Characterization of the activity of purified recombinant human 5 ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 8, Issue of March 15, pp. 50725079 1991 Printed in d.S.A.

Characterization of the Activity of Purified Recombinant Human 6-Lipoxygenase in the Absence and Presenceof Leukocyte Factors* (Received for publication, August 21,1990)

Danielle Denis*, Jean-Pierre FalgueyretS, Denis RiendeauSQ,and MarkAbramovitzgn From the Departments of SBwchemistrvand TMoleculur Biology, Merck Frosst Centre for Therapeutie Research, Pointe Claire-Dorval, Quebec H9R 4PBCanuda

Purified recombinant human 5-lipoxygenase was used to investigatethe catalytic propertiesof the protein in thepresence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 X 10’ cells) and a specific activity of 3-6 rtmol of 6-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to theselective 5-lipoxygenase inhibitors BW756C (ICao= 13MM) and L-656,224(ICao = 0.8 p ~ ) .The reaction products of arachidonic acid oxidation were 6-HPETE and 6-trans- and 12-epi-6tram-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4),indicating that the purified protein expressed both the 6-oxygenase and leukotriene A4 synthase activities (ratio 6:l). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+,and phosphatidylcholine. These factors were found to stabilize 6-lipoxygenase during preincubation of the enzyme at 37OC with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect againstenzyme inactivation butare not essential for enzyme activity.

The 5-lipoxygenase from leukocytes is the first enzyme involved in theconversion of arachidonic acid to leukotrienes. The enzyme catalyzes both the oxygenation of arachidonic

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 To whom correspondence and reprint requests should be addressed.

acid to5-HPETE’ (5-oxygenase activity)and the further conversion of 5-HPETE to unstable allylic epoxide LTA, (LTA, synthase activity) (Yamamoto, 1989; Samuelsson and Funk, 1989). LTA, then serves as substrate for enzymatic hydrolysis to LTB, and conjugation with glutathione to yield cysteinyl-peptidoleukotrienesor can be nonenzymatically degraded to trans-LTB, isomers (Borgeat et aZ., 1985). LTA, synthesis by 5-lipoxygenase represents the first step in the biosynthesis of leukotrienes, a group of mediators which has been implicated in the pathophysiology of inflammatory and allergic reactions (Brain and Williams, 1990; Lewis and Austen, 1984; Ford-Hutchinson, 1989). Mammalian 5-lipoxygenases have been shown to require ATP and Ca2+for activity (Yamamoto, 1989) unlike other lipoxygenases from plants and animals. The rat and human 5-lipoxygenases have been shown to translocate from the soluble to themembrane fraction duringleukocyte activation (Rouzer and Kargman, 1988; Wong et al., 1988),and, recently, a membrane-bound 18-kDa protein has been identified as an essential protein for leukotriene biosynthesis (Miller et al., 1990). The cDNA for human 5-lipoxygenase has been cloned (Dixon et al., 1988; Matsumoto et al., 1988) and encodes a 674-amino acid protein of molecular weight 78,000. The amino acid sequence shows 93% identity with that of rat 5-lipoxygenase and also indicates the presence of two regions of weak homology to a 17-amino acid consensus sequence of Ca2+dependent membrane-binding proteins (Balcarek et al., 1988). A hydrophobic region with homology to other lipoxygenases has been identified which contains conserved histidine residues and is postulated to represent an iron-binding domain (Shibata etal., 1988).No ATP-binding sitecould bepredicted from the sequence information. Procedures have been described for the purification of 5lipoxygenase from rat basophilic leukemic cells (Goetze et al., 1985;Hogaboom et al., 1986), pig leukocytes (Ueda et al., 1986), mouse mastocytoma cells (Shimizu et al., 1986) and human leukocytes (Rouzer and Samuelsson, 1985), and more recently from bacteria and yeast expressing the human 5The abbreviations used are: 5-HPETE, B(S)-hydroperoxy-6trans-8,11,14-cis-eicosatetraenoicacid; ATP-agarose, adenosine 5’triphosphate-agarose;(5S,GR)-diHETE, (5S,GR)-dihydroxy-7,9trans-ll,14-cis-eicosatetraenoic acid 13-HOD, 13(S)-hydroxy-9-cis11-trans-octadecadienoic acid; 13-HPOD, 13(S)-hydroperoxy-9-cis11-trans-octadecadienoic acid; HEPES, N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid; LTA., (5S)-trans-5,6-oxido-7,9-trans11,14-cis-eicosatetraenoicacid; LTB,, (5S,12R)-dihydroxy-6,14-cis8,lO-trans-eicosatetraenoicacid; 6-trans-LTBa, (5S,12R)-dihydroxy6,8,10-trans-14-cis-eicosatetraenoicacid; 12-epi-6-trans-LTB4, (5S,12S)-dihydroxy-6,8,lO-trans-l4-cis-eicosatetraenoic acid; PC, La-phosphatidylcholine; P-100, 100,000 X g pellet; RP-HPLC, reverse phase-high pressure liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; S-100, 100,000 X g supernatant, kb, kilobaseb); bp, base pairb).

5072

Properties of Purified Recombinant

Human 5-Lipoxygenme

5073

phate method (Summers and Smith, 1987). After 7 days, the cellular debris was spun down, and the supernatantwas used as thesource of recombinant virus for the first plaque assay. Plaque assays were carried out in culture dishes (100 X 15 mm) as previously described (Summers and Smith, 1987). The infected cells were overlaid with 10 ml of 10% SeaPlaque agarose (FMC Corp., Marine Colloid Div., Rockland, ME) diluted in Grace's complete medium plus 100 pg/ml Bluo-Gal (Bethesda Research Laboratories) (50 mg/ml in dimethylformamide). Blue plaques indicative of the presence of @-galactosidase were visible within 4-6 days. The plaques were picked with a Pasteur pipette and placed in 1 mlof Grace's complete medium. The virus was allowed to elute from the agarose plug overnight at 4°C. From this initial plaque assay, 12 blue plaques were subjected to dot-blot analysis (see below) and probed with a fragment of the human 5lipoxygenase cDNA. Seven of the twelve plaques (58%) which expressed @-galactosidaseactivity also contained the 5-lipoxygenase cDNA (data not shown). Recombinant virus from three of the seven 5-lipoxygenase positive plaques was used for the second round of plaque assays and performed with 1-, lo2-, and103-folddilutions of virus, from which pure recombinant virus containing both 5-lipoxygenase and &galactosidase was obtained. Usually, two to three rounds of plaque assays were sufficient in order to purify recombinant virus from wild type virus. The recombinant virus was used to infect Sf9 cells so as to produce viral stocks with titers of10' to lo9 plaqueforming units/ml. All three separate isolates of recombinant virus, upon infection of Sf9 cells, produced virtually equivalent amounts of 5-lipoxygenase (data not shown).One of them, designated rvH5L0(8MATERIALS ANDMETHODS I), was used in subsequent experiments. Nucleic AcidDot-Blot Hybridizations-24-Well culture plates were Cells and Virus-Spodoptera frugiperda (Sf91 insect cells and Autographa californica nuclear polyhedrosis virus were obtained from C. seeded with 3-5 X lo5 cells in 1 ml of Grace's complete medium and Richardson, Biotechnology Research Institute,Montreal, Quebec. infected with virus derived from blue plaques. Infected cells from week-old cultures were used for the dot-blot assay according to a Cell counts and percent cell viability were determined in0.2% trypan published method (Summers and Smith, 1987). The probe, an 813blue. Cells were cultured in Grace's complete medium (Gibco Labobp ClaI-BamHI 5-lipoxygenase cDNA fragment (Dixon et al., 1988) ratories) supplemented with 10% fetal bovine serum (Flow Laborawas labeled with [a-32P]dCTPusing the Multiprime labeling system tories Inc.), TC Yeastolate and TC lactalbumin hydrolysate (Difco (Amersham, Canada) to specific activities of >lo9 dpm/pg. The Laboratories), 50 pgof gentamicin sulfateper ml and 2.5pg of membranes were exposed to X-Omat AR film (Kodak) for 2-4 h at amphotericin B (Fungizone) per ml in either Falconware T flasks - 70 "C. (Becton Dickinson Labware) or spinner flasks (Bellco Glass, Inc.) at Polyacrylamide Gel Electrophoresis and Immunoblots-Various 28°C following the procedures of Summers and Smith (1987). Esch- protein samples were prepared in electrophoresis loading buffer (0.1 erichia coli, JM107, was obtained from Bethesda Research Labora- M Tris-HC1, pH 6.8, 4% SDS, 10% @-mercaptoethanol,30% glycerol, tories. The bacterial cells were transformed according to published 0.05% bromphenol blue) and were applied to 10% polyacrylamide gels methods (Hanahan, 1985). according to themethod of Laemmli (1970).For immunoblot analysis, Chemicals and Reagents-Restriction endonucleases were pur- proteins were electrophoretically transferred to nitrocellulose chased from Pharmacia LKB Biotechnology Inc., and Bethesda Re- (Schleicher & Schuell/Baxter, Montreal, Quebec) and probed with search Laboratories. DNA polymerase I, Klenow fragment, and T4 the rabbit anti-5-lipoxygenase LO-33 antiserum, generated against DNA ligasewere obtained from Bethesda Research Laboratories. the 5-lipoxygenase purified from human leukocytes according to the Radioisotopes '261-labeledprotein A (10 mCi/mg) and [CY-~'P]~CTPprocedure of Rouzer and Samuelsson (1985). The antigen-antibody (3,000 Ci/mmol) were from Du Pont-New England Nuclear. Adeno- complexes were detected with "'1-protein A (Towbin et al., 1979). sine 5'-triphosphate-agarose (attachedto beaded agarose through C- The nitrocellulose membrane was exposed to X-Omat AR film (East8 with a 6-carbon spacer, 2.4 pmol/ml of gel), L-a-phosphatidylcholine man Kodak Co.) with intensifying screens for 2-6 h at -70 "C. (PC) (type 111 from egg yolk), and arachidonic acid were purchased Preparation of Lysates from Infected Cells-Sf9 cells were grown at from Sigma. LTB., BW755C, and L-656,224 were synthesized at the 27 "C in 100-ml spinner flasks to a cell density of1.5-2 X lo6 cells/ Dept. of Medicinal Chemistry, Merck Frosst. 6-trans-LTB4, 12-epi- ml and infected for 44-48 h with rvH5L0(8-1). The cells were then 6-trans-LTB4, 13-HPOD, 13-HOD, 5-HPETE, 5-HETE, and collected by centrifugation (900 X g for 10 min, at 20"C), washed (5S,GR)-diHETE were obtained from Biomol Research Laboratories twice with Dulbecco's phosphate-buffered saline (pH 7.4) (25 m1/2 X Inc., Plymouth Meeting, PA. 10' cells) and resuspended a t 1.2 X lo7 cells/ml in a homogenization Construction of Recombinant Transfer Vector pJVETLZH5LObuffer containing50 mM potassium phosphate (pH 7.9) 2 mM EDTA, The baculovirus expression vector pJVETLZ was generously provided 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 60 by J. Vialard and C. Richardson (Biotechnology Research Institute, pg/ml soybean trypsin inhibitor. The cells were then lysed by soniMontreal, Quebec). This vector differed from the recently published cation at 4 "C using a Cole Parmer (4710 Series) Ultrasonic homogevector pJV(Nhe1) (Vialard et al., 1990) in that theP10 promoter was nizer (3 to5 burstsof 10 s with 30-5 lags,pulse mode, 70%duty cycle, replaced by the ETL promoter (Crawford and Miller, 1988) of wild and output setting at 3). The preparations were examined under the type baculovirus. The vector, pGBT-H5LO, was provided by T. microscope to achieve efficient cell lysis (>go%) with minimal soniNguyen, Merck Frosst Centre for Therapeutic Research. A 2.1-kb cation. The lysate was then centrifuged at 100,000 X g for 1 h EcoRI-StuI restrictionfragment from pGBT-H5LO containing11bp (Beckman L5-65, 60 Ti rotor) at 4 "C and the resulting supernatant of 5'-noncoding, the entire coding region, and 85 bp of 3'-noncoding (S-100 fraction) brought to 24 pg/ml PC by the addition of a 250-fold sequence of the human 5-lipoxygenase cDNA (Dixon et al., 1988) was concentrated solution in ethanol. The S-100 fraction (1-3 mg/ml) blunt-end-ligated into NheI-cut pJVETLZ. Miniprep DNA from was stable for several hours at 4 "C and could be stored for several JM107 transformants was isolated, and theorientation of the inserted months at -70 "C in 20% ethylene glycol with about 50% recovery of fragment with respect to the polyhedrin promoter was confirmed by activity. digestion with EcoRV. The 16.1-kb vector, PJVETLZ-H~LO,was Affinity Purification of Recombinant Human 5-Lipoxygenme-A isolated from minipreps and purified on NACS columns (Bethesda chromatography column (0.7 X 15 cm) containing 2 mlof ATPResearch Laboratories) and was subsequently used for co-transfec- agarose gel was equilibrated a t 4 'C in 0.1 M Na+-HEPES buffer (pH tion. 7.31, containing 1 mM EDTA, 1 mM dithiothreitol, and 24 pg/ml PC. DNA Transfections and Plaque Assays-Wild type A. californica The S-100 fraction (8 ml) was applied at a flow rate of 5 ml/h, and nuclear polyhedrosis virus DNA (1 pg) was mixed with pJVETLZ- the column was then washed at a flow rate of 10 ml/h with 2 ml of H5LO (2 pg) and co-transfected into Sf9 cells by the calcium phos- the equilibration buffer. After a high salt wash (10 ml of 0.5 M NaCl

lipoxygenase cDNA (Noguchi et al., 1989; Nakamura et al., 1990). However, studies on purified human 54ipoxygenase have proven to be extremely difficult because of the tediousness of the purification procedures, the low recoveries of activity (1-4%), and the extreme lability of the purified enzyme. Furthermore, previous studies have shown that the purified protein from human leukocytes had very low levels of activity unless a membrane fraction and other fractions generated during the purification procedure were added to the assay mixtures (Rouzer and Samuelsson, 1985, 1990). Thus, most of our information on the reaction catalyzed by human 5-lipoxygenasehas been obtained from activity measurements in leukocyte extracts or in the presence of stimulatory fractions and inference from studies of the soybean lipoxygenase and purified 5-lipoxygenase from other sources. In this study, we describe the isolation and characterization of recombinant human 5-lipoxygenase from a baculovirus high expression system. The purified recombinant human 5lipoxygenase was found to be catalytically active, which has allowed for the study of the reaction catalyzed by the isolated protein and a re-examinationof the effect of various protein stimulatory factors from leukocytes.

5074

Properties Purified of

Recombinant Human 5-Liprygenase

in equilibration buffer), the column was re-equilibrated with 5 ml of buffer before elution of 5-lipoxygenase with 20 mM ATP in equilibration buffer (Wiseman, 1989). All buffers were filtered through 0.22pm membranes (Millex-GS, nonsterile, Millipore) and purged with helium gas. Fractions containing 5-lipoxygenase activity were identified using the spectrophotometric assay and pooled. The enzyme could be stored as aliquots in 20% ethylene glycol a t -70°C for several months with approximately 50% recovery of activity. RP-HPLC Assay for S-Lipoxygenase Actiuity-The enzyme reaction was performed in 0.025 M Na'/K*-phosphate buffer (pH 7.3) containing 0.5 mM CaCI,, 1 mM ATP, 24 pg/ml PC, and 20 p M arachidonic acid in a final volume of 200 pl. Arachidonic acid was added as 2 pl of a 2 mM solution in ethanol prior to the initiation of the reaction with the addition of enzyme. After an incubation of 5 min a t room temperature, the reaction was stopped by mixing with 0.8 ml of diethyl ether/methanol/l M citric acid (304:l) containing 100 ng of 13-HOD as internalstandard. The mixture was centrifuged at 1,000 X g for 5 min. The upper phase was collected and dried under NZ, and theresidue was redissolved in 100 pl of the RP-HPLCsolvent system. This sample was injected on aNova-Pak C18column (Waters) eluted isocratically with acetonitrile/water/acetic acid (6535:O.l) a t 2 ml/min. The effluent was monitored a t 235 nm for the detection of 5-HETE and 5-HPETE which eluted a t 3.0 and 3.4 min, respectively. These producta were quantitated from an absorbance area relative to the 13-HOD standard andwere corrected for a low level of background using control samples incubated in the absence of enzyme or arachidonic acid. The reaction products were 5-HPETE for the purified enzyme and a mixture of 5-HPETE and 5-HETE for assays of the S100 fraction or for the purified enzyme in the presence of protein factors. The 5-lipoxygenase activity is expressed as the maximal amount of 5-H(P)ETE accumulated after incubation (Rouzer and Samuelsson, 1985) under conditions where arachidonic acid is not limiting (