Human 5-lipoxygenase (EC 1.13.11.34), the key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has ...
Proc. Nail. Acad. Sci. USA Vol. 86, pp. 2592-2596, April 1989 Biochemistry
Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system (iron-binding domain/leukocyte/site-directed mutagenesis)
COLIN D. FUNK*, HANS GUNNEt, HAKAN STEINERt, TAKASHI IZUMI*,
AND BENGT SAMUELSSON* *Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden; and tDepartment of Microbiology, University of Stockholm, S-106 91 Stockholm, Sweden
Contributed by Bengt Samuelsson, January 17, 1989
entiated HL-60 cell (10) cDNA libraries and has provided some information about the structure of the enzyme. The mature enzyme consists of 673 amino acids and shares specific homologies with the plant lipoxygenases in the carboxyl-terminal region (11-15). All lipoxygenases are believed to contain a non-heme iron atom, and it has been postulated that one specific homologous region containing conserved histidine and acidic and basic residues could represent an iron-binding domain (13). The regulation of 5-lipoxygenase expression is poorly understood. We have recently undertaken steps to clarify this situation by characterizing the human 5-lipoxygenase gene and putative promoter region (11). Further means to enhance our understanding of 5-lipoxygenase and its gene expression requires a reliable expression system. In the present study we show that 5-lipoxygenase overexpressed in a baculovirus/ insect cell system (16) offers excellent advantages over human leukocytes in furthering our comprehension of this essential enzyme involved in leukotriene biosynthesis.
Human 5-lipoxygenase (EC 1.13.11.34), the ABSTRACT key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus (3B6) carrying the human 5lipoxygenase coding sequence downstream of the strong polyhedrin protein promoter was isolated. Approximately 48 hr after infection of Spodoptera frugiperda cells with the recombinant baculovirus, maximal intracellular enzyme activity and protein levels were dptected. The recombinant 5-lipoxygenase in 10,000 x g supernatant fractions was able to synthesize large amounts of 5-hydroperoxy-6,8,10,14-icosatetraenoic acid, together with smaller amounts of the nonenzymatic hydrolysis products of leukotriene A4, and exhibited a dependence on Ca2l and ATP for maximal activity. Immunoblot analysis of supernatant proteins from human leukocytes and recombinant virus-infected cells indicated the presence of indistinguishable '80-kDa bands. However, 5-lipoxygenase protein in recombinant-infected cells was found to be present in amounts 50-200 times that present in leukocytes on a per-cell basis. Histidine362 and histidine-372, potential iron-atom ligands within a putative iron-binding domain, were changed to serine residues. Recombinant baculoviruses carrying the mutations were isolated and used to infect insect cells. Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity was not substantially altered, suggesting the nonessential nature of these histidines in binding iron at the putative ferric catalytic site.
MATERIALS AND METHODS Plasmids, Cells, and Viruses. The plasmid pVL941 and baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV strain E2) were generously provided by Max D. Summers (Texas A & M University). Spodoptera frugiperda (Sf-9) cells, obtained from the American Type Culture Collection, were infected at a multiplicity of -10 plaque-forming units per cell with recombinant and wild-type baculoviruses. Construction of Recombinant Transfer Vector pVL9415BSm. The EcoRI-Bcl I fragment of the clone Ap15BS, which contains the cDNA sequence of human 5-lipoxygenase (9), was first inserted into the EcoRI and BamHI sites of the plasmid pERAT 308 (17). The cDNA insert of Apl5BS contains a 51-base-pair (bp) cloning artifact within a Xma IXma I fragment (9). The corresponding fragment of clone Ap19AS (9), a partial-length 5-lipoxygenase cDNA clone without the artifact, was inserted into pERAT308-plSBS after removal of its Xma I-Xma I fragment (now called pERAT308-pl5BSm). The orientation of the inserted fragment was checked by digestion with Pvu II. A 2.2-kilobase (kb) EcoRI-Sal I fragment of pERAT308-pl5BSm was isolated, treated with BamHI methylase (New England Biolabs) to protect the internal BamHI site, and then ligated with excess EcoRI/BamHI and Sal I/BamHI converters. After BamHI digestion and agarose gel purification, the fragment was ligated into BamHI-digested pVL941 transfer vector (18). The orientation of the insert with respect to the polyhedrin promoter was checked by Xho I and Pst I
The leukotrienes constitute a group of compounds formed from arachidonic acid that are implicated in the pathophysiology of inflammatory and immediate hypersensitivity reactions (1, 2). The first step in the biosynthesis of these compounds is the oxygenation at position C-5 of 20:4 to yield 5-hydroperoxy-6,8,11,14-icosatetraenoic acid (5-HPETE). This step, as well as the ensuing transformation of 5-HIPETE to the allylic epoxide, 5,6-oxido-7,9,11,14-icosatetraenoic acid (leukotriene A4, LTA4), is carried out by the enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) (3-6). LTA4 can be subsequently hydrolyzed by the enzyme LTA4 hydrolase to form the granulocyte chemotactic factor 5,12-dihydroxy-6,8,10,14-icosatetraenoic acid (leukotriene B4, LTB4) or conjugated with cysteine by a specific glutathione S-transferase to yield leukotriene C4, which together with the metabolites leukotriene D4 and leukotriene E4 constitutes the slow-reacting substance of anaphylaxis. Human 5-lipoxygenase is a complex enzyme requiring Ca2+, ATP, and certain unidentified soluble and membranebound proteins for maximal activity (3-8). The cDNA for 5-lipoxygenase has been cloned from placenta (9) and differ-
Abbreviations: 5-UPETE, 5-hydroperoxy-6,8,11,14-icosatetraenoic acid; 5-HETE, 5-hydroxy-6,8,11,14-icosatetraenoic acid; LTA4, leukotriene A4; LTB4, leukotriene B4; AcNPV, Autographa californica nuclear polyhedrosis virus.
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digestion. A 12-kb construct, pVL941-5BSm, was purified by CsCl gradient centrifugation and used for cotransfection. Selection of Recombinant Virus. Wild-type AcNPV DNA and transfer vector pVL941-5BSm were cotransfected into Sf-9 cells by the calcium phosphate method as described (19). Recombinant viruses were detected in a plaque assay, initially by hybridization with 32P-labeled 5-lipoxygenase cDNA probes and subsequently by visual screening for the absence of nuclear occlusion bodies (19, 20). The plaque-purified recombinant virus used in these studies is designated 3B6. 5-Lipoxygenase Assays of Recombinant Virus-Infected Cells. Monolayer Sf-9 cells (-5 x 106) in 25-cm2 flasks were inoculated with virus stock for 1 hr in complete medium [TNM-FH supplemented with 10% fetal bovine serum, 100 units of penicillin per ml, and 100 ,ug of streptomycin sulfate per ml (19)]. The medium was replaced with 5 ml of fresh medium and the cells were incubated at 270C. At various times after infection, cells were removed for counting, centrifuged at 400 x g, and resuspended in 0.5 ml of KPB1 buffer [50 mM potassium phosphate/0.1 M NaCI/1 mM EDTA/1 mM dithiothreitol/5 mM phenylmethylsulfonyl fluoride, pH 7.1, containing 60 ,Ag of soybean trypsin inhibitor per ml (7)]. The cells were disrupted by sonication (Branson S125 Sonifier, setting 4) for two pulsatile 1-sec bursts in an ice/water bath. The sonicates were centrifuged at 10,000 x g for 10 min. The supernatant was removed and aliquots were taken for enzyme assay, NaDodSO4/PAGE-immunoblot analysis, and protein analysis by the method of Bradford (21). The standard enzyme assay mixture consisted of 90 ,ul of KPB1 buffer containing various amounts of protein and 10 Al of 20 mM CaCl2/20 mM ATP. The reaction was initiated by the addition of a mixture of arachidonic acid (16 nmol) and 13-hydroperoxy-9,11-octadecadienoic acid (0.5 nmol) in 3 Al of ethanol and was allowed to proceed for 10 min at 37°C. Two hundred microliters of stop solution (acetonitrile/ methanol/acetic acid, 350:150:3, vol/vol) containing the internal standard, 16-hydroxy-9,12,14-henicosatrienoic acid (0.5 nmol), was added and proteins were precipitated on ice for 10 min. After centrifugation the supernatants were analyzed directly by HPLC (LDC/Milton Roy system with variable-wavelength UV detector coupled with Waters 712 WISP automatic injector and Waters 745 data module; 5-,um Nucleosil C18, 250 x 4.6-mm column) with UV detection at 235 nm, an acetonitrile/methanol/water/acetic acid (350: 150:250:1) mobile phase, and a flow rate of 1.5 ml/min. In some cases, dihydroxy compounds from incubations with arachidonic acid or 5-HPETE were measured by UV detection at 270 nm, with a mobile phase consisting of methanol/ water/acetic acid (70:30:0.01) at a flow rate of 1 ml/min. RNA Preparation and Blot Analysis. Total RNA (-70 ,g) from 5 x 106 wild-type- and 3B6-infected Sf-9 cells was prepared by the method of Chomczynski and Nicoletta (22). RNA was size-fractionated by electrophoresis in 1% agarose gels containing 0.7% formaldehyde (23) and transferred to nitrocellulose. Hybridization and washing conditions were as described (24). Site-Directed Mutagenesis and Construction of Mutant 5Lipoxygenase Transfer Vectors. Site-directed mutagenesis was performed by the method of Taylor et al. (25) on the Xma I-Xma I fragment of pERAT308-pl5BSm subcloned into the phage vector M13mpl8, using a kit from Amersham. The
oligodeoxyribonucleotides 5'-TC-CAC-GTC-JL.C-CAGACC-AT-3' and 5'-TG-CGA-ACA-KT-CTG-GTG-TC-3' (altered nucleotides are underlined, and hyphens indicate the reading frame) were used to change codons 362 and 372, respectively, from histidine to serine. The changes were verified by sequencing the entire fragment by the dideoxy chain-termination method (26). The mutated Xma I-Xma I
fragments were subcloned into pVL941-5BSm, after removal of the nonmutated Xma I-Xma I fragment. The orientation of
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Proc. Natl. Acad. Sci. USA 86 (1989)
Biochemistry: Funk et al.
the inserted fragment was checked by digestion with Pvu II, and the authenticity of the mutation in the transfer-vector constructs was verified by DNA sequence analysis. Cotransfection experiments and screening procedures to isolate pure recombinant mutant virus were carried out as described above. Miscellaneous. The internal standard, 16-hydroxy9,12,14-henicosatrienoic acid, was synthesized from 8,11,14icosatrienoic acid by an Arndt-Eistert synthesis and subsequent reaction with soybean lipoxygenase (27). Densitometric analysis of 5-lipoxygenase bands on immunoblots was carried out with a Shimadzu CS-930 scanner either in the reflection mode for nitrocellulose membranes or in the transmission mode for double negatives or autoradiograms.
RESULTS Isolation of Recombinant Baculovirus Encoding Human 5-Lipoxygenase. A 2.2-kb cDNA encoding human 5lipoxygenase, including 34 bp of 5' noncoding and 162 bp of 3' noncoding DNA, was introduced into the transfer vector, pVL941 (18), downstream from the polyhedrin promoter (pVL941-5BSm). The fusion gene contained no ATG codons upstream of the authentic 5-lipoxygenase translation initiation site. Sf-9 insect cells were cotransfected with pVL9415BSm and wild-type AcNPV DNA (19). Recombinant baculoviruses produced by allelic replacement of the polyhedrin gene by homologous recombination were isolated by a combination of hybridization and plaque-morphology screening. Cells infected with the recombinant virus clone 3B6 were found to express large amounts of enzymatically active 5-lipoxygenase; thus 3B6 was used in all subsequent studies. Time Course of 5-Lipoxygenase Expression in Sf-9 Cells. At various times after infection with recombinant virus 3B6, intracellular levels of 5-lipoxygenase enzyme activity and protein were assayed. Enzyme activity was readily detected 24 hr postinfection (Fig. 1) and reached maximal levels =2 days after infection. Protein detected on immunoblots was barely detectable at 24 hr and reached maximal levels 2-3 days postinfection (data not shown). Between 3 and 6 days after infection, many cells lost viability and lysed, resulting in a decrease of intracellular 5-lipoxygenase enzyme activity and protein. 30
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TIME (hours) FIG. 1. Time course of production of 5-lipoxygenase enzyme activity in recombinant baculovirus-infected cells. Sf-9 cells (3.3 x 106) seeded into 25-cm2 flasks were infected with recombinant baculovirus. At various times postinfection, cells were harvested and 5-lipoxygenase enzyme activity was assayed in aliquots of 10,000 x g supernatants. Points represent the average of duplicate determinations; duplicates varied by
