The N-terminal Domain of 5-Lipoxygenase Binds Calcium and Mediates Calcium Stimulation 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. 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 into the of Pharmacia To construct the vector initial in the recombinant E. lipoxygenase proteins is number the the from the construct Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) was by the and and and into of To construct the vector the from the construct Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) were by the and and and into of the vector the human from the plasmid T. S. Rådmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) was by the and and and into of the vector the from the construct T. Rådmark O. Samuelsson B. J. Biochem. 1993; PubMed Scopus Google Scholar) were by the and and into the site of The were by and by on an the Life by at the The N-terminal (amino acids of 5-LO was into the known structure of rabbit reticulocyte (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar) the were calculated with PubMed Scopus Google Scholar). fusion proteins were for the and for the of containing was with of and the were at was by to a of another the were by at for The was in a and at to the was and in of 2 soybean and at for by on were by a at and at for The containing fusion protein in was by in of by at for The was with and in 2 and at for at 2 The was and the was Protein was as described by Anal. Biochem. PubMed Scopus Google Scholar) a protein assay from with bovine serum in as the The of the fusion proteins by this was for 45Ca2+ overlay GST a fusion as a in the was from the E. and GST was to the PubMed Scopus Google Scholar) from and PVDF was as described (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). the membrane was first with containing and for in of a containing and (specific of The membrane was with of in and on at for 2 to at for In two with the same of were and membrane was and the other was amino acid 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 were the from The of were by as described and were into E. and proteins were at as described T. Y.Y. B. Rådmark O. Samuelsson B. J. Biochem. PubMed Scopus Google Scholar). 5-LO proteins were purified from to the chromatography T. Y.Y. B. Rådmark O. Samuelsson B. J. Biochem. PubMed Scopus Google Scholar), by chromatography (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). activity assay of 5-LO were were by in of in the presence of 2 soybean and for at the proteins in the were with on for The protein were by for at in and Amersham Pharmacia into the same The of and 5-LO proteins were by Protein were to a and the The proteins were to Amersham Pharmacia that been on a and were to proteins as described T. Y.Y. B. Rådmark O. Samuelsson B. J. Biochem. PubMed Scopus Google Scholar). 5-LO protein was at in in a of or A substrate containing phosphatidylcholine and in was in a for the of 5-LO and The concentrations in the incubation are in and were from in The the incubation was the incubation was by the of of and acid containing a of the The was and at for and of the was a with acid at The was at and the enzyme activity was calculated from the of the of 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and were in a at and formation of lipoxygenase products containing a was at The incubation of was the same as for the assay, and the concentrations of the are in the The was by the of 5-LO to the A model of the N-terminal amino acids of based the structure of rabbit reticulocyte (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 in In reticulocyte the N-terminal β-barrel is composed of β-strands, the at the is and occur amino acids and in and amino acids and in 5-LO the are to be B. Protein 1999; PubMed Scopus Google Scholar), and was to a structure Refs. R. S. T. M. J. Proteins Struct. Funct. Genet. 1997; PubMed Scopus (68) Google and Proteins Struct. Funct. Genet. 1997; PubMed Scopus Google Scholar). In of the conserved 5-LO and as well as are found in the loop which connect the Our 5-LO which be described as a β-sandwich and resembles of C2 in O. S. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and E.A. J. Biol. 1998; PubMed Scopus Google In the two in the 5-LO model at an to the in the domain are more is that the 5-LO model of the two that have been described for C2 domains E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). is 5-LO and the 5-LO β-sandwich model is similar to the calcium-binding C2 and also the 5-LO model structure This to the 5-LO N-terminal region as a potential Ca2+-binding of the 5-LO N-terminal domain. acid of human 5-LO is with the of rabbit reticulocyte and human and with the human C2 domain. The is to on the 5-LO in are that are conserved in the are that are in human 5-LO and cPLA2. The and the in our 5-LO model and in the C2 human are the and putative to mutagenesis are with O. S. M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and the of these are The are as human rabbit human and The of this was with the of the 5-LO N-terminal β-sandwich domain. is the model in from another 5-LO and two putative domains N-terminal β-sandwich, and the C-terminal were as GST fusion proteins in E. All fusion proteins were in The of the fusion proteins to bind Ca2+ was determined by 45Ca2+ on the protein purification Calmodulin, which can bind Ca2+ was as a for the 45Ca2+ overlay in our to PVDF membrane at with the same as recombinant 5-LO a GST GST bind Ca2+ that the calcium binding of was by the 5-LO part of the fusion bound as the fusion a much weaker Thus, of a of about the same as of and We the of as it be of other proteins that bind calcium by the binding (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). In a the calcium binding for be by the of to overlay that the calcium binding by the overlay was for the The calcium binding of the N-terminal part of 5-LO was with that of the of GST fusion proteins of the N-terminal of (amino acids and of human (amino acids were in E. and in In the calcium binding of these fusion proteins is with that of can be up to of the proteins the of the protein to bind of the fusion proteins was on the overlay In to C2 domain proteins, we the ligands in 5-LO to be located in the loops that connect the are and are in the model and in the 5-LO proteins were in which Ca2+ present in of the loops located on the of the β-sandwich to the were to In E. at showed about the same as 5-LO as by 6 The for Ca2+ of the 5-LO enzyme of the with that of are shown in 6 A. The loop 2 showed a Ca2+ also mutations in the other loops the higher Comparison primarily with led to on the loops on the to the the loop the and β-strands, on the other of the β-sandwich, was also to This to Ca2+ as 5-LO of putative Ca2+ ligands in the N-terminal region of in a The loop 2 was This protein was in and purified the described T. Y.Y. B. Rådmark O. Samuelsson B. J. Biochem. PubMed Scopus Google Scholar). The purified loop 2 showed a decreased Ca2+ with 5-LO. activity was determined in the presence of high concentrations of and similar were in the presence or of It is important to that the concentrations of and be present if these lipids were in the incubation were and the concentrations the of these the activity of 5-LO a maximum at μm the loop 2 maximum activity first at present higher calcium concentrations were to maximum enzyme We that one for this is the of calcium by the activity decreased at μm and determined in the of this be to the of Ca2+ on the of and at high J. Chem. 2000; PubMed Scopus Google Scholar), an effect that have the activity also of the loop 2 at the present this was the of the of 5-LO and the loop 2 to at a in with a high of the of the protein in were and for 5-LO and the loop 2 μm μm and the stimulatory effect of was determined μm and the of was the loop 2 and the and of of at It was described that the effect of Ca2+ on 5-LO activity was in concentrations of and that a basal activity K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) Google Scholar, Biochemistry. 1998; 37: PubMed Scopus Google Scholar). We determined enzyme activity assay, in the presence of concentrations of and these assay Ca2+ the of and Ca2+ as high activity for 5-LO as the loop 2 the initial and of were with the at μm calcium at a high calcium the loop 2 a that was similar in to the obtained for 5-LO at μm substrate was observed for the enzyme and for the loop 2 higher activity than μm in the assay at μm Ca2+ and The same was observed with the loop 2 at higher Ca2+ concentrations μm and indicate that the loop 2 mutations the catalytic site of 5-LO. We recently that concentrations of also stimulate 5-LO K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) Google Scholar). In the of 5-LO and the loop 2 is the of the loop 2 was the that and Ca2+ bind to the same site on 5-LO. The to was for Ca2+ K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) Google Scholar). the was at μm Ca2+ is for 5-LO the obtained with was to Ca2+ to that the to calcium of the loop 2 was to calcium binding this protein was to 45Ca2+ overlay μm shown in association of Ca2+ with purified loop 2 protein and was 5-LO a This well with the activity particularly at concentrations of and were obtained for another of the loop 2 2 was was Thus, the GST fusion of the C-terminal domain of 5-LO a higher for Ca2+ than the loop 2 the C-terminal In it is the C-terminal part of 5-LO binds 5-Lipoxygenase enzyme activity is by and 5-LO was found to bind calcium with a of μm (3Hammarberg T. Rådmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (68) Google Scholar). we that an N-terminal part of 5-LO (amino acids which the putative 5-LO β-barrel β-sandwich, amino acids can bind to the same extent as the intact enzyme. A model of the 5-LO N-terminal domain was based on the structure for rabbit reticulocyte (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar). This model was similar to calcium-binding C2 in and structural 5-LO and C2 domain proteins. A 5-LO and was observed amino acid located in the loops the as calcium ligands in 5-LO. of in loop 2 of the 5-LO β-sandwich in in Ca2+ binding and in the calcium This the calcium of the 5-LO β-sandwich to the calcium of enzyme and based on these we suggest that the putative β-sandwich of 5-LO functions as a calcium-binding C2 domain. C2 domains are of two with E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). the β-barrel domain (6Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. 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