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Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human Nipoxygenase Satoshi Ishii, Masato Noguchi, Masashi Miyano, Takashi Matsumoto and Masana Noma Life Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Midori-ku, Yokohama, Kanagawa 227, Japan
Received
January
9, 1992
Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron. 0 1992 Academic Press,
Inc.
Lipoxygenase is ubiquitously distributed in plants and animals. They all catalyze the incorporation of a molecular oxygen into polyunsaturated fatty acid containing a cis, cis- 1,4pentadiene moiety. Human 5-lipoxygenase (EC 1.13.11.34) catalyzes the oxygenation of arachidonic acid at carbon 5 to yield its hydroperoxide 5-HPETE and the subsequent dehydration of the hydroperoxide to form leukotriene A4 (LTAa), which are the early steps in the biosynthesis of leukotrienes [for review, see ref. 11. LTA4 can then be converted to leukotriene B4 (LTB4) or be conjugated with glutathione to form leukotriene C4 (LTG). LTC2, together with its metabolic
Abbreviations: 5-HETE, SS-hydroxy-6-truns-8,11,14-cis-eicosatetraenoic
acid; 5HPETE, 5S-hydroperoxy-6-truns-8,11,14-cis-eicosatetraenoic acid; 13-HODE, 13Shydroxy-9-cis-l l-truns-octadecadienoic acid; Leukotriene A4 (LTAS, 5,6-trunsoxido-7,9-truns11,14-cis-eicosatetraenoic acid; Leukotriene B4 (LTBa), 5S,12Rdihydroxy-6,14-cis-8,10-truns-eicosatetraenoic acid; Leukotriene C4 (LTCa), 5Shydroxy-6R-S-glutathionyl-7,9-truns-ll,14-cis-eicosatetraenoic acid; Leukotriene D4 (LTDd), 5S-hydroxy-6R-S-cysteinylglycyl-7,9-tran~-1l,14-cis-eicosatetraenoic acid; Leukotriene E4 (LT&), SS-hydroxy-6R-S-cysteinyl-7,9-truer11,16eicosatetraenoic acid; EGTA, ethylene glycol bis(2-aminomethyl ether) tetraacetic acid; HPLC, high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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products, leukotriene Ds(LTD4) and leukotriene & (LTE4), is known to be one of the slow-reacting substances of anaphylaxis. As LTB4 and the three cysteinyl leukotrienes have been postulated to have a role in allergic and inflammatory diseases, detailed characterizations of 5lipoxygenase, especially human enzyme, may provide clues for the design of anti-allergic and anti-inflammatory drugs. The 5-lipoxygenases have been isolated from human and porcine leukocytes, murine mast cells, and rat basophilic leukemia cells. Human 5-lipoxygenase is a labile 78kDa protein and the peptide sequence has been deduced from the cloned cDNA [2]. For maximal activity, ATP and Ca2+ are required as cofactors [3]. The enzyme contains non-heme iron and the iron seems to be essential for the activity [4,5]. The role of the iron in soybean lipoxygenase has been studied so extensively that conversion of the high-spin state of ferrous ion to that of ferric ion was found to result in the activation of the enzyme . The change in the valence of the iron has been reported by ESR [6], Miissbauer spectra [7], and magnetic susceptibility measurements [8]. The iron environment is extensively studied on the soybean enzyme. Its extended X-ray absorption fine structure and Mossbauer spectra are consistent with a six-coordinate iron, and four histidine and two acidic amino acid residues are proposed to ligate in a roughly octahedral field of symmetry [7,9]. There are 6 histidines which are completely conserved among the lipoxygenases sequenced to date (Figure 1) [2,10-181. Five residues of them are located in a highly 358
352
367
372
376
190
h5LO r5LO hl2LO P12LO h15LO rbl5LO sbLO1 sbLO2 sbL03 peL0
351 351 344 345 344 344 482 511 502 506
KIWVRSSDFHVEQTITELLRTELVSEVFGIAMYRQLPAVEPIFKLLVAEVR KIWVRSSDFHVEQTITELLRTELVSEVFGIAMYRQLPAVBPPFKLLVAEVR KSWVRNSDFQLHEIQYELLNTELV~VIAVATMRCLPGLEP~FKFPIPEIR KCWVRSSDFQLEELHSELLRGELM~VIAVATMRCLPSIEPIFKLLIPBFR KCWVRSSDFQLEELQSELLRGBLMAEVIWATMRCLPSIBPIFKLIIPELR KCWVRSSDFQVBELNSHLLRGELMAEVFTVATMRCLPSIEPVFKLIVPELR KAYVIVNDSCYHQIMSEWLNT~PFVIATHRHLSVLEPIYKLLTPEYR KAYVVVNDSCYEQLMSBWLNTEAVIEPFIIATNRHLSALBPIYKLLTPEYR KAYVWNDSCYEQLVSEWLNTEAWEPFIIATNRHLSVVEPIYKLLHPBYR KAYVIVNDSCYEQLVSEWLNTBAVVBiPFVIATNRHLSCLHPIYKLLYPEYR
h5LO K5LO h12LO p12LO h15LO rbl5LO sbLO1 sbL02 sbL03 peL0
427
TGGGGEVQMVQRA TGGGGEVQMVQRA TGGGGEVQLLRRA TGGGGBVELLRRA TGGGGEVQLLKQA TGGGGEVQLLQQA
(32
421
420 421 420 420
399
401 401 394 395 394 394 532 561 552 556 550
439 439 432 433 432 432
542 542 532 533 532 532 681 710 701 705
VIFTASAQEAAVNFGQYDWC VIFTASAQEAAVNFGQYDWC CVFTCTAQEAAINQGQLDWY CIFTCTGQESSNHIGQLDWY CIFTCTGQEASVHIGQLDWY CIFTCTGQESSIHLGQLDWF IIWIASALEAAVNFGQYPYG IIWTASALEAAVNFGQYPYG IIWTASALEAAVNFGQYPYG VIWTASALEAAVNFGQYSYG
561 561 551 552 551 551 700 729 720 724
Figure 1. Alignment of the amino acid sequences of lipoxygenases. The conserved histidine and acidic amino acid residues are represented by hold letters. Amino acid residues in human 5lipoxygenase sequence are numbered from the first base of the ATG initiator codon. h5L0, human S-lipoxygenase (2); r5LO. rat 5 lipoxygenase (9); h12L0, human 12-lipoxygenase (10); p12L0, porcine 12lipoxygenase (11); h15L0, human 15-lipoxygenase (12); rblSL0, rabbit 15lipoxygenase (13); sbLO1, soybean lipoxygenase 1 (14); sbL02. soybean lipoxygenase 2 (15); sbL03, soybean lipoxygenase 3 (16); peL0, pea lipoxygenase (17). 1483
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homologous region among the lipoxygenases (amino acid residues 351 to 401 of the human enzyme), which has been presumed to be the iron-binding domain in the catalytic site [13,16,19]. The other conserved histidine residue is located at position 550. Between amino acid residues 546 to 558, there are 12 out of 13 residues identical in the 5-lipoxygenases and the plant lipoxygenases. As far as the animal lipoxygenases, a histidine residue at position 432 is also conserved. Furthermore, an aspartic acid residue at position 358 and a glutamic acid residue at position 376 are conserved in the homologous region. They are also candidates for the iron ligands. In order to determine which amino acid residues are involved in the iron-binding and the activity, the conserved histidine and acidic amino acid residues were individually replaced by means of site-directed mutagenesis. Replacement of the essential amino acid residues for iron binding is presumed to result in elimination of the activity. Iron ligands were determined on the basis of this criterion.
MATERIALS
AND
METHODS
Materials Restriction enzymes were purchased from either Takara Shuzo or Nippon Gene, except for XmaI from New England Biolabs. Bacterial alkaline phosphatase was from Toyobo. [c@P]dCTP (30OOCi/mmol) was purchased from Du Pont-New England Nuclear. Arachidonic acid (Nu-Chek Prep.) was dissolved in ethanol and stored at -80°C in an atmosphere of nitrogen. Phenylmethylsulfonyl fluoride (Wako) was dissolved in isopropylalcohol and stored at -20°C. Dithiothreitol (Wake) was dissolved in each buffer just before use. Sonicated phosphatidylcholine (Sigma) was also prepared just before use from its aqueous solution. Other chemicals were of reagent grade. The oligonucleotides used as primers for preparing mutants and for DNA sequencing were synthesized with an Applied Biosystems DNA synthesizer Model 392 and purified by OPC columns (Applied Biosystems). Construction of Mutant Plasmid The EcoRI-XbaI fragment of plasmid pH5LOKC [20], which had been constructed for expression of human 5-lipoxygenase cDNA in Escherichia coli, was inserted into the cloning vector pBluescript SK(-) previously treated with EcoRI, XbaI and bacterial alkaline phosphatase. Ligation reaction was carried out using a DNA Ligation Kit (Takara Shuzo). The resultant plasmid is called pBSK(-)SLO. Site-directed mutagenesis was performed on single-stranded pBSK(-)SLO using a Mu&Gene kit (Bio-Rad). The single-stranded pBSK(-)SLO was prepared by using helper phage VCSM13. Table I shows the oligonucleotides used in the preparation of each of the mutants. The changes of the cDNAs were detected either by the fluorescent dideoxy sequencing system, Genesis 2000 (Du Pont), using the oligonucleotide 5’-954CAACAAGATTGTCCCCA970-3’ as a sequencing primer, or by restriction enzyme mapping. The XmaI-XmaI fragment containing the mutation was ligated between the two XmaI sites of pH5LOKC to obtain the mutant 5lipoxygenase cDNA. The mutation at codon 550 was carried out with the &“-Not1 fragment. The mutation was confkmed by dideoxy sequencing using a PGquencing kit (Pharmacia). The sequencing primer for the XmaI-XmaI fragment was 5’954CAACAAGATTGTCCCCA970-3’ and that for the SfiI-Not1 fragment was 5’‘*ssGGCGGTGGGCACGTGCAGATGis”s-3’. 1484
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Table I. Summary of oligonucleotides used for site-directed mutagenesis of human 5-lipoxygenase. Superscripts indicate the nucleotide number of the cDNA for human 5-lipoxygenase. Codon for the replaced amino acid residue is denoted by bold letters. The mismatched position in each oligonucleotide is underlined. Introduction of the mutation was confiied by appearance or disappearance of a restriction site, which is designated by the mark “+” or “-” before the name of the restriction enzyme. n indicates that the mutagenic oligonucleotide did not change restriction pattern of the cDNA so that introduction of the mutation was confirmed by the dideoxy sequencing. Mutant Intact H362N H362.S H367N H367A H372N H372S H3WN* H39OA H399N H399A H432N H432A H55ON H55OA D358N E376Q
Activity (%) 100 15.9 34.3 N.D. N.D. N.D. N.D.
Yield (%) 100 90.2 49.8
Slight NfD. 59.8 N.D. 61.9 94.1 N.D. N.D. 78.0 N.D.
24.0 111 113 83.3
Relative activity (8) 100 18 69 0 0 0 0 + 0 249 0 2: 0 0 94 0
Preparation of E. coli Lysate Containing Human 5-Lipoxygenase E. coli harboring the mutated or intact plasmid was grown at 26°C for 24 hours
in SOOml of TYSG medium (1% Bacto Tryptone (Difco); 0.5% Yeast Extract (Difco); 2% NaCl; 2%(w/v) glycerol and 50% tap water (pH7.8)) with 50mg/l ampicillin (Wako). The cells were harvested by centrifugation (65OOxg, lOmin, 0°C) and rinsed in 50ml of 0.9% NaCl. After re-centrifugation (45OOxg, lOmin, OOC), the pellets were suspended in KPG buffer containing 50mM potassium phosphate (pH7.5), 1OOmMNaCl, 1mM EGTA (Sigma) and lO%(w/v) glycerol at 6.25ml per gram cell and stored at -80°C. The frozen suspension was thawed and treated with O.Smg/ml lysozyme (Wake), 0.5mM phenylmethylsulfonyl fluoride and 0.5mM dithiothreitol on ice for 1 hour. The cells were then disrupted by a Branson Sonifier Model 450 with the microtip at power level of 2.5 for three 20 second periods separated by an interval of 40 seconds. The sonication was carried out in an ice/water bath. The lysate was centrifuged at 174OOxgfor 20 minutes at 0°C and the resultant supematant was used for 5-lipoxygenase assay. Assay for 5-Lipoxygenase
Activity
The standard mixture for 5-lipoxygenase assay (total volume lOOp1) contained 1OOmM Tris-HCl (pH8.0), 2mM CaC12, 2mM ATP, 157pg/ml sonicated phosphatidylcholine and 1mM arachidonic acid. The reaction was initiated by addition of an aliquot of the lysate. After incubation at 30°C for 10 minutes, the reaction was terminated by addition of 3OOpl of cold methanol (-20°C) containing 0.2nmol of 13-HODE (Cayman Chemical) as an internal standard, and lpi of 1M acetic acid. Following centrifugation, the supematant was analyzed by HPLC (Shimadzu) as reported previously [20], except for using methanol/water/acetic acid (80:25:0.01 v/v) as a solvent. 5-lipoxygenase activity was determined from a ratio of sum of 5HPETE and 5HETE peak areas to that of the internal standard. 1485
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Measurement of S-Lipoxygenuse Yield Measurements of the expressed lipoxygenase yields were carried out by the enzyme-linked immunosorbent assay (ELISA). The microtiter plates were coated with adequately diluted lysates containing each of the mutant or the intact enzyme by incubating overnight at 4°C. Anti-5-lipoxygenase rabbit antiserum and alkaline phosphatase-labeled anti-rabbit IgG antibody from goat (Zymed) were used to generate a colored reaction product of the substrate, p-nitrophenyl phosphate (lmg/ml). The color reaction was evaluated from the absorbance at 405nm. The lysate of E. coli transformed with pKC [20] was used as a control. Immunoblot Analysis Proteins of E. coli transformed with the mutated or the intact plasmid were separated by 10% SDS-PAGE and transferred to Immobilon membranes (0.45pm, Millipore). The membranes were incubated with anti-5-lipoxygenase rabbit antiserum. After washing, the membranes were incubated with a goat anti-rabbit IgG serum conjugated with peroxidase (Grganon Teknika). The color reaction was developed in 20mM Tris-HCI (pH7.5), 0.03% hydrogenperoxide and SOpg/ml 3,3’diaminobenzidine tetrahydrochloride.
RESULTS AND DISCUSSION The plasmids containing a mutated cDNA were constructed and used to transform E. coli. Then the lysates of these transformants were assayed for 5Relative enzyme activities of the human 5-lipoxygenase lipoxygenase activity. mutants are summarized in Table II. Expression of the mutated cDNAs was
Table II. Relative activities of the mutant human 5-lipoxygenases. Measurements of the activity and the expressed enzyme yields in lysates prepared from E. coli transformed with the mutated or the intact plasmid were performed as described in Materials and Methods. The data presented here are from one experiment in which duplicate measurements on three or four dilutions of lysate were performed, with intact lipoxygenase as a control. The relative activity of mutated lipoxygenase is designated by the ratio of the activity to the expressed enzyme yield. As for the mutants without activity, the yields were not measured. The experiments were repeated a total of three times with substantially the same results. N.D.; not detected. *Though H390N were active, which is designated by the mark “+I’, the yield was too low to determine. Mutant H362N H362s
H367N H367A H372N H372s H39ON H39OA H399N H399A H432N H432A H55ON H55OA D358N E376Q
Mutagenic
oligonucleotide
5’-l”78TTCCACGTCAACXAGACCATC”‘=3’ S’-1075GACTTCCACGTmCCAGACCATCA’“-3 5’-‘“ACCATCACCMCCTTCTGCGA”‘3-3’
Restriction ll
+PttKZCl n
+SpeI -ApaLI -ApaL
5’-‘m7ATCTGGGTGCGcTCGAGTJ@CTTCCACGTC1’=3’ 5’-1119TCTGGTGTCTL;1rGGTTTTTGG113v-3’
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Figure 2. Immunoblot analysis. Proteins (2Opg each) of E. coli transformed with the mutated or the intact plasmid were separated by 10% SDS-PAGE and transferred to membrane. The membrane was incubated with anti-5-lipoxygenase rabbit antiserum.
confirmed by immunoblot analysis (Figure 2). All mutants were yielded enough for determination of the activity. Three mutated human 5lipoxygenases whose histidine residue was replaced by asparagine residue at a position 367, 372 or 550 (H367N, H372N and H550N)* obviously lacked the enzyme activity. The other asparagine substituted mutants retained the catalytic function to convert arachidonic acid into 5-HPETE at varying levels. Serine residue was also used to substitute for His-362 and His-372. H372S was devoid of the activity like H372N. We concluded that His-372 is essential for 5lipoxygenase activity, whereas Funk et al. reported that the mutation of a histidine residue at a position 362 or 372 to serine residue using the baculovirus/insect cell expression system did not affect the enzyme activity [19]. With respect to the histidine residues other than His-362 and His-372, alanine substituted mutants were constructed to confirm the results obtained with the asparagine substituted mutants. H367A, H432A and H550A exhibited the same characteristics as the corresponding asparagine substituted mutants. Therefore His367 and His-550 are determined to be indispensable amino acid residues for the catalysis of the oxygenation. The other alanine substituted mutants, H390A and H399A, had no activity, while the corresponding mutants replaced by asparagine residue were in active forms. His-390 and His-399 mutants were produced apparently less than the other mutants (Figure 2). Two proline residues (Pro-387 and Pro-391), which are considered to be configurationally “rigid” amino acids, are located in the vicinity of His-390, suggesting steric stringency for amino acid replacements around the proline residues. Thus His-390, maybe also His-399, would play important roles to keep the
*For brevity’s sake, mutations are designated using the one-letter code for the original amino acid and for the replaced one before and after the residue number, respectively. 1487
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folding structure of 5lipoxygenase. The asparagine residues of H39ON and H399N seemed to be able to substitute for the histidine residues because of comparable molecular size of side chain of asparagine residue to that of histidine residue with a corresponding Na atom. Asp-358 and Glu-376 were replaced with an asparagine and a glutamine residue, respectively, since carboxyl groups of these residues also could coordinate to the iron. E376Q lost the activity, while D358N retained. The results indicate that Glu376 might ligate to the iron. Human 5lipoxygenase has 9 conserved amino acid residues as candidates for iron ligands. The putative iron ligands were changed by site-directed mutagenesis. Three histidine residues (His-367, His-372 and His-550) and one glutamic acid residue (Glu-376) were revealed to be essential for the activity. Two histidine residues (His-390 and His-399) seemed to be important for the folding structure of human lipoxygenase. According to reports on the three-dimensional structure of the other non-heme enzymes, it is unlikely that each substituted amino acid residue (asparagine, serine or alanine) directly ligates to the iron [21-281. We propose that the four residues coordinate to the essential iron at least. Two carboxyl oxygen atoms of Glu-376 might provide two ligands to the iron such as other non-heme proteins whose structural analysis have been reported [21-231. It is obscure that coordination system of the iron in human 5-lipoxygenase is similar to the symmetric six-coordination one proposed in soybean enzyme 191, since our preliminary ESR data suggested that the ligand field of human enzyme was different from that of soybean enzyme (unpublished data). A few ligands bound to the iron in human 5lipoxygenase might not be from amino acid residue because many non-heme proteins have proved to possess one or more non-proteinaceous ligands including substrate [25]. For example, azidomyohemerythrin possesses an oxide anion other than an azide anion [22] and ribonucleotide reductase does an oxide anion and two water molecules [23] to complete the iron coordination. Lactoferrin has a carbonate anion
WI. Three amino acid residues (His-367, His-372 and Glu-376) in the homologous region were specified as iron ligands. Although the ligands exist at rather short distances within 10 amino acid sequence, they seems to be able to coordinate to the iron without steric hindrance. In fact, the three dimensional structure of azurin, a blue copper protein, revealed that three of five amino acid ligands (Cys-112, His117 and Met-121), which lie in the interstrand loop, exist at just the same distances as that of human 54ipoxygenase [29]. Our results would help to define the coordination of the non-heme iron of the lipoxygenases, however it remains to confirm the concomitant loss of enzyme activity and iron content in each purified mutated protein. During preparation of this manuscript, a paper appeared reporting evaluation of the role of the conserved amino acid residues [30]. We specified His-550 and Glu1488
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376 as iron ligands other than His-367 and His-372 that were shown to be important for 5lipoxygenase activity in the paper.
1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Lewis, R.A., Austen, K.F. and Soberman, R.J. (1990) N. Engl. J. Med. 323, 645-655. Matsumoto, T., Funk, C.D., R&hnark, O., HGiig, J.-O., Jomvall, H. and Samuelsson, B. (1988) Proc. Natl. Acad. Sci. USA 85,26-30 and correction (1988) 85,3406. Rouzer, C.A. and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82,60406044. Matsumoto, T., Noguchi, M., Nakamura, M. and Ishii, S. (1991) in Proceedings of Yamada Conference XXVII: International Symposium on Oxygenases and Oxygen Activation, pp 5 l-54, Y amada Science Foundation, Osaka. Percival, M. D. (1991) J. Biol. Chem. 266, 10058-10061. de Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) B&him. Biophys. Acta 377,71-79. Dunham, W.R., Carroll, R.T., Thompson, J.F., Sands, R.H. and Funk, M.O. (1990) Eur. J. Biochem. 190, 611-617. Petersson, L., Slappendel, S., Feiters, M.C. and Vliegenthart, J.F.G. (1987) Biochim. Biophys. Acta 913, 228-237. Navaratnam, S., Feiters, M.C., Al-Hakim, M., Allen, J.C., Veldink, G.A. and Vliegenthart, J.F.G. (1988) B&him. Biophys. Acta 956, 70-76. Balcarek, J.M , Theisen, T.W., Cook, M.N., Varrichio, A., Hwang, S.-M., Strohsacker, M.W. and Crooke, S.T. (1988) J. Biol. Chem. 263, 13937-13941. Funk, C.D., Furci, L. and FitzGerald, G.A. (1990) Proc. Natl. Acad. Sci. USA 87,5638-5642. Yoshimoto, T., Suzuki, H., Yamamoto, S., Takai, T., Yokoyama, C. and Tanabe, T. (1990) Proc. Natl. Acad. Sci. USA 87, 2142-2146. Sigal, E., Craik, C.S., Highland, E., Grunberger, D., Costello, L.L., Dixon, R.A.F. and Nadel J.A. (1988) B&hem. Biophys. Res. Commun. 157,457464. Fleming, J., Thiele, B.J., Chester, J., G’Prey, J., Janetzki, S., Aitken, A., Anton, I.A., Rapoport,S.M. and Harrison, P.R. (1989) Gene 79, 181-188. Shibata, D., Steczko, J., Dixon, J.E., Hermodson, M., Yazdanparast, R. and Axelrod B. (1987) J. Biol. Chem. 262, 10080-10085. Shibata, D., Steczko, J., Dixon, J.E., Andrews, P.C., Hermodson, M. and Axelrod, B. (1988) J. Biol. Chem. 263, 6816-6821. Yenofsky, R.L., Fine, M. and Liu, C. (1988) Mol. Gen. Genet. 211, 215-222. Ealing, P.M. and Casey R. (1989) B&hem. J. 264,929-932. Funk, C.D., Gunne, H., Steiner, H., Izumi, T. and Samuelsson, B. (1989) Proc. Natl. Acad. Sci. USA 86,2592-2596. Noguchi, M., Matsumoto, T., Nakamura, M. and Noma, M. (1989) FEBS Lett. 249, 267-270. Michel, H., Epp, 0. and Diesenhofer, J. (1986) EMBO J. 5, 2445-2451. Sheriff, S., Hendrickson, W.A. and Smith, J.L. (1987) J. Mol. Biol. 197,273296. Nordlund, P., SjSberg, B.-M. and Elkund, H. (1990) Nature 345, 593-598. 1489
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Ohlendolf, D.H., Lipscomb, J.D. and Weber, P.C. (1988) Nature 336,403405. Stoddard, B.L., Howell, P.L., Ringe, D. and Petsko, G.A. (1990) Biochemistry 29, 8885-8893. Anderson, B.F., Baker H.M., Dodson, E.J., Norris, G.E., Rumball, S.V., Waters, J.M. and Baker, E.N. (1987) Proc. Natl. Acad. Sci. USA 84, 17691773. Bailey, S.B., Evans, R.W., Garratt, R.C., Gorinsky, B., Hasnain, S., Horsburgh, C., Jhoti, H., Lindley, P.F., Mydin, A., Sarra, R. and Watson, J.L. (1988) Biochemistry 27, 5804-5812. Lawson, D.M., Artymiuk, P.J., Yewdall, S.J., Smith, J.M.A., Livingstone, J.C., Treffry, A., Luzzago, A., Levi, S., Arosio, P., Cesareni, G., Thomas, C.D., Shaw, W.V. and Harrison, P.M. (1991) Nature 349,541-544. Norris, G.E., Anderson, B.F. and Baker, E.N. (1986) J. Am. Chem. Sot. 108, 2784-2785. Nguyen, T., Falgueyret, J.-P., Abramovitz, M. and Riendeau, D. (1991) J. Biol. Chem. 266, 22057-22062.
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Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human Nipoxygenase Satoshi Ishii, Masato Noguchi, Masashi Miyano, Takashi Matsumoto and Masana Noma Life Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Midori-ku, Yokohama, Kanagawa 227, Japan
Received
January
9, 1992
Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron. 0 1992 Academic Press,
Inc.
Lipoxygenase is ubiquitously distributed in plants and animals. They all catalyze the incorporation of a molecular oxygen into polyunsaturated fatty acid containing a cis, cis- 1,4pentadiene moiety. Human 5-lipoxygenase (EC 1.13.11.34) catalyzes the oxygenation of arachidonic acid at carbon 5 to yield its hydroperoxide 5-HPETE and the subsequent dehydration of the hydroperoxide to form leukotriene A4 (LTAa), which are the early steps in the biosynthesis of leukotrienes [for review, see ref. 11. LTA4 can then be converted to leukotriene B4 (LTB4) or be conjugated with glutathione to form leukotriene C4 (LTG). LTC2, together with its metabolic
Abbreviations: 5-HETE, SS-hydroxy-6-truns-8,11,14-cis-eicosatetraenoic
acid; 5HPETE, 5S-hydroperoxy-6-truns-8,11,14-cis-eicosatetraenoic acid; 13-HODE, 13Shydroxy-9-cis-l l-truns-octadecadienoic acid; Leukotriene A4 (LTAS, 5,6-trunsoxido-7,9-truns11,14-cis-eicosatetraenoic acid; Leukotriene B4 (LTBa), 5S,12Rdihydroxy-6,14-cis-8,10-truns-eicosatetraenoic acid; Leukotriene C4 (LTCa), 5Shydroxy-6R-S-glutathionyl-7,9-truns-ll,14-cis-eicosatetraenoic acid; Leukotriene D4 (LTDd), 5S-hydroxy-6R-S-cysteinylglycyl-7,9-tran~-1l,14-cis-eicosatetraenoic acid; Leukotriene E4 (LT&), SS-hydroxy-6R-S-cysteinyl-7,9-truer11,16eicosatetraenoic acid; EGTA, ethylene glycol bis(2-aminomethyl ether) tetraacetic acid; HPLC, high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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products, leukotriene Ds(LTD4) and leukotriene & (LTE4), is known to be one of the slow-reacting substances of anaphylaxis. As LTB4 and the three cysteinyl leukotrienes have been postulated to have a role in allergic and inflammatory diseases, detailed characterizations of 5lipoxygenase, especially human enzyme, may provide clues for the design of anti-allergic and anti-inflammatory drugs. The 5-lipoxygenases have been isolated from human and porcine leukocytes, murine mast cells, and rat basophilic leukemia cells. Human 5-lipoxygenase is a labile 78kDa protein and the peptide sequence has been deduced from the cloned cDNA [2]. For maximal activity, ATP and Ca2+ are required as cofactors [3]. The enzyme contains non-heme iron and the iron seems to be essential for the activity [4,5]. The role of the iron in soybean lipoxygenase has been studied so extensively that conversion of the high-spin state of ferrous ion to that of ferric ion was found to result in the activation of the enzyme . The change in the valence of the iron has been reported by ESR [6], Miissbauer spectra [7], and magnetic susceptibility measurements [8]. The iron environment is extensively studied on the soybean enzyme. Its extended X-ray absorption fine structure and Mossbauer spectra are consistent with a six-coordinate iron, and four histidine and two acidic amino acid residues are proposed to ligate in a roughly octahedral field of symmetry [7,9]. There are 6 histidines which are completely conserved among the lipoxygenases sequenced to date (Figure 1) [2,10-181. Five residues of them are located in a highly 358
352
367
372
376
190
h5LO r5LO hl2LO P12LO h15LO rbl5LO sbLO1 sbLO2 sbL03 peL0
351 351 344 345 344 344 482 511 502 506
KIWVRSSDFHVEQTITELLRTELVSEVFGIAMYRQLPAVEPIFKLLVAEVR KIWVRSSDFHVEQTITELLRTELVSEVFGIAMYRQLPAVBPPFKLLVAEVR KSWVRNSDFQLHEIQYELLNTELV~VIAVATMRCLPGLEP~FKFPIPEIR KCWVRSSDFQLEELHSELLRGELM~VIAVATMRCLPSIEPIFKLLIPBFR KCWVRSSDFQLEELQSELLRGBLMAEVIWATMRCLPSIBPIFKLIIPELR KCWVRSSDFQVBELNSHLLRGELMAEVFTVATMRCLPSIEPVFKLIVPELR KAYVIVNDSCYHQIMSEWLNT~PFVIATHRHLSVLEPIYKLLTPEYR KAYVVVNDSCYEQLMSBWLNTEAVIEPFIIATNRHLSALBPIYKLLTPEYR KAYVWNDSCYEQLVSEWLNTEAWEPFIIATNRHLSVVEPIYKLLHPBYR KAYVIVNDSCYEQLVSEWLNTBAVVBiPFVIATNRHLSCLHPIYKLLYPEYR
h5LO K5LO h12LO p12LO h15LO rbl5LO sbLO1 sbL02 sbL03 peL0
427
TGGGGEVQMVQRA TGGGGEVQMVQRA TGGGGEVQLLRRA TGGGGBVELLRRA TGGGGEVQLLKQA TGGGGEVQLLQQA
(32
421
420 421 420 420
399
401 401 394 395 394 394 532 561 552 556 550
439 439 432 433 432 432
542 542 532 533 532 532 681 710 701 705
VIFTASAQEAAVNFGQYDWC VIFTASAQEAAVNFGQYDWC CVFTCTAQEAAINQGQLDWY CIFTCTGQESSNHIGQLDWY CIFTCTGQEASVHIGQLDWY CIFTCTGQESSIHLGQLDWF IIWIASALEAAVNFGQYPYG IIWTASALEAAVNFGQYPYG IIWTASALEAAVNFGQYPYG VIWTASALEAAVNFGQYSYG
561 561 551 552 551 551 700 729 720 724
Figure 1. Alignment of the amino acid sequences of lipoxygenases. The conserved histidine and acidic amino acid residues are represented by hold letters. Amino acid residues in human 5lipoxygenase sequence are numbered from the first base of the ATG initiator codon. h5L0, human S-lipoxygenase (2); r5LO. rat 5 lipoxygenase (9); h12L0, human 12-lipoxygenase (10); p12L0, porcine 12lipoxygenase (11); h15L0, human 15-lipoxygenase (12); rblSL0, rabbit 15lipoxygenase (13); sbLO1, soybean lipoxygenase 1 (14); sbL02. soybean lipoxygenase 2 (15); sbL03, soybean lipoxygenase 3 (16); peL0, pea lipoxygenase (17). 1483
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homologous region among the lipoxygenases (amino acid residues 351 to 401 of the human enzyme), which has been presumed to be the iron-binding domain in the catalytic site [13,16,19]. The other conserved histidine residue is located at position 550. Between amino acid residues 546 to 558, there are 12 out of 13 residues identical in the 5-lipoxygenases and the plant lipoxygenases. As far as the animal lipoxygenases, a histidine residue at position 432 is also conserved. Furthermore, an aspartic acid residue at position 358 and a glutamic acid residue at position 376 are conserved in the homologous region. They are also candidates for the iron ligands. In order to determine which amino acid residues are involved in the iron-binding and the activity, the conserved histidine and acidic amino acid residues were individually replaced by means of site-directed mutagenesis. Replacement of the essential amino acid residues for iron binding is presumed to result in elimination of the activity. Iron ligands were determined on the basis of this criterion.
MATERIALS
AND
METHODS
Materials Restriction enzymes were purchased from either Takara Shuzo or Nippon Gene, except for XmaI from New England Biolabs. Bacterial alkaline phosphatase was from Toyobo. [c@P]dCTP (30OOCi/mmol) was purchased from Du Pont-New England Nuclear. Arachidonic acid (Nu-Chek Prep.) was dissolved in ethanol and stored at -80°C in an atmosphere of nitrogen. Phenylmethylsulfonyl fluoride (Wako) was dissolved in isopropylalcohol and stored at -20°C. Dithiothreitol (Wake) was dissolved in each buffer just before use. Sonicated phosphatidylcholine (Sigma) was also prepared just before use from its aqueous solution. Other chemicals were of reagent grade. The oligonucleotides used as primers for preparing mutants and for DNA sequencing were synthesized with an Applied Biosystems DNA synthesizer Model 392 and purified by OPC columns (Applied Biosystems). Construction of Mutant Plasmid The EcoRI-XbaI fragment of plasmid pH5LOKC [20], which had been constructed for expression of human 5-lipoxygenase cDNA in Escherichia coli, was inserted into the cloning vector pBluescript SK(-) previously treated with EcoRI, XbaI and bacterial alkaline phosphatase. Ligation reaction was carried out using a DNA Ligation Kit (Takara Shuzo). The resultant plasmid is called pBSK(-)SLO. Site-directed mutagenesis was performed on single-stranded pBSK(-)SLO using a Mu&Gene kit (Bio-Rad). The single-stranded pBSK(-)SLO was prepared by using helper phage VCSM13. Table I shows the oligonucleotides used in the preparation of each of the mutants. The changes of the cDNAs were detected either by the fluorescent dideoxy sequencing system, Genesis 2000 (Du Pont), using the oligonucleotide 5’-954CAACAAGATTGTCCCCA970-3’ as a sequencing primer, or by restriction enzyme mapping. The XmaI-XmaI fragment containing the mutation was ligated between the two XmaI sites of pH5LOKC to obtain the mutant 5lipoxygenase cDNA. The mutation at codon 550 was carried out with the &“-Not1 fragment. The mutation was confkmed by dideoxy sequencing using a PGquencing kit (Pharmacia). The sequencing primer for the XmaI-XmaI fragment was 5’954CAACAAGATTGTCCCCA970-3’ and that for the SfiI-Not1 fragment was 5’‘*ssGGCGGTGGGCACGTGCAGATGis”s-3’. 1484
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Table I. Summary of oligonucleotides used for site-directed mutagenesis of human 5-lipoxygenase. Superscripts indicate the nucleotide number of the cDNA for human 5-lipoxygenase. Codon for the replaced amino acid residue is denoted by bold letters. The mismatched position in each oligonucleotide is underlined. Introduction of the mutation was confiied by appearance or disappearance of a restriction site, which is designated by the mark “+” or “-” before the name of the restriction enzyme. n indicates that the mutagenic oligonucleotide did not change restriction pattern of the cDNA so that introduction of the mutation was confirmed by the dideoxy sequencing. Mutant Intact H362N H362.S H367N H367A H372N H372S H3WN* H39OA H399N H399A H432N H432A H55ON H55OA D358N E376Q
Activity (%) 100 15.9 34.3 N.D. N.D. N.D. N.D.
Yield (%) 100 90.2 49.8
Slight NfD. 59.8 N.D. 61.9 94.1 N.D. N.D. 78.0 N.D.
24.0 111 113 83.3
Relative activity (8) 100 18 69 0 0 0 0 + 0 249 0 2: 0 0 94 0
Preparation of E. coli Lysate Containing Human 5-Lipoxygenase E. coli harboring the mutated or intact plasmid was grown at 26°C for 24 hours
in SOOml of TYSG medium (1% Bacto Tryptone (Difco); 0.5% Yeast Extract (Difco); 2% NaCl; 2%(w/v) glycerol and 50% tap water (pH7.8)) with 50mg/l ampicillin (Wako). The cells were harvested by centrifugation (65OOxg, lOmin, 0°C) and rinsed in 50ml of 0.9% NaCl. After re-centrifugation (45OOxg, lOmin, OOC), the pellets were suspended in KPG buffer containing 50mM potassium phosphate (pH7.5), 1OOmMNaCl, 1mM EGTA (Sigma) and lO%(w/v) glycerol at 6.25ml per gram cell and stored at -80°C. The frozen suspension was thawed and treated with O.Smg/ml lysozyme (Wake), 0.5mM phenylmethylsulfonyl fluoride and 0.5mM dithiothreitol on ice for 1 hour. The cells were then disrupted by a Branson Sonifier Model 450 with the microtip at power level of 2.5 for three 20 second periods separated by an interval of 40 seconds. The sonication was carried out in an ice/water bath. The lysate was centrifuged at 174OOxgfor 20 minutes at 0°C and the resultant supematant was used for 5-lipoxygenase assay. Assay for 5-Lipoxygenase
Activity
The standard mixture for 5-lipoxygenase assay (total volume lOOp1) contained 1OOmM Tris-HCl (pH8.0), 2mM CaC12, 2mM ATP, 157pg/ml sonicated phosphatidylcholine and 1mM arachidonic acid. The reaction was initiated by addition of an aliquot of the lysate. After incubation at 30°C for 10 minutes, the reaction was terminated by addition of 3OOpl of cold methanol (-20°C) containing 0.2nmol of 13-HODE (Cayman Chemical) as an internal standard, and lpi of 1M acetic acid. Following centrifugation, the supematant was analyzed by HPLC (Shimadzu) as reported previously [20], except for using methanol/water/acetic acid (80:25:0.01 v/v) as a solvent. 5-lipoxygenase activity was determined from a ratio of sum of 5HPETE and 5HETE peak areas to that of the internal standard. 1485
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Measurement of S-Lipoxygenuse Yield Measurements of the expressed lipoxygenase yields were carried out by the enzyme-linked immunosorbent assay (ELISA). The microtiter plates were coated with adequately diluted lysates containing each of the mutant or the intact enzyme by incubating overnight at 4°C. Anti-5-lipoxygenase rabbit antiserum and alkaline phosphatase-labeled anti-rabbit IgG antibody from goat (Zymed) were used to generate a colored reaction product of the substrate, p-nitrophenyl phosphate (lmg/ml). The color reaction was evaluated from the absorbance at 405nm. The lysate of E. coli transformed with pKC [20] was used as a control. Immunoblot Analysis Proteins of E. coli transformed with the mutated or the intact plasmid were separated by 10% SDS-PAGE and transferred to Immobilon membranes (0.45pm, Millipore). The membranes were incubated with anti-5-lipoxygenase rabbit antiserum. After washing, the membranes were incubated with a goat anti-rabbit IgG serum conjugated with peroxidase (Grganon Teknika). The color reaction was developed in 20mM Tris-HCI (pH7.5), 0.03% hydrogenperoxide and SOpg/ml 3,3’diaminobenzidine tetrahydrochloride.
RESULTS AND DISCUSSION The plasmids containing a mutated cDNA were constructed and used to transform E. coli. Then the lysates of these transformants were assayed for 5Relative enzyme activities of the human 5-lipoxygenase lipoxygenase activity. mutants are summarized in Table II. Expression of the mutated cDNAs was
Table II. Relative activities of the mutant human 5-lipoxygenases. Measurements of the activity and the expressed enzyme yields in lysates prepared from E. coli transformed with the mutated or the intact plasmid were performed as described in Materials and Methods. The data presented here are from one experiment in which duplicate measurements on three or four dilutions of lysate were performed, with intact lipoxygenase as a control. The relative activity of mutated lipoxygenase is designated by the ratio of the activity to the expressed enzyme yield. As for the mutants without activity, the yields were not measured. The experiments were repeated a total of three times with substantially the same results. N.D.; not detected. *Though H390N were active, which is designated by the mark “+I’, the yield was too low to determine. Mutant H362N H362s
H367N H367A H372N H372s H39ON H39OA H399N H399A H432N H432A H55ON H55OA D358N E376Q
Mutagenic
oligonucleotide
5’-l”78TTCCACGTCAACXAGACCATC”‘=3’ S’-1075GACTTCCACGTmCCAGACCATCA’“-3 5’-‘“ACCATCACCMCCTTCTGCGA”‘3-3’
Restriction ll
+PttKZCl n
+SpeI -ApaLI -ApaL
5’-‘m7ATCTGGGTGCGcTCGAGTJ@CTTCCACGTC1’=3’ 5’-1119TCTGGTGTCTL;1rGGTTTTTGG113v-3’
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Figure 2. Immunoblot analysis. Proteins (2Opg each) of E. coli transformed with the mutated or the intact plasmid were separated by 10% SDS-PAGE and transferred to membrane. The membrane was incubated with anti-5-lipoxygenase rabbit antiserum.
confirmed by immunoblot analysis (Figure 2). All mutants were yielded enough for determination of the activity. Three mutated human 5lipoxygenases whose histidine residue was replaced by asparagine residue at a position 367, 372 or 550 (H367N, H372N and H550N)* obviously lacked the enzyme activity. The other asparagine substituted mutants retained the catalytic function to convert arachidonic acid into 5-HPETE at varying levels. Serine residue was also used to substitute for His-362 and His-372. H372S was devoid of the activity like H372N. We concluded that His-372 is essential for 5lipoxygenase activity, whereas Funk et al. reported that the mutation of a histidine residue at a position 362 or 372 to serine residue using the baculovirus/insect cell expression system did not affect the enzyme activity [19]. With respect to the histidine residues other than His-362 and His-372, alanine substituted mutants were constructed to confirm the results obtained with the asparagine substituted mutants. H367A, H432A and H550A exhibited the same characteristics as the corresponding asparagine substituted mutants. Therefore His367 and His-550 are determined to be indispensable amino acid residues for the catalysis of the oxygenation. The other alanine substituted mutants, H390A and H399A, had no activity, while the corresponding mutants replaced by asparagine residue were in active forms. His-390 and His-399 mutants were produced apparently less than the other mutants (Figure 2). Two proline residues (Pro-387 and Pro-391), which are considered to be configurationally “rigid” amino acids, are located in the vicinity of His-390, suggesting steric stringency for amino acid replacements around the proline residues. Thus His-390, maybe also His-399, would play important roles to keep the
*For brevity’s sake, mutations are designated using the one-letter code for the original amino acid and for the replaced one before and after the residue number, respectively. 1487
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folding structure of 5lipoxygenase. The asparagine residues of H39ON and H399N seemed to be able to substitute for the histidine residues because of comparable molecular size of side chain of asparagine residue to that of histidine residue with a corresponding Na atom. Asp-358 and Glu-376 were replaced with an asparagine and a glutamine residue, respectively, since carboxyl groups of these residues also could coordinate to the iron. E376Q lost the activity, while D358N retained. The results indicate that Glu376 might ligate to the iron. Human 5lipoxygenase has 9 conserved amino acid residues as candidates for iron ligands. The putative iron ligands were changed by site-directed mutagenesis. Three histidine residues (His-367, His-372 and His-550) and one glutamic acid residue (Glu-376) were revealed to be essential for the activity. Two histidine residues (His-390 and His-399) seemed to be important for the folding structure of human lipoxygenase. According to reports on the three-dimensional structure of the other non-heme enzymes, it is unlikely that each substituted amino acid residue (asparagine, serine or alanine) directly ligates to the iron [21-281. We propose that the four residues coordinate to the essential iron at least. Two carboxyl oxygen atoms of Glu-376 might provide two ligands to the iron such as other non-heme proteins whose structural analysis have been reported [21-231. It is obscure that coordination system of the iron in human 5-lipoxygenase is similar to the symmetric six-coordination one proposed in soybean enzyme 191, since our preliminary ESR data suggested that the ligand field of human enzyme was different from that of soybean enzyme (unpublished data). A few ligands bound to the iron in human 5lipoxygenase might not be from amino acid residue because many non-heme proteins have proved to possess one or more non-proteinaceous ligands including substrate [25]. For example, azidomyohemerythrin possesses an oxide anion other than an azide anion [22] and ribonucleotide reductase does an oxide anion and two water molecules [23] to complete the iron coordination. Lactoferrin has a carbonate anion
WI. Three amino acid residues (His-367, His-372 and Glu-376) in the homologous region were specified as iron ligands. Although the ligands exist at rather short distances within 10 amino acid sequence, they seems to be able to coordinate to the iron without steric hindrance. In fact, the three dimensional structure of azurin, a blue copper protein, revealed that three of five amino acid ligands (Cys-112, His117 and Met-121), which lie in the interstrand loop, exist at just the same distances as that of human 54ipoxygenase [29]. Our results would help to define the coordination of the non-heme iron of the lipoxygenases, however it remains to confirm the concomitant loss of enzyme activity and iron content in each purified mutated protein. During preparation of this manuscript, a paper appeared reporting evaluation of the role of the conserved amino acid residues [30]. We specified His-550 and Glu1488
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376 as iron ligands other than His-367 and His-372 that were shown to be important for 5lipoxygenase activity in the paper.
1 2 3 4
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