membrane topology of LTC4S based on hydropathy analysis consists of three transmembrane domains of 25-31 Pre-Immune Peptide residues separated by two hydrophilic loops, with the N and Sera Antisera C termini on opposite sides ofthe membrane (Fig. 3) (29). The orientation of LTC4S in the membrane and the cellular FIG. 1. Immunoprecipitation of hiuman LTC4S from THP- cells. membrane to which the enzyme is localized are not yet A partially purified preparation of LT C4S was incubated with protein known. However, based on the localization of other proteins A-Sepharose that was preadsorbed wiith peptide antisera (preimmune involved in LT biosynthesis to the nuclear membrane (30), it sera control). After incubation, the L.TC4S bioactivity in the soluble is possible that LTC4S is integrated into this membrane as (hatched bars) and insoluble (solid bazrs) fractions was determined by well.

the formation of LTC4 in incubatiorn mixtures containing reduced Comparison of the sequence of LTC4S with the sequences glutathione and the free acid of LTAL4 of other proteins in the Swiss-Prot data base (Version 28) indicated that the amino acid sequence for human LTC4S was THP-1 cDNA library using oligronucleotides based on the unique and that it showed no similarity to other glutathione primer-independent RT-PCR anfnplified sequence. The nu- S-transferases. Interestingly, LTC4S is most similar to FLAP cleotide sequence contains an olpen reading frame that en- at the amino acid sequence level (31% identity, 53% similar- codes a protein of 150 amino acids having a predicted ity). LTC4S and FLAP contain 2 cysteine residues in their molecular weight of 16,568 (Fig. 2). This molecular weight 150- and 161-amino acid primary sequences, respectively, compares favorably with that det "ermined by SDS/PAGE for and the alignment between these proteins does not require the purified protein (20). The decduced amino acid sequence the introduction of any gaps or overlaps. This high degree of contains 94 hydrophobic and 34 polar but uncharged amino similarity is also reflected by initl = 236, initn = 236, and

1 M 1 GAATTCGGCACGAGGAGCAGCAGACGGGGCTAAGCGTTCCCCAGCTCGCCTTCACACACAGCCCGTGCCACCACACCGACGGTACCATG

2 K D E V A L L A A V T LL G V L L Q A Y F S L Q V I S A RI R 90 AAGGACGAGGTAGCTCTACTGGCTGCTGTCACCCTCCTGGGAGTCCTGCTGCAAGCCTACTTCTCCCTGCAGGTGATCTCGGCGCGCAGG

32 A F R V S P PL T T G P P E F E R V Y R A Q V| N C) S] E Y F P 180 GCCTTCCGCGTGTCGCCGCCGCTCACCACCGGCCCACCCGAGTTCGAGCGCGTCTACCGAGCCCAGGTGAACTGCAGCGAGTACTTCCCG

62 L F L A T L W V A G I F F H E G AA A L (J) G L V Y L F A R L

270 CTlGTTlClC lLt.GCCACGCTCTlIGTlCGLCCGGCA TCL''1''lATAAGGCG CCCTG l' WL;G'1'C bLGUCALLC''('lt,,(CC'T

92 R YF Q G Y A R S A Q L R L A P L Y A S A RI A L W L L V A L 360 CGCTACTTCCAGGGCTACGCGCGCTCCGCGCAGCTCAGGCTGGCACCGCTGTACGCGAGCGCGCGCGCCCTCTGGCTGCTGGTGGCGCTG

122 A A L G L L A H F LP A A L R A A L L G R L R T LL P W A * 450 GCTGCGCTCGGCCTGCTCGCCCACTTCCTCCCGGCCGCGCTGCGCGCCGCGCTCCTCGGACGGCTCCGGACGCTGCTGCCGTGGGCCTGA

540 GACCAAGGCCCCCGGGCCGACGGAGCCGGGAAAGAAGAGCCGGAGCCTCCAGCTGCCCCGGGGAGGGGCGCTCGCTTCCGCATCCTAGTC

630 TCTATCATTAAAGTTCTAGTGACCGAGAAAAAAAAAAAAAAAAAAAAAAA FIG. 2. Nucleotide and deduced amino acid sequence of cDNA clone for human LTC4S. Amino acid residues are numbered beginning with the initiator methionine. Predicted transmembrane segments are underlined. The protein kinase C consensus sequences (SAR) are boxed, as is the NCS sequence, which may serve as a site for N-linked glycosylation. Cysteine residues are circled. Downloaded by guest on September 27, 2021 9748 Biochemistry: Welsch et A Proc. Natl. Acad. Sci. USA 91 (1994)

FIG. 3. Proposed membrane topology of human LTC4S based on hydropathy analysis is indicated. Horizontal lines represent membrane boundaries. The single-letter amino acid code is used. Shaded residues indicate amino acids that demonstrate identity with human FLAP. optimization = 261 scores determined as reported (31). The FLAP in this region and maintains a negative charge at the model proposed for the structure of LTC4S can be closely position equivalent to Asp62 of FLAP (Glu58 of LTC4S). The aligned with that reported for FLAP (32). Most of the FLAP antagonist MK-886 (35) was tested for its ability to similarity between LTC4S and FLAP is localized to their inhibit LTC4 production from THP-1 cells incubated with N-terminal regions; LTC4S and FLAP are 41 and 10%o LTA4 and was found to dose-dependently inhibit LTC4 identical over the N-terminal two-thirds (41 of 100 residues) biosynthesis with an IC50 value of =10 ,uM (Fig. 4). While and C-terminal one-third (5 of50 residues) of LTC4S, respec- MK-886 in this LTC4S assay is about three orders of mag- tively (Fig. 3). nitude less potent than when assayed for FLAP inhibitory Immunoprecipitation, site-directed mutagenesis, and pho- activity (Fig. 4), this result may indicate a functional dem- toaffinity labeling studies have identified the C-terminal half onstration of the homology between these two proteins. of the first hydrophilic loop of FLAP to be important in the Expression of Human LTC4S. The cDNA encoding human binding of LT biosynthesis inhibitors (32-34). Additionally, LTC4S was subcloned into appropriate vectors and subse- site-directed mutagenesis studies have demonstrated the quently expressed in bacterial, insect, and mammalian cell importance of a negative charge at amino acid 62 (Asp62) of systems. The LTC4S activity relative to total cellular protein FLAP for inhibitor binding (32). LTC4S is =60% identical to was determined for mock- and cDNA-transfected cell lysates by quantitation ofthe LTC4 produced in incubation mixtures 120 containing LTA4 and reduced glutathione (Fig. 5). It is apparent from these data that enzymatically active LTC4S 100 can be generated in mammalian and nonmammalian cells and in prokaryotes. Relative expression ofactive enzyme appears 0 80 best in baculovirus but absolute expression levels of protein have not been determined; however, preliminary examina- U 60 \ tion by Western blot analysis suggests that expression of LTC4S in bacteria is poor. Nevertheless, expression of LTC4S in baculovirus-infected cells should provide sufficient 40 \ material for further structural and functional analyses. In conclusion, we report the molecular cloning of LTC4S 20 from the human monocytic cell line THP-1 and expression of active enzyme in bacterial, insect, and mammalian cells. LTC4S appears to be a unique glutathione S-transferase that 0.01 0.1 1 10 100 is strikingly similar to FLAP, another protein involved in MK-886 (PM) of arachidonic acid. Elucidation of the sequence FIG. 4. Inhibition of LTC4 biosynthesis by the FLAP antagonist of LTC4S and generation of immunological reagents specific MK-886. The amount of LTC4 generated in response to calcium for this enzyme should provide useful tools for the delinea- ionophore (A23187 0.5 pg/ml) stimulation of ASFTL-3 cells (2.5 x tion of its expression and function. 104 cells per ml) (e) and LTA4 (1 AM) stimulation ofTHP-1 cells (2.5 x 1iO cells per ml) (0) was measured as a function of MK-886 We thank Leslie Askonas, Rory Finn, Pegg, Shaukat Rang- concentration in the incubation mixture. Control LTC4 production wala, Christina Schmidt, Mark Walker, and Mark Zupec for their was defined as that synthesized in the absence of inhibitor. contributions to this work. 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