Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, FL, USA. (Received 14 February 1996) - EJB 96 020712.
Eur. J. Biochem. 238, 606-612 (1996) 0 FEBS 1996
Molecular cloning, expression and characterization of mouse leukotriene C, synthase Bing K. LAM’, John F. PENROSE’, Joshua ROKACH’, Kongyi XU’, Mathew H. BALDASARO’ and K. Frank AUSTEN’ ’ Department of Medicine, Harvard Medical School, and Division of Rheumatology and Immunology, Brigham and Women’s Hospital, Boston MA, USA ’ Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, FL, USA (Received 14 February 1996) - EJB 96 020712
Leukotriene C, synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A, to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C, synthase shares substantial amino acid identity in its consensus N-terminal two-thirds with 5-lipoxygenase-activating protein and has a region (residues 37- 58) that exhibits 46 % amino acid identity with a domain of this protein (residues 41-62) to which an inhibitor binds. We have now cloned mouse leukotriene C, synthase cDNA using the polymerase chain reaction to screen a mouse pcDNA3 expression library with oligonucleotide primers based on the translated human leukotriene C, synthase cDNA sequence. Mouse leukotriene C, synthase cDNA is 667 bp in length, including the poly(A)-rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150amino-acid sequence of mouse leukotriene C, synthase differs from the human enzyme by only 1 8 amino acids, of which 9 reside at the C terminus. The potential N-glycosylation site, two protein kinase C phosphorylation sites, the two cysteine residues, and the putative inhibitor-biding domain (substitutions Thr414Ser and TyrSO-Phe) were conserved in mouse leukotriene C, synthase. Northern blot analysis indicated that the leukotriene C, synthase RNA transcript is widely distributed. The K,, values for leukotriene A, methyl ester, leukotriene A, free acid and GSH were 7.6 pM, 3.6 pM and 1.6 mM, respectively, for purified human recombinant enzyme, and 10.3 pM, 2.5 pM and 1.9 mM, respectively, for purified recombinant mouse enzyme; the corresponding V,,;,, values were 2.5, 1.3 and 2.7 pmol . min-’ . mg-’ protein, respectively, for human enzyme, and 2.3, 1.2 and 2.2 pmol . min . m g ’ protein, respectively, for mouse enzyme. The 5-lipoxygenase-activating-protein inhibitor, MK-886, was active against both human and mouse recombinant leukotriene C, synthase with IC,,, values of 3.1 pM and 2.7 pM, respectively. These findings confirm that the leukotriene C, synthases belong to a gene family that includes the 5-lipoxygenase-activating protein and suggest that the C-terminal domain of leukotriene C, synthase may not be critical for its conjugation function.
Keywords: cloning; enzyme; kinetics; leukotriene.
Leukotriene C, is the biosynthetic parent of the receptoractive cysteinyl leukotrienes, leukotriene D, and leukotriene E,, which induce bronchial smooth muscle contraction in many species including humans [ I , 21. The cysteinyl leukotrienes also stimulate mucus secretion from bronchial epithelial cells in vitro and increase pulmonary vascular permeability through stimulation of endothelial cell contraction at the post-capillary venules in vivo [3, 41. The pathobiological role of the cysteinyl leukotrienes in bronchial asthma in general and in asthma caused by idiosyncratic activation of the 5-lipoxygenase pathway by nonsteroidal anti-inflammatory agents is established by the measured benefit observed with inhibitors of the biosynthetic pathway or with receptor antagonists [ S , 61. The formation of leukotriene C, is initiated by cell activation with release of arachidonic acid from membrane phospholipid Correspondence to B. K. Lam, Seeley G. Mudd Building, Room 63 0, 250 Longwood Avenue, Boston, MA 021 15. USA AbbrcJviutions.GSH, glutathione; SB, Super Broth medium. Etzzytnes. Leukotriene C, synthase (EC 2.5.1.37); 5-lipoxygenase (EC 1.13.11.12). Note. The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned the accession number U27195.
by the action of cytosolic phospholipase A, [7]. The arachidonic acid is translocated to an integral perinuclear membrane protein, the 5-lipoxygenase-activating protein, and is presented to Slipoxygenase that has also been translocated to the perinuclear membrane from the cytosol or nucleoplasm [S-IO]. 5-Lipoxygenase metabolizes arachidonic acid in two sequential steps to form 5-hydroperoxy-eicosatetraenoic acid and then the epoxide intermediate leukotriene A, [I I]. The integral perinuclear membrane leukotriene C, synthase conjugates leukotriene A, with reduced glutathione (GSH) to form the intracellular product, leukotriene C, [ 12, 131. After carrier-mediated cellular transport of leukotriene C, [14, 151, sequential cleavage of Glu and Gly provides the extracellular, receptor-active derivatives, leukotriene D, and leukotriene E, [16, 171, respectively. The recent molecular cloning of the cDNA for human leukotriene C, synthase from human leukemic cell libraries revealed that the nucleotide and deduced amino acid sequences had no significant similarity to the GSH S-transferases, either cytosolic or microsomal [18, 191. Instead there was 31 % amino acid sequence identity with human 5-lipoxygenase-activating protein, which increased to 4 4 % for the N-terminal two-thirds of these proteins. The oligonucleotides encoding these regions, amino
Lain et al. ( E m J. Biochem. 238)
acids 5-99 for leukotriene C, synthase and 9-103 for 5-lipoxygenase-activating protein, were 52% identical. Furthermore, these proteins had a related functional domain in that the inhibitors of the arachidonic-acid-binding domain of 5-lipoxygenaseactivating protein, termed 5-lipoxygenase-activating protein inhibitors, suppressed the conjugation by leukotriene C, synthase of leukotriene A, with reduced GSH. There was also an apparent structural similarity in that the predicted amino acid sequence for each protein provided three hydrophobic putative transmembrane domains with intervening hydrophilic loops of essentially the same sizes. The 5-lipoxygenase-activating protein was initially recognized because a group of agents that blocked stimulus-initiated 5-lipoxygenase function in intact cells had no inhibitory action in subcellular preparations in which arachidonic acid was directly available to the 5-lipoxygenase [20]. After chromatographic isolation of the 5-lipoxygenase-activating protein and cloning of an encoding cDNA, the putative inhibitor-binding domain was localized by three approaches. The binding of radiolabelled photoaffinity analogs of the inhibitors to fragments derived by chemical cleavage of internal Met or Trp residues and analysis by immunoblot with antiserum to defined peptide sequences identified the first hydrophilic loop between the putative first and second transmembrane domains [21]. Comparison of the consensus amino acid sequences for human S-lipoxygenaseactivating protein and that from other species revealed significant amino acid substitutions in the N-terminal half of this hydrophilic loop, prompting attention to the C-terminal half. Deletion mutants in the C-terminal but not the N-terminal portions of this hydrophilic loop impaired the binding of radiolabelled inhibitors, thereby further localizing the critical binding domain to the C-terminal half of the first hydrophilic loop of 5-lipoxygenase-activating protein [21]. The putative inhibitor-binding domain of leukotriene C, synthase is predicted by strong similarity with a stretch in which 8 of 11 amino acid residues are identical in the C-terminal region of the first hydrophilic loop to those in 5-lipoxygenase-activating protein [18, 221. Because the 5-lipoxygenase-activating-protein inhibitor, MK-886, is active against the recombinant human leukotriene C, synthase, the inhibitor-binding domain of this integral perinuclear membrane protein, unlike 5-lipoxygenase activating protein, must serve a single protein by providing a binding function that supports a conjugation/catalytic function. To determine if the inhibitor-binding domain in human leukotriene C, synthase is conserved by similar sequence and function, we obtained a cDNA for this enzyme in a second species. In the present study we used PCR to screen and clone mouse leukotriene C, synthase cDNA from a mast cell expression library. The deduced amino acid sequence displayed 88% similarity to the human enzyme. The putative inhibitor-binding domain was retained with two substitutions, whereas the 18 amino acids of the C terminus of mouse leukotriene C, synthase displayed only 50% amino acid identity to the human enzyme. The purified recombinant mouse leukotriene C, synthase exhibited nearly identical kinetics and susceptibility to the 5-lipoxygenaseactivating-protein inhibitor, MK-886, when compared with purified recombinant human enzyme. This finding supports the hypothesis that the sequence of leukotriene C, synthase which is similar to the 5-lipoxygenase-activating-protein inhibitor-binding domain is critical for the conjugation function of the enzyme.
MATERIALS AND METHODS Materials. Recombinant mouse c-kit ligand was obtained from supernatants of cultured COS-7 cells (American Type Cul-
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ture Collection) transfected with plasmid (pcDNA1) containing a cDNA encoding the soluble form of c-kit ligand (J. Flanagan, Harvard Medical School, Boston MA) [23]. Nu-serum Plus (Collaborative Research, New Bedford MA); MK-886 (A. W. Ford-Hutchinson, Quebec) ; Tuq polymerase and dNTP (Pharmacia) ; dimethylsulfoxide (Me,SO), chloroquine, DEAEdextran (molecular mass > 500 kDa), BSA, GSH, fetal calf serum (Sigma) and acetonitrile and methanol (Burdick & Jackson, Muskegan MI) were obtained as noted. Leukotriene A, methyl ester was synthesized as described [24]. Cell cultures. Bone-marrow-derived mast cells were obtained by culture of bone marrow from male BALB/cJ mice (Jackson Laboratory, Bar Harbor ME) for 3-6 weeks in 50% enriched medium (RPMI-1640 medium containing 10% fetal calf serum, 100 pg/ml streptomycin, 100 units/ml penicillin, 10 yg/ml gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acid, and 50 pM 2-mercaptoethanol), 100 ng/ml c-kit ligand, and 50% WEHI-3 cell-conditioned medium under a humidified atmosphere of 5 % CO, and 95 % air at 37 "C. COS-7 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and 15 pg/ml gentamicin under identical culture conditions. Construction of a pcDNA3 library. mRNA was prepared from 3X108 mast cells derived from mouse bone marrow using a FastTrack mRNA (InVitrogen) isolation kit according to the manufacturer's instruction. A pcDNA3 library was constructed from the mRNA by InVitrogen in the usual sequence. The firststrand cDNA was synthesized from mRNA using NotT priming (InVitrogen). cDNAs were sized by agarose gel electrophoresis, and those > 500 bp were ligated with an EcoRIIBstXI adaptor and cloned into pcDNA3 mammalian expression vector. The pcDNA3 library was then used to transform Escherichiu coli strain Top 10F' with a complexity of 1.54X10' original clones. Screening of a pcDNA3 library. A mast cell pcDNA3 mammalian expression library derived from mouse bone marrow was screened by PCR with three oligonucleotides designed from the nucleotide sequence of human leukotriene C, synthase cDNA; primer A corresponded to nucleotides 55-82, primer B to nucleotides 181 -207, and primer C (antisense oligonucleotide) to nucleotides 325-305. 1.2X105 clones were divided into 24 pools of 5000 colonies each; the pools were each cultured in 100 y1 Super Broth medium (SB; 32 g tryptone, 20 g yeast extract, 5 g NaCl, and 2.5 ml of 2 M NaOH in 1 I water) in a 96-well microtiter plate overnight at 37°C. Then, 10-pl samples from each well were pelleted by microcentrifugation at 12000 rpm, and the pellets were resuspended in 20 y1 water and boiled for 5 min. The boiled samples were centrifuged and a 10-yl sample of each supernatant was removed. These samples were used as templates for PCR with primers B and C. Positive pools, defined as those providing a 145-bp PCR product, were subdivided into smaller pools of 200 colonies each which were cultured in 100 p1 SB at 37°C for 1.5 h in a 96-well microtiter plate. Samples (50 pl) were taken from each pool for use as templates for PCR as described above with the same set of primers. Once the positive pools were identified by the provision of a 145-bp product, the remaining 50-y1 samples of the 200-colony pools, which had been held at 4"C, were resuspended in a final volume of 1 ml and plated into 20 SB agar plates. After overnight growth, a replicate plate was made by overlaying nitrocellulose membranes and then transferring each of the membranes to empty plates. Each membrane was submerged in 10 ml SB for 10 min with intermittent agitation by pipeting SB to elute the bacterial colonies. Bacterial colonies eluted from the nitrocellulose membranes were boiled in 200 y1 water and 10-y1 samples of each were used as PCR templates with primers B and C
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to define positive plates by generation of the 145-bp product. The original plates were cultured at 37°C for an additional 6 h to replenish the bacterial colonies removed by the nitrocellulose membranes. Those for which the replicate membrane-derived colonies were positive were subjected to a second PCR with primers A and C ; confirmation was defined by the generation of a 270-bp PCR product. Individual colonies from the plates that were positive for both sets of primers were then picked and grown in SB at 37°C. After overnight culture, plasmids were prepared from each colony and were used as templates for both sets of primers. Plasmids from colonies generating both 145-bp and 270-bp products with primers B and C and with primers A and C, respectively, were then used to transfect COS-7 cells for the expression of leukotriene C, synthase. PCR were performed with a Perkin Elmer GeneAmp PCR system 9600. The samples were denatured at 94°C for 30 s, annealed at 60°C for 30 s, and extended at 72°C for 1 min for 35 cycles. Northern blot analysis. Mouse multiple tissue Northern blots (Clontech) were used to determine the tissue distribution of leukotriene C, synthase for which probes were generated by random priming of its full-length cDNA using [cr-"PIdCTP. Heat-denatured probes were hybridized to the multiple tissue Northern blots under high stringency conditions. Blots were then developed by autoradiography using Kodak XAR film. Transfection and analysis of the expressed proteins. COS7 cells were transfected with either human or cloned mouse leukotriene C, synthase cDNA by DEAE-dextran transfection [18]. Three days after transfection, the cells were harvested by treatment with trypsin and suspended in microsomal buffer (SO mM Hepes pH 7.9 containing 2 mM EDTA and 10 mM 2-mercaptoethanol) at 5x10' cells/ml. The cells were lysed by sonication with a Branson microtip sonicator at a setting of 7. Cell lysates were centrifuged at 100000Xg for 60 min at 4"C, and the pellets (microsomes) were solubilized by the addition of 0.4% Triton X-102, 0.4% sodium deoxycholate, and 1 0 % glycerol, and stirring at 4°C for 45 min. Recombinant leukotriene C, synthases were purified by Shexyl GSH agarose affinity chromatography. Solubilized microsomes were loaded onto an open bed (2.5X1.5 cm) of S-hexyl GSH resin equilibrated with buffer A (SO mM Hepes, 1 mM EDTA, 5 mM 2-mercaptoethanol, 0.1 % Triton X-102, 1 0 % glycerol, pH 7.6) at 4°C. The column was washed sequentially with 5 vol. each of buffer A/0.3 M NaC1/20 mM GSH/O.I % sodium deoxycholate, buffer A/2.5 mM S-hexyl GSH/2.5 mM Soctyl CSH/O.I % sodium deoxycholate, and buffer A/O. 1 % sodium deoxycholate; and the enzyme was eluted with five fractions of 1 vol. each of 15 mM probenecid in buffer A containing 0.1 % sodium deoxycholate. The enzyme was purified as determined by the presence of a single protein band in silver-stained SDS/PAGE gels; the average yield was 14.8 2 2 . 6 % (mean 2 SD, n = 4). After sequential concentration and dilution of the enzyme with a Microsep 10 K centrifugal concentrator (Filtron Technology Corp., Northborough MA) to remove the probenecid, the protein concentration was estimated by Coomassie staining of a SDSiPAGE gel of the purified enzyme as compared to a standard amount of BSA resolved i n the same gel. The purified enzymes were then stored at -20°C i n 50% glycerol for 2448 h. To examine the enzyme kinetics of recombinant leukotriene C, synthase, 75 - 100-ng portions of purified enzyme were incubated i n 200 p1 SO mM Hepeh pH 7.6, containing 10 mM MgC1, with either 20 pM leukotriene A, methyl ester and various concentrations of GSH or with 10 mM CSH and various concentrations ofleukotriene A , methyl ester for 2 min at 24°C. The reactions were terminated by the addition of 2 vol. methanol contain-
ing 200 ng prostaglandin B,. Samples were then analyzed for leukotriene C, methyl ester by reverse-phase HPLC. Effect of the 5-lipoxygenase-activating-proteininhibitor MK-886 on leukotriene C, activity. To examine the effect of a 5-lipoxygenase-activating-proteininhibitor on human and mouse leukotriene C, synthase, 2 - ~ 1portions of COS cell lysate containing either human or mouse recombinant leukotriene C, synthase were each incubated with 10 pM leukotriene A, methyl ester and 10 mM GSH in the presence of various concentrations of MK-886 for 10 min. Reactions were terminated and analyzed as above. Reverse-phase HPLC. HPLC was performed with a model 126 dual pump system and model 167 scanning ultraviolet detector (Beckman Instruments) controlled by an IBM PS2/50 computer using Beckman System Gold software. Samples were applied to a 5-pm C18 Ultrasphere reverse-phase column (4.6X250 mm; Beckman Instruments) equilibrated with a solvent of methanol/acetonitrile/water/acetic acid (1 0: 15 : 100: 0.2, by vol.), pH 6.0 (solvent A). After injection of the sample, the column was eluted at a flow rate of 1 ml/min with a programmed concave gradient (System Gold curve 6) to 1 5 % solvent A and 85% pure methanol (solvent B) over 0.2 min. Beginning at 3 min, solvent B was increased linearly to 100% over 2 min and was maintained at this level for 10 niin more. The absorbance at 280 nm and the ultraviolet spectra were recorded simultaneously. The retention times for prostaglandin B2 and leukotriene C, methyl ester were 7.1 min and 7.9 min, respectively. Leukotriene C, methyl ester was quantitated by calculating the ratio of peak area to the area of the internal standard prostaglandin B2. DNA sequencing. Plasmids were prepared with a Nucleobond isolation kit (Nest, Southboro MA) and were sequenced as described by Sanger et al. [25] using dye-labelled dideoxynucleotides as terminators. Samples were analyzed on an Applied Biosystems model 373A automated DNA sequencer 1261.
RESULTS Cloning of the cDNA for mouse leukotriene C, synthase. Plasmids from the 24 pools (5000 colonies each) of the mouse bone-marrow-derived mast cell pcDNA3 library were used as templates for PCR with primers B and C. Plasmids from two pools of bacterial colonies gave a 145-bp PCR product as would be expected for a positive result with these primers. One pool was carried forward for further rounds of screening by subdividing it into smaller pools until individual colonies were identified that provided the 145-bp product with primers B and C. Individual colonies were rescreened with primers A and C, with positive colonies defined by a 270-bp PCR product. Those colonies providing the 145-bp product with primers B and C and the 270-bp product with primers A and C were used to transfect COS cells for the expression of leukotriene C, synthase. Five individual colonies gave the two appropriate PCR products by size, but only two of these were truly positive as determined by the ability of their plasmids to express leukotriene C, synthase when transfected into the COS-7 cells. Double-strand sequencing was then performed on a single clone (AY-Hll-P12-C9) with cycle sequencing 1261. Nucleotide and deduced amino acid sequence of mouse leukotriene C, synthase cDNA. Clone A9-HI 1-P12-C9 contained a 667-bp cDNA insert with an open reading frame of 4.50 bp terminated by a TGA rtop codon (Fig. 1). The 5' untranslated region ir 36 bp long. The 3' 171-bp untranslated region includes a 69-bp poly(A)-rich tail and an ATTAAA polyadenylylation
609
Lain et al. ( E U LJ . Biochem. 238) Mouse Human
ooo 1
AGAACACCRAAGCTAATTCTGCCTGTGGCTGGCAAC ATG AAG GAC GAA
48
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66
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49 GTG GCT CTT CTG GCT ACC GTC ACC CTC GTG GGA GTT CTG TTG CAA GCC TAC TTC 102
Human
67 GTA GCT CTA CTG GCT GCT GTC ACC CTC CTG GGA GTC CTG CTG CAA GCC TAC TTC 120
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Mouse 103 TCC CTA CAG GTG ATC TCT GCA CGA AGG GCT TTC CAC GTG TCG CCG CCG CTC ACC 156 Ill II I l l I l l I l l I1 I 1 I I Ill II I l l I I Ill I l l I l l I l l I l l Ill Human 121 TCC CTG CAG GTG ATC TCG GCG CGC AGG GCC TTC CGC GTG TCG CCG CCG CTC ACC 174 Mouse
157 TCT GGC CCT CCC GAG TTC GAG CGC GTC TTC CGA GCC CAG GTA AAC TGC AGC GAG 210 I i l l I I Ill i l l Ill Ill 1 1 1 I l l 1 1 Ill I I I Ill I I I l l I l l I l l Ill
Human 175 ACC GGC CCA CCC GAG TTC GAG CGC GTC TAC CGA GCC CAG GTG AAC TGC AGC GAG 228 B *
Mouse 211 TAC TTT CCG CTG TTC CTC GCC ACA CTC TGG GTC GCC GGC ATC TTC TTC CAC GAA 264 I l l It I l l I l l Ill I l l I l l II I l l I l l I l l Ill Ill Ill Ill II II Ill Human 229 TAC TTC CCG CTG TTC CTC GCC ACG CTC TGG GTC GCC GGC ATC TTC TTT CAT GAA 282 Mouse 265 GGA GCC GCA GCC CTG TGC GGA CTG TTC TAC CTG TTC GCG CGC CTC CGC TAT TTC 318 II II II Ill Ill Ill II Ill II I l l I l l Ill I l l I l l I l l I l l I I I l l Human 283 GGG GCG GCG GCC CTG TGC GGC CTG GTC TAC CTG TTC GCG CGC CTC CGC TAC TTC 336
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319 CAG GGA TAC GCG CGC TCA GCG CAA CTC AGG CTG ACT CCC CTA TAC GCG AGC GCG 372 I l l II Ill Ill Ill II Ill II I l l I l l I l l I I1 I 1 I t 1 I l l I l l I l l Human 337 CAG GGC TAC GCG CGC TCC GCG CAG CTC AGG CTG GCA CCG CTG TAC GCG AGC GCG 390
Mouse
373 CGC GCA CTC TGG CTG CTG GTG GCG ATG GCT GCA CTG GGC TTG CTA GTC CAC TTC 426 Ill II Ill Ill Ill Ill Ill Ill II Ill II II I l l II II I I Ill Ill
Human 391 CGC GCC CTC TGG CTG CTG GTG GCG CTG GCT GCG CTC GGC CTG CTC GCC CAC TTC 444 Mouse 427 CTC CCC GGC ACG CTA CGG ACT GCG CTC TTC AGA TGG CTC CAG ATG CTC CTG CCG 480 I l l I1 I I I I I I I1 I I l l I l l II II II I l l I I I I I 1 Ill Ill Human 445 CTC CCG GCC GCG CTG CGC GCC GCG CTC CTC GGA CGG CTC CGG ACG CTG CTG CCG 498 I..
Mouse
481 ATG GCC TGAGGCGAAGGCCCTCCGAATCAGCAGAACTGGAGATCTTCGAAC~CGCTGGGGTGCCCG 549 I I I I I IIII I Ill IIII I IIIIIII I I I I Ill I Human 499 TGG GCC TGAGACCAAGGCCCCCGGGCCGACGGAGCCGGGRAAGAAGAGCCGGAGCCTCCAGCTGCCCCG 567
Mouse 550 CCCCCGAATCCCAGTTTTTAATTAAAGTTCCCACGACGG (POlY A+) I I II 7 I l l Human 568 GGGAG~GGCGCTCGCTTCCGCATccTAGTcTcTATCATTRAAGTTcTAGTGAccG (poiy A+
588 622
Fig. 1. Comparison of the nucleotide sequences of mouse and human leukotriene C, synthase. The two cDNAs are presented by aligning the corresponding open reading frames. Numbering begins with the first base of the cDNA and proceeds to the poly(A)-rich tails (poly A ' ) . Identical nucleotides are depicted by vertical lines. The circles above the nucleotides indicate the ATG translation initiation codon. The asterisks indicate the stop codon. The polyadenylylation sites are double underlined. Oligonucleotides that were used for PCR screening of the mouse pcDNA3 library are underlined, with the arrow representing the orientation of the nucleotides.
HUMAN R A Q V N C S E I Y F P L F L A T L W V A G I F F H E G A A A L C G L V Y L F A I R L R Y F Q G Y A R S
HUMAN A Q L R L A P L Y A S A R A L W L L V A L A A L G L L A H F L P A A MOUSE - - - - - T - - - - - - - - - - - - - - M - - - - - - v -
signal (Fig. 1). The insert encodes a protein of 150 amino acids with a calculated molecular mass of 16800 Da and a PI of 10.8. The deduced amino acid sequence of mouse leukotriene C, synthase contains two protein kinase C phosphorylation sites at residues 28 and 111 and a potential N-linked glycosylation site at residue 55 (Fig. 2). Hydrophobic plot analysis of the secondary structure predicts three membrane-spanning regions with two hydrophilic loops (Fig. 2).
Tissue distribution of leukotriene C, synthase. Multiple tissue Northern blot analysis of mouse leukotriene C , synthase message identified a band of approximately 700 bp in length (Fig. 3). Leukotriene C, synthase transcript was prominent in lung and kidney and, to a lesser extent, in skeletal muscle, heart, and brain. The message was slightly detectable in spleen but
RAALLGRLRTLLPWA - - T - -G -T F R W - Q M - - - M -
not detectable in liver. I n human multiple tissue Northern blots, leukotriene C, synthase was detected in heart, placenta, lung, skeletal muscle, kidney and pancreas and, to a lesser extent, in brain and liver (data not shown).
Enzyme kinetics of purified human and mouse recombinant leukotriene C, synthase. To determine the K,,, for leukotriene A, methyl ester, 75 - 100-ng portions of the purified mouse and human recombinant enzymes were incubated with 10 mM GSH and 0-40 pM leukotriene A, methyl ester for 2 min. There was a substrate-dependent increase in formation of the product (leukotriene C , methyl ester) that plateaued at about 15 pM with either human or mouse recombinant leukotriene C, synthase (Fig. 4). Using a double-reciprocal plot of l l u vs l/[S] (data not shown), the K,, and V,,,,,,values for human recombinant enzyme
Lam et al. (Eur J. Biochern. 238)
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Fig.3. Northern blot analysis of the tissue distribution of mouse leukotriene C, synthase. Each lane contains approximately 2 Fg poly(A)rich RNA from mouse tissues as indicated. (A) The blot was probed with 32P-labelledmouse leukotriene C, synthase cDNA under high stringency conditions and exposed to X-ray film at - 80°C for 24 h. The size standard of the band is indicated on the left. (B) The same blot was stripped and probed with "P-labelled /!-actin cDNA under identical conditions and was exposed to X-ray film at -80°C for 1 h.
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Fig. 6. Enzyme kinetics of purified recombinant human (0)and mouse (0) leukotriene C, synthase with leukotriene A, free acid as substrate. LT, leukotriene.
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Fig. 7. Effect of the 5-lipoxygenase-activating-protein inhibitor MK886 on mouse (0) and human (m) recombinant leukotriene C, synthase activity with the transfected COS-7 cell lysates used as enzyme sources. Representative data from one of three experiments. LTC4-ME. leukotriene C, methyl ester.
0"
's
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Fig. 4. Enzyme kinetics of purified recombinant human (0)and mouse (0) leukotriene C, synthase with leukotriene A, methyl ester as substrate. Representative data from one of two experiments. LTC,ME, leukotriene C, methyl ester
Mouse 10
a double-reciprocal plot of l / u vs l/[S] (data not shown), the K,, and V,,,,i, values for GSH were determined to be 1.6 mM and 2.7 pmol . min-' . mg-' protein, respectively, for human recombinant enzyme and 1.9 mM and 2.2 pmol . min ' . r n g ' protein, respectively, for mouse recombinant enzyme (mean, n = 2 ) . In a separate experiment in which leukotriene A, methyl ester was replaced by the unstable native substrate, leukotriene A, free acid, each assay was performed for 1 min at 24°C. Again, there was a substrate-dependent increase in product formation (Fig. 6). The K,,, and V,,,,, values were calculated to be 3.6 pM and 1.3 pmol . mingl . mgg' protein, respectively, for the human recombinant enzyme and 2.5 pM and 1.2 pmol . min- ' . mg -' protein, respectively, for the mouse recombinant enzyme (single experiment; data not shown).
20
GSH ( m M ) Fig. 5. Enzyme kinetics of purified recombinant human (0)and mouse (0) leukotriene C, synthase with GSH as substrate. Representative data from one of two experiments. LTC,-ME, leukotriene C, methyl ester.
were calculated to be 7.6 pM and 2.5 pmol . min ' . mg ' protein, respectively, and for mouse recombinant enzyme were 10.3 pM and 2.3 pmol . min-l . mgg' protein, respectively (mean, ~r'= 2). To determine the K,,, and V,,,,, values for GSH, 75- 100-ng portions of purified enzymes were incubated with 20 pM leukotriene A, methyl ester and 0- 20 mM GSH for 2 min. There was a substrate-dependent increase in product formation that peaked at 10 mM with either enzyme (Fig. 5). Using
Effect of a 5-lipoxygenase-activating-proteininhibitor on human and mouse leukotriene C, synthase. Transfected COS cell lysates were incubated with 1 0 pM leukotriene A, methyl ester and 10 mM GSH at 24OC for 10 min in the presence of various concentrations of MK-886. As shown in a typical experiment (Fig. 7), MK-886 inhibited both human and mouse recombinant leukotriene C, synthase in a dose-related manner. The mean IC,,, values for the human and mouse recombinant enzymes were 3.1 ? 1 .O pM and 2.7 ? 0.8 pM (mean ? SD, n = 3). respectively.
~
DISCUSSION Human leukotriene C, synthase is a novel integral membrane protein whose deduced amino acid sequence shares significant
Lam et al. (ELMJ . Biachem. 238)
sequence identity with 5-lipoxygenase activating protein, another protein in the 5-lipoxygenase pathway. We cloned the mouse leukotriene C, synthase to determine the extent of the overall amino acid sequence similarity between human and mouse leukotriene C, synthase, the presence of a putative 5lipoxygenase-activating-protein-like inhibitor-binding domain in mouse leukotriene C, synthase, and the kinetic parameters of the purified human and mouse recombinant enzymes. The strategy for the cloning of the mouse enzyme began by utilizing three oligonucleotides from the open reading frame of human leukotriene C, synthase cDNA for PCR screening of a mouse bone marrow mast cell pcDNA3 library to isolate the double positive clones. Since this selection was based upon generating the appropriately sized inserts, final identification of the cDNA for mouse leukotriene C, synthase was based upon function after transfection into COS-7 cells. We isolated two clones that expressed leukotriene C, synthase when their plasmids were transfected into COS cells. The nucleotide sequence of the cloned mouse cDNA (clone A9-Hll-P!2-C9) shares 87 % similarity with the human cDNA within the open reading frame of the two cDNAs (Fig. 1). In contrast, there was little similarity in the untranslated regions of the cDNAs of the human and mouse enzymes. This is consistent with our failure to clone the mouse enzyme by PCR using the two oligonucleotide primers corresponding to nucleotides 1-21 and 622-594 of human leukotriene C, cDNA (Lam et al., unpublished results), as these two oligonucleotides represent untranslated sequences largely outside of the mouse cDNA. The deduced amino acid sequence of mouse leukotriene C, synthase shares 88% similarity with the human enzyme (Fig. 2), both of which are composed of 150 amino acids. The calculated molecular mass and PI for mouse leukotriene C, are similar to those of the human enzyme [18]. Western blot analysis of the SDS/PAGE of the recombinant mouse enzyme with rabbit anti(human leukotriene c, synthase) serum raised against purified human lung enzyme [! 31 showed that it migrated as a 18-kDa polypeptide identical in position to recombinant human leukotriene C, synthase (data not shown). Northern blot analysis indicated that both human and mouse enzyme transcripts were widely distributed (Fig. 3). The potential N-glycosylation site and the two potential protein kinase C phosphorylation sites recognized in human leukotriene C, synthase are conserved in the mouse enzyme, as are Cys56 and Cys82. The putative 5-lipoxygenase-activating-protein-like inhibitor-binding domain was located in the C-terminal region of the first hydrophilic loop of mouse leukotriene C, synthase. The only modifications in this domain are the substitutions Thr41-Ser and T y r S h P h e . That the mouse recombinant enzyme is inhibited in a dose-dependent manner by MK-886 with an IC,,, of 2.7 pM, very similar to that of the IC,,, MK-886 for the human enzyme, implies that these two amino acid substitutions allow comparable function (Fig. 7). Nine of the 18 amino acid differences for the entire human and mouse proteins occurred i n the C-terminal 18 residues (Fig. 2). That these differences at the C terminus do not affect function is apparent in the kinetic analyses of the purified revalues combinant human and mouse enzymes. The K,, and for human recombinant enzyme are 7.6 pM and 2.5 pmol . min-' . mg-', respectively, for leukotriene A, methyl ester, 3.6 pM and 1.3 pmol . min-' . mg-l, respectively, for leukotriene A, free acid, and 1.6 mM and 2.7 pmol . min-' . mg- ', respectively, for GSH (Figs 4-6). These values are in close agreement with those published for leukotriene A, free acid and GSH using enzymes purified from human leukemic cells [27]. The K,,, and V,,,, values for purified recombinant mouse leukotriene C, synthase are 10.3 pM and 2.3 pmol . min-I . mg-' protein for leukotriene A, methyl ester, 2.5 pM and 1.2 pmol . min-' . mg-' for leukotriene
v,,,,,,
61 1
A, free acid, and 1.9 mM and 2.2 pmol . min ' . mg-' protein for GSH, respectively (Figs 4-6). These results suggest that the C terminal of leukotriene C, synthase may not be important in either the binding or the catalytic function of the enzyme. The isolation of the cDNA for mouse leukotriene C, synthase reveals that the putative inhibitor-binding amino acid domain is conserved between human and mouse enzymes. This finding supports our previous suggestion that leukotriene C, synthase and 5-lipoxygenase-activating protein belong to a distinct gene family of lipid-binding proteins that present selective substrates for catalytic processing. Furthermore, the amino acid sequence similarity and enzyme kinetic comparisons between human and mouse leukotriene C, synthase suggest that the C terminus may not be important for the conjugation of leukotriene A, and reduced GSH. The authors thank Ms Arlene Stolper Simon for expert editorial help and Ms Joanne Miccile for secretarial assistance in the preparation of the manuscript. This work was supported by National Institutes of Health grants AI22531, AI31599, AR-36308, DK44730, ES06105, HL-03208, and HL36110. J. F. P. is the recipient of a fellowship from the Arthritis Foundation.
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