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The N-terminal Domain of 5-Lipoxygenase Binds Calcium and Mediates Calcium Stimulation of Enzyme Activity

2000; Elsevier BV; Volume: 275; Issue: 49 Linguagem: Inglês

10.1074/jbc.m006136200

1083-351X

Tove Hammarberg, Patrick Provost, Bengt Persson, Olof Rádmark,

Genomics, phytochemicals, and oxidative stress

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca2+-binding site to the N-terminal region of the enzyme. Thus, in a 45Ca2+ overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathioneS-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120–673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles β-sandwich C2 domains of other Ca2+-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A2suggested a number of amino acids, located in the loops that connect the β-strands, as potential Ca2+ ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca2+ binding and a requirement for higher Ca2+ concentrations to stimulate enzyme activity. Our data indicate that an N-terminal β-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity. Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca2+-binding site to the N-terminal region of the enzyme. Thus, in a 45Ca2+ overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathioneS-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120–673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles β-sandwich C2 domains of other Ca2+-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A2suggested a number of amino acids, located in the loops that connect the β-strands, as potential Ca2+ ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca2+ binding and a requirement for higher Ca2+ concentrations to stimulate enzyme activity. Our data indicate that an N-terminal β-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity. 5-lipoxygenase cytosolic phospholipase A2 polyvinylidene difluoride glutathioneS-transferase 13(S)-hydroperoxy-9,11-octadecadienoic acid high performance liquid chromatography phosphatidylcholine The leukotrienes are important mediators in asthma as well as in other inflammatory and allergic disorders. 5-Lipoxygenase (5-LO1; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes two initial steps in the cellular production of leukotrienes. Thus, 5-LO converts arachidonic acid into 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently into the unstable epoxide leukotriene A4, which, in turn, is the precursor of the biologically active leukotrienes B4, C4, D4, and E4 (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2327) Google Scholar). Leukotriene B4 stimulates adherence of leukocytes to the vessel wall and is a potent chemotactic agent for these cells. The cysteinyl leukotrienes C4, D4, and E4 increase vascular permeability and are effective constrictors of bronchial smooth muscle. 5-LO also participates in the formation of lipoxins, another group of arachidonate-derived bioactive lipids that are implicated in inflammatory and vascular events (2Serhan C.N. Takano T. Gronert K. Chiang N. Clish C.B. Clin. Chem. Lab. Med. 1999; 37: 299-309Crossref PubMed Scopus (33) Google Scholar). Calcium is a well known 5-LO activator (for reviews, see Refs. 3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar, 4Reddy K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) Google Scholar, 5Rådmark O. Folco G. Samuelsson B. Murphy R.C. Novel Inhibitors of Leukotrienes. Birkhäuser Verlag, Basel, Switzerland1999: 1-22Crossref Google Scholar). In brief, stimuli that elevate the intracellular Ca2+levels were shown to induce cellular 5-LO activity, and several reports have described Ca2+-induced translocation of 5-LO from the cytosol to cellular membranes. More detailed analyses showed an association primarily with the nuclear envelope, where the membrane-bound 5-LO-activating protein (FLAP) is also found and where the substrate arachidonic acid can be released from membrane lipids by cytosolic phospholipase A2 (cPLA2). The stimulatory effect of Ca2+ is evident also for purified 5-LO. The basal enzyme activity, which is observed in the presence of a membrane fraction or lipids, increases up to 10-fold if micromolar concentrations of Ca2+ are included in the assay mixture. 5-LO catalysis has been shown to occur at the lipid/water interface, and Ca2+-dependent binding of 5-LO to phospholipid vesicles has been reported. By several experimental approaches, we have recently demonstrated that 5-LO binds Ca2+ in a reversible manner (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). A Kdclose to 6 μm was determined by equilibrium dialysis, and the stoichiometry of maximum binding averaged around two Ca2+ ions/5-LO molecule. We also showed that binding of calcium increased the hydrophobicity of 5-LO. Thus, a present conception is that calcium stimulates 5-LO activity and leukotriene production by promoting membrane association. The first structural determination of a mammalian 15-lipoxygenase (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar) revealed that, similar to soybean lipoxygenases (7Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (456) Google Scholar, 8Minor W. Steczko J. Stec B. Otwinowski Z. Bolin J.T. Walter R. Axelrod B. Biochemistry. 1996; 35: 10687-10701Crossref PubMed Scopus (394) Google Scholar, 9Skrzypczak-Jankun E. Amzel L.M. Kroa B.A. Funk Jr., M.O. Proteins Struct. Funct. Genet. 1997; 29: 15-31Crossref PubMed Scopus (150) Google Scholar), it is composed of two major domains: a C-terminal domain containing the catalytic site and an N-terminal β-barrel domain. It seems reasonable that this is the overall structure also for 5-LO. The capability of 5-LO to bind more than one calcium ion and the calcium-dependent binding to phospholipids make 5-LO functionally similar to a group of calcium-binding proteins known as C2 domain proteins. The C2 domain is a conserved structural motif that forms an eight-stranded anti-parallel β-sandwich, and C2 domains have been identified in some 70 membrane-interacting proteins, including protein kinase C, synaptotagmin, and cPLA2. C2 domains mediate binding to a variety of ligands such as divalent cations, phospholipids, and proteins (for reviews, see Refs. 10Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar, 11Rizo J. Südhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 12Grobler J.A. Hurley J.H. Nat. Struct. Biol. 1997; 4: 261-262Crossref PubMed Scopus (14) Google Scholar). In this report, we suggest that the N-terminal domain of 5-LO functions as a Ca2+-binding C2 domain. This is based on a model structure, calcium binding analysis, and site-directed mutagenesis of putative calcium ligands in the 5-LO N-terminal domain. All chemicals were of analytical grade and obtained from Merck, unless stated otherwise. Calmodulin, imidazole, phosphatidylcholine (P-3556), soybean lipoxygenase, and detergents were from Sigma. PVDF membranes, bovine serum albumin, and Ready-Gels were from Bio-Rad. Vectors, the GST purification kit,45CaCl2 (specific activity of 10–40 mCi/mg), ATP-agarose, and other chromatography products were from Amersham Pharmacia Biotech. 17(S)-Hydroxy-(13Z,19Z,15E)-docosatrienoic acid was a kind gift from Dr. Mats Hamberg (Karolinska Institutet). 13(S)-Hydroperoxy-9,11-octadecadienoic acid (13-HPOD) was prepared by incubation of linoleic acid with soybean lipoxygenase (13Hamberg M. Anal. Biochem. 1971; 43: 515-526Crossref PubMed Scopus (219) Google Scholar). Linoleic and arachidonic acids were from Nu-Chek Prep Inc. (Elysian, MN). Oligonucleotides were from Cyber Gene (Huddinge, Sweden) and Life Technologies, Inc. To construct the vector pGEX-5X-1-5LO, 5-LO cDNA from the plasmid pT3-5LO (14Zhang Y.Y. Rådmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 485-489Crossref PubMed Scopus (71) Google Scholar) (EcoRI/SalI fragment) was cloned into the EcoRI/SalI restriction sites of pGEX-5X-1 (Amersham Pharmacia Biotech). To construct the vector pGEX-5X-1-5LO(0–128), 2The initial methionine residue in the recombinant E. coli lipoxygenase proteins is designated number 0 throughout the paper. the nucleotide fragment encoding 5-LO-(0–128) from the construct pGBT9-5LO (15Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar) was amplified by polymerase chain reaction using the primers 5′-TACTGAATTCCCCTCCTACACGGTCACCGTGGCCACT-3′ and 5′-GATACTGTCGACTTGCTTGAGAATGTGAATTTGGTCAT-3′, cleaved withEcoRI and SalI, and cloned into theEcoRI/SalI restriction sites of pGEX-5X-1. To construct the vector pGEX-5X-1-5LO(120–673), the nucleotides encoding 5-LO-(120–673) from the construct pGBT9-5LO (15Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar) were amplified by polymerase chain reaction using the primers 5′-GCTCGAATTCGATGACCAAATTCACATTCTCAAGC-3′ and 5′-GATACTGTCGACGATGGCCACACTGTTCGGAATCCGGTCT-3′, cleaved withEcoRI and SalI, and cloned into theEcoRI/SalI restriction sites of pGEX-5X-1. For the vector pGEX-5X-1-hum12LO(0–109), the nucleotide fragment encoding human platelet 12-LO-(0–109) from the plasmid CDM8plT3 (16Izumi T. Hoshiko S. Rådmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7477-7481Crossref PubMed Scopus (101) Google Scholar) was amplified by polymerase chain reaction using the primers 5′-ACTAGGATCCATATGGGCCGCTACCGCATCCGCGT-3′ and 5′-GCGCGCGTCGACTCACTCGGGCAGGCTCAGGATGTCCTCGCCCT-3′, cleaved withBamHI and SalI, and cloned into theBamHI/XhoI restriction sites of pGEX-5X-1. For the vector pGEX-5X-1-rat12LO(0–110), the nucleotides encoding rat brain 12-LO-(0–110) from the construct pT3-12LO (17Watanabe T. Medina J.F. Haeggström J.Z. Rådmark O. Samuelsson B. Eur. J. Biochem. 1993; 212: 605-612Crossref PubMed Scopus (96) Google Scholar) were amplified by polymerase chain reaction using the primers 5′-TATCAGGTCGACCAATGGGTGTCTACCGCATCCGCGTCTCCA-3′ and 5′-GCGCGCGTCGACTCACTCAGGGAGGCTCAGGATGCTTCTGCCCT-3′, cleaved withSalI, and cloned into the SalI restriction site of pGEX-5X-1. The constructs were verified by restriction analysis and by DNA sequencing on an Applied Biosystems Prism 377 sequencer using the Applied Biosystems Prism Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer Life Sciences) carried out by KISeq, Core Facilities at the Karolinska Institutet. The N-terminal sequence (amino acids 1–114) of 5-LO was modeled into the known structure of rabbit reticulocyte 15-LO (Protein Data Bank code 1LOX) (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar) using the program ICM (Version 2.7, Molsoft LLC, San Diego, CA). Multiple sequence alignments were calculated with ClustalW (18Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). Plasmids encoding fusion proteins were transformed intoEscherichia coli cells (BL21 for the GST/5-LO inserts and JM101 for the GST/12-LO inserts). Typically, 250 ml of 2× TY (tryptone-yeast) medium containing ampicillin (150 μg/ml) was inoculated with 2.5 ml of overnight culture, and the cells were grown at 37 °C. At A600 = 0.6–0.9 (after ∼3 h), overexpression was induced by adding isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 200 μm. After another 2.5 h, the cells were harvested by centrifugation at 7700 × g for 10 min. The cell pellet was snap-frozen in a dry ice/ethanol bath and kept at −20 °C until used. Prior to homogenization, the pellet was thawed and incubated in 20 ml of lysis solution (50 mm triethanolamine HCl (pH 8.0), 5 mm EDTA, 2 mm dithiothreitol, 60 μg/ml soybean trypsin inhibitor, and 500 μg/ml lysozyme) at room temperature for 5 min, followed by 25 min on ice. Lysed cells were homogenized by sonication (2 × 15 s) using a Branson sonicator at level 4 and centrifuged at 20,000 × g for 15 min. The pellet, containing fusion protein in inclusion bodies, was washed twice by resuspending in 10 ml of 1% Triton X-100, followed by centrifugation at 15,000 × g for 15 min. The pellet was rinsed with phosphate-buffered saline and then solubilized in urea buffer (50 mm Tris-HCl (pH 7.5), 8 m urea, 2 mm EDTA, 4 mm and dithiothreitol) at room temperature for at least 2 h. The solution was centrifuged, and the remaining non-solubilized material was discharged. Protein concentration was estimated as described by Bradford (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using a protein assay kit from Bio-Rad with bovine serum albumin in urea buffer as the standard. The purity of the fusion proteins achieved by this procedure was sufficient for 45Ca2+ overlay experiments. GST without a fusion partner (used as a control) did not appear in the inclusion bodies, but was recovered from the E. coli supernatant after lysis and homogenization. GST was affinity-purified using GSH-Sepharose (Amersham Phamacia Biotech) according to the manufacturer's instructions. After conventional SDS-polyacrylamide gel electrophoresis (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) using 4–15% pre-cast gradient gels from Bio-Rad (Ready-Gels) and electrotransfer onto PVDF membrane, 45Ca2+overlay was performed as described previously (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). Briefly, the membrane was first washed (3 × 10 min) with 5 mmimidazole buffer (pH 7.4) containing 1 mg/ml octaethylene glycol dodecyl ether (C12E8) and then incubated for 30 min in 15 ml of a solution containing 5 mm imidazole buffer (pH 7.4), 1 mg/ml C12E8, 60 mm KCl, 5 mm MgSO4, and 10 μm45CaCl2 (specific radioactivity of 1 mCi/μmol). The membrane was washed three times with 15 ml of 30% (v/v) ethanol in deionized water and dried on Whatman filter paper at room temperature for 2 h before exposure to Fuji RX film at −70 °C for 35–50 h. In general, two gels with the same set of samples were run and blotted simultaneously. One membrane was Coomassie Blue-stained, and the other was subjected to45Ca2+ overlay. Selected amino acid codons (in plasmid pT3-5LO (14Zhang Y.Y. Rådmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 485-489Crossref PubMed Scopus (71) Google Scholar)) were mutated using the QuikChangeTMkit from Stratagene. The DNA sequences of mutated plasmids were confirmed by sequence analysis as described above. Mutant and wild-type DNAs were transformed into E. coli MV1190, and proteins were expressed at 27 °C as described previously (21Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar). 5-LO proteins were purified from 1-liter cultures according to the ATP affinity chromatography procedure (21Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar), followed by MonoQ anion exchange chromatography (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). For activity assay of crude 5-LO samples, 10-ml cultures were grown. Cells were homogenized by sonication in 1 ml of 50 mmtriethanolamine (pH 8.0) in the presence of 2 mmdithiothreitol, 60 μg/ml soybean trypsin inhibitor, and 0.5 mg/ml lysozyme. After centrifugation for 20 min at 16,000 ×g, the proteins in the supernatant were precipitated with 60% ammonium sulfate on ice for 45 min. The protein precipitates were collected by centrifugation for 25 min at 16,000 × g, resuspended in Chelex-treated Tris-HCl (50 mm, pH 7.5), and gel-filtered through Sephadex G-25 (NAP-5 columns, Amersham Pharmacia Biotech) into the same buffer. The expression levels of wild-type and mutant 5-LO proteins were analyzed by Western blotting. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis using a Mini-Protean system (Bio-Rad) and the 4–15% pre-cast gels. The separated proteins were then transferred to nitrocellulose membranes (Hybond-C, Amersham Pharmacia Biotech). Rabbit anti-5-LO antiserum that had been affinity-purified on a 5-LO-Sepharose column and alkaline phosphatase-conjugated goat anti-rabbit IgG were used to visualize 5-LO-immunoreactive proteins as described previously (21Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar). 5-LO protein was incubated at room temperature in Eppendorf tubes in a total volume of 50 or 100 μl. A substrate mixture containing arachidonate, 13-HPOD, phosphatidylcholine (PC), and ATP in 50 mm Tris-HCl (pH 7.5) was mixed (by immersion in a sonication bath for 1 min) before the addition of EDTA/EGTA, 5-LO protein, and CaCl2. The final concentrations in the incubation solution are specified in each figure legend. Arachidonate, 13-HPOD, and PC were added from stock solutions in ethanol. The final ethanol concentration during the incubation was 3.5% (v/v). After 10 min, the incubation was terminated by the addition of 3 volumes of cold stop solution (67% acetonitrile, 33% water, and 0.2% acetic acid containing a 3.3 μmconcentration of the internal standard 17(S)-hydroxy-(13Z,19Z,15E)-docosatrienoic acid). The sample was mixed and centrifuged at 15,000 ×g for 10 min, and 100 μl of the supernatant was injected onto a C18 reverse-phase HPLC column (Waters Nova Radial Pak) eluted with acetonitrile/water/acetic acid (65:35:0.2, v/v) at 1.2 ml/min. The eluate was monitored at 234 nm, and the enzyme activity was calculated from the sum of the peak areas of 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and 5(S)-hydroxyeicosatetraenoic acid. Incubations were performed in a quartz cuvette at room temperature, and formation of lipoxygenase products containing a conjugated diene was monitored at 236 nm. The incubation solution (total volume of 500 μl) was the same as for the HPLC assay, and the concentrations of the various components are given in the figure legends. The reaction was started by the addition of 5-LO (0.25 μg) to the cuvette. A model of the N-terminal 114 amino acids of 5-LO, based upon the crystal structure of rabbit reticulocyte 15-LO (Protein Data Bank code 1LOX) (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar), is presented in Fig. 1. In reticulocyte 15-LO, the N-terminal β-barrel is composed of eight β-strands, the last ending at residue 111. Since the sequence identity is 34% and only few insertions/deletions occur between amino acids 1 and 111 in 15-LO and amino acids 1 and 114 in 5-LO (see Fig. 3), the structures are expected to be homologous (cf. Ref. 22Rost B. Protein Eng. 1999; 12: 85-94Crossref PubMed Scopus (1221) Google Scholar), and molecular modeling was therefore expected to give a plausible structure (cf. Refs. 23Abagyan R. Batalov S. Cardozo T. Totrov M. Webber J. Zhou Y. Proteins Struct. Funct. Genet. 1997; Suppl. 1: 29-37Crossref PubMed Scopus (68) Google Scholar and 24Martin A.C. MacArthur M.W. Thornton J.M. Proteins Struct. Funct. Genet. 1997; Suppl. 1: 14-28Crossref PubMed Scopus (105) Google Scholar). In support of the model, residues not conserved between 5-LO and 15-LO, as well as gaps, are found mainly in the loop regions, which connect the β-strands. Our 5-LO model, which may be described as a β-sandwich (Fig. 1 (upper) and Fig. 2), resembles reported structures of C2 domains, e.g. in cPLA2 (Fig. 1,lower) (Protein Data Bank codes 1RLW (25Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) and 1BCI(26Xu G.Y. McDonagh T., Yu, H.A. Nalefski E.A. Clark J.D. Cumming D.A. J. Mol. Biol. 1998; 280: 485-500Crossref PubMed Scopus (100) Google Scholar)). In comparison, the two β-sheets in the 5-LO model cross at an angle close to 45°, whereas the β-sheets in the cPLA2C2 domain are more parallel. Another difference is that the 5-LO model did not exhibit any of the two distinct topologies that have been described for C2 domains (10Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). There is no pronounced sequence similarity between 5-LO and cPLA2 (Fig. 3); nevertheless, the 5-LO β-sandwich model is similar to the calcium-binding C2 domains, and also the 5-LO model structure contains apparent ligand-binding loops. This encouraged us to examine the 5-LO N-terminal region as a potential Ca2+-binding domain.Figure 3Sequence alignment of the 5-LO N-terminal domain. Amino acid sequence 1–114 of human 5-LO (hum5LO) is aligned with the corresponding sequences of rabbit reticulocyte 15-LO (rab15LO), rat brain 12-LO (rat12LO), and human platelet 12-LO (hum12LO) and with the human cPLA2 C2 domain. The cPLA2sequence is aligned according to its optimal three-dimensional superposition on the 5-LO model. Highlighted in gray are residues that are conserved in the lipoxygenases. Highlighted inblack are residues that are identical in human 5-LO and cPLA2. The lines above and below the sequences represent β-strands in our 5-LO model and in the cPLA2 C2 domain, respectively. For human 5-LO, residue numbers are denoted above the sequence, and putative Ca2+ligands subjected to mutagenesis are marked with arrows; For cPLA2, arrows denote reported Ca2+ligands (25Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and the positions of these residues are given. The GenBankTM /EBI Data Bank accession numbers are as follows: human 5-LO, J03571; rabbit 15-LO, M27214; rat 12-LO, L06040; human 12-LO, M38792; and cPLA2, M72393. The alignment of this figure was created with the program PRALIN.View Large Image Figure ViewerDownload (PPT)Figure 2Model of the 5-LO N-terminal β-sandwich domain. Shown is the model in Fig. 1(upper) viewed from another angle.View Large Image Figure ViewerDownload (PPT) 5-LO and its two putative domains (the N-terminal β-sandwich, 5-LO(N), and the C-terminal domain, 5-LO(C)) were expressed as GST fusion proteins in E. coli (Fig. 4 A). All three fusion proteins were recovered in insoluble inclusion bodies after cell homogenization. The ability of the fusion proteins to bind Ca2+ was determined by 45Ca2+ overlay, performed directly on the solubilized inclusion bodies without further protein purification (Fig. 4 B). Calmodulin, which can bind four Ca2+ ions/protein molecule, was used as a positive control for the 45Ca2+ overlay procedure (Fig. 4 B, lane 7). However, in our hands, calmodulin (up to 0.5 nmol) blotted onto PVDF membrane stained weakly (or not at all) with Coomassie Blue. GST/5-LO gave the same response as recombinant 5-LO without a GST tag (lanes 4 and5). GST alone did not bind Ca2+ (lane 3), confirming that the calcium binding of GST/5-LO was caused by the 5-LO part of the fusion protein. GST/5-LO(N) bound Ca2+as strongly as the entire GST/5-LO fusion protein, whereas GST/5-LO(C) gave a much weaker response (lanes 1, 2, and4). Thus, 0.7 nmol of GST/5-LO(C) did not give a detectable signal, but 2.1 nmol of GST/5-LO(C) gave about the same response as 0.7 nmol of GST/5-LO (lanes 2, 5, and 8). We consider the response of GST/5-LO(C) as weak, but it may be significant since comparable amounts of other proteins that do not bind calcium gave bright spots, probably by reducing the background binding (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). In a control experiment, the calcium binding for GST/5-LO(N) could be competed by the addition of cold CaCl2 to the45Ca2+-containing overlay buffer, indicating that the calcium binding visualized by the overlay technique was not unique for the 45Ca isotope (data not shown). The calcium binding of the N-terminal part of 5-LO was compared with that of the corresponding regions of 12-lipoxygenases. GST fusion proteins of the N-terminal parts (cf. Fig. 2) of rat 12-LO (amino acids 0–110) and of human platelet 12-LO (amino acids 0–109) were expressed in E. coli and recovered in inclusion bodies. In Fig. 5, the calcium binding of these 12-LO fusion proteins is compared with that of GST/5-LO(N). As can be seen, up to 2.6 nmol of the 12-LO(N) proteins did not share the ability of the 5-LO(N) protein to bind calcium. However, when ∼4 nmol (or more) of the 12-LO fusion proteins was loaded, dark spots appeared on the overlay films (data not shown). In analogy to documented C2 domain proteins, we expected the Ca2+-chelating ligands in 5-LO to be located in the loops that connect the β-sheets. Several ligand candidates are present. Among these, Asp and Glu residues are depicted ingray in the model (Fig. 1, upper) and marked witharrows in the 5-LO sequence (Fig. 3). Four mutant proteins were produced in which Ca2+ ligand candidates present in each of the four loops located on the side of the β-sandwich opposite to the N terminus were mutated to Ala (TableI). In E. coli cells grown at 27 °C, all four mutants showed about the same expression levels as wild-type 5-LO as judged by Western blotting (Fig. 6 B). The dose responses for Ca2+ stimulation of the 5-LO enzyme activities of the mutants, compared with that of wild-type 5-LO, are shown in Fig. 6 A. The loop 2 mutant showed a clearly reduced Ca2+ response, but also mutations in the other loops shifted the response slightly toward higher Ca2+concentrations. Comparison primarily with cPLA2 (see Fig. 3) led us to focus on the loops on the side opposite to the N terminus. However, the loop between the fourth and fifth β-strands, on the other side of the β-sandwich, was also subjected to mutagenesis. This mutant (D58A/E59A/E60A/E63A) responded to Ca2+ stimulation as did wild-type 5-LO (data not shown).Table IMutants of putative Ca2+ -chelating ligands in the N-terminal region of 5-LOCa2+ ligand candidatesMutationsLoop 1Asp18, Asp19D18A/D19ALoop 2Asn43, Asp44, Glu46N43A/D44A/E46ALoop 3Asp78, Asp79D78A/D79ALoop 4Asp106, Glu108D106A/E108A Open table in a new tab The loop 2 mutant was studied further. This protein was expressed in l-liter scale and purified using the previously described ATP-agarose procedure (21Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar). The purified loop 2 mutant showed a markedly decreased Ca2+ response compared with wild-type 5-LO. When activity was determined in the presence of high concentrations of PC (250 μg/ml) and arachidonate (100 μm), similar patterns were seen in the presence or absence of 1 mm ATP (Fig. 7). It is important to note that the given concentrations of arachidonate and PC would be present if these lipids were truly dissolved in the incubation mixtures. Instead, micelles were formed, and the indicated concentrations reflect the compositions of these micelles. Without ATP (Fig. 7 A), the activity of wild-type 5-LO reached a maximum at ∼2–10 μm added Ca2+, whereas the loop 2 mutant reached maximum activity first at 100 μmCa2+. With ATP present (Fig. 7 B), higher added calcium concentrations were required to reach maximum enzyme activation. We believe that one reason for this is the chelation of calcium by ATP. For wild-type 5-LO, the activity then decreased substantially at 100 μm and above when determined in the absence of ATP (Fig. 7 A). Possibly, this could be related to the effects of Ca2+ on the aggregation of PC and arachidonate at high levels (27Lopez-Nicolas J.M. Martinez R.B. Garcia-Carmona F. J. Agric. Food Chem. 2000; 48: 292-296Crossref PubMed Scopus (3) Google Scholar), an effect that might have reduced the activity also of the loop 2 mutant at the highest Ca2+concentrations. With ATP present (Fig. 7 B), this tendency was less pronounced. At the highest concentration of Ca2+(1 mm), the activities of wild-type 5-LO and the loop 2 mutant tended to become quite similar. Also at a low PC concentration in combination with a high concentration of Ca2+, the specific activities of the protein preparations used in Fig. 7 were similar: 21 and 16 μmol/mg/10 min for wild-type 5-LO and the loop 2 mutant, respectively (25 μg/ml PC, 100 μm arachidonate, 10 μm 13-HPOD, 1.9 mm CaCl2, 1.2 mm EDTA, 5 mm ATP, and 15 mm2-mercaptoethanol). When the stimulatory effect of ATP alone was determined (25 μg/ml PC, 100 μm arachidonate, 1 mm EDTA, and 0–1 mm ATP, without the addition of