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Neutrophil-activating Peptide-2 and Melanoma Growth-stimulatory Activity Are Functional as Monomers for Neutrophil Activation

1997; Elsevier BV; Volume: 272; Issue: 3 Linguagem: Inglês

10.1074/jbc.272.3.1725

1083-351X

Krishna Rajarathnam, Cyril M. Kay, Béatrice Dewald, Marlene Wolf, Marco Baggiolini, Ian Clark‐Lewis, Brian D. Sykes,

S100 Proteins and Annexins

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH → NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 μM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of ∼5 μM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers. Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH → NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 μM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of ∼5 μM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers. INTRODUCTIONRecruitment and accumulation of circulating leukocytes at the site of inflammation are mediated in part by a family of small molecular weight proteins called chemokines (1Miller M.D. Krangel M.S. Crit. Rev. Immunol. 1992; 12: 17-46PubMed Google Scholar, 2Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2258) Google Scholar, 3Furie M.B. Randolph G.J. Am. J. Pathol. 1995; 146: 1287-1301PubMed Google Scholar). They have four conserved cysteines and are called either CXC or CC chemokines, depending on whether the first two cysteines are separated by one amino acid (CXC) or are adjacent (CC). Functionally, the CXC chemokines predominantly attract neutrophils, whereas CC chemokines attract monocytes, lymphocytes, and eosinophils.Three well studied proteins of the CXC chemokine family are interleukin-8 (IL-8) 1The abbreviations used are: IL-8interleukin-8MGSAmelanoma growth-stimulatory activityNAP-2neutrophil-activating peptide-2RANTESregulated upon activation, normal T-cell expressed, and presumably secreted. , melanoma growth-stimulatory activity (MGSA), and neutrophil-activating peptide-2 (NAP-2). The structure of IL-8 has been solved by both NMR and x-ray methods, and both approaches indicated it to be a dimer (4Clore G.M. Appella E. Yamada M. Matsushima K. Gronenborn A.M. Biochemistry. 1990; 29: 1689-1696Crossref PubMed Scopus (413) Google Scholar, 5Baldwin E.T. Weber I.T. Charles R. Xuan J.-C. Appella E. Yamada M. Matsushima K. Edwards B.F.P. Clore G.M. Gronenborn A.M. Wlodawer A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 502-506Crossref PubMed Scopus (334) Google Scholar). NMR studies of MGSA (6Kim K.-S. Clark-Lewis I. Sykes B.D. J. Biol. Chem. 1994; 269: 32909-32915Abstract Full Text PDF PubMed Google Scholar, 7Fairbrother W.J. Reilly D. Colby T.J. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar) and x-ray studies of a variant of NAP-2 (M6L) (8Malkowski M.G. Wu J.Y. Lazar J.B. Johnson P.H. Edwards B.F.P. J. Biol. Chem. 1995; 270: 7077-7087Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) indicated them to be a dimer and tetramer, respectively. The tertiary and quaternary structures of IL-8 and MGSA are similar, and NAP-2 adopts a similar structure while showing an additional level of complexity by forming a tetramer. The tertiary structure consists of a series of turns and loops in the N-terminal region followed by three β-strands and a C-terminal α-helix. In the dimeric structure, the three β-strands from each monomer form a six-stranded β-sheet with the helices transversing the β-sheet. On the basis of the dimeric structure of IL-8, it was previously thought that dimerization is essential for function. However, we subsequently demonstrated that an analog in which Leu-25 was substituted with N-methylleucine (L25NMe) was monomeric and had similar functional properties as the native IL-8 (19Clark-Lewis I. Dewald B. Loetscher M. Moser B. Baggiolini M. J. Biol. Chem. 1994; 269: 16075-16081Abstract Full Text PDF PubMed Google Scholar). The structure of this synthetic IL-8 monomer was solved by 1H NMR spectroscopy and was shown to be largely similar to that of the monomeric unit in the NMR and x-ray structures of the native dimer (16Clark-Lewis I. Schumacher C. Baggiolini M. Moser B. J. Biol. Chem. 1991; 266: 23128-23134Abstract Full Text PDF PubMed Google Scholar). Taken together, these observations suggested that dimerization of IL-8 is essential neither for structural integrity nor for functional activation.Two receptors (IL-8R1 and IL-8R2) that bind CXC chemokines have been identified and characterized in neutrophils, and they belong to the superfamily of seven transmembrane domain-containing proteins that bind to G-proteins. IL-8R2 binds IL-8, MGSA, and NAP-2 with equal affinity. In contrast, IL-8R1 binds only IL-8 with high affinity, but it does bind MGSA and NAP-2 with low affinity (9Rajarathnam K. Sykes B.D. Kay C.M. Dewald B. Geiser T. Baggiolini M. Clark-Lewis I. Science. 1994; 264: 90-92Crossref PubMed Scopus (282) Google Scholar, 10Rajarathnam K Clark-Lewis I. Sykes B.D. Biochemistry. 1995; 34: 12983-12990Crossref PubMed Scopus (87) Google Scholar, 11Gayle III, R.B. Sleath P.R. Srinivason S. Birks C.W. Weerawarna K.S. Cerretti D.P. Kozlosky C.J. Nelson N. Bos T.V. Beckmann M.P. J. Biol. Chem. 1993; 268: 7283-7289Abstract Full Text PDF PubMed Google Scholar). The N-terminal residues Glu-4, Leu-5, and Arg-6 2The numbering corresponds to the sequence in IL-8. The corresponding positions are 2, 3 and 4 in NAP-2 and 6, 7 and 8 in MGSA. (the "ELR" motif) (Fig. 1) are conserved and are absolutely essential for receptor binding in MGSA, NAP-2, and IL-8 (12LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar, 13Schumacher C. Clark-Lewis I. Baggiolini M. Moser B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10542-10546Crossref PubMed Scopus (148) Google Scholar, 14Hesselgesser J. Chitnis C.E. Miller L.H. Yansura D.G. Simmons L.C. Fairbrother W.J. Kotts C. Wirth C. Gillece-Castro B.L. Horuk R. J. Biol. Chem. 1995; 270: 11472-11476Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Differential binding to the two receptors has been attributed to a region of about 10 residues immediately preceding the first β-strand, and these residues are similarly located away from the interface (17Hébert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar, 18Schraufstätter I.U. Ma M. Oades Z.G. Barritt D.S. Cochrane C.G. J. Biol. Chem. 1995; 270: 10428-10431Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Mutation of charged residues at the dimer interface in IL-8 had no affect on activity (18Schraufstätter I.U. Ma M. Oades Z.G. Barritt D.S. Cochrane C.G. J. Biol. Chem. 1995; 270: 10428-10431Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Thus, functional data indicate that the residues at the dimer interface are not directly involved in receptor binding.In order to address the relevance of higher order states observed in structures of NAP-2 and MGSA, monomeric analogs of NAP-2 and MGSA were synthesized using a similar strategy employed in the synthesis of the monomeric IL-8 (19Clark-Lewis I. Dewald B. Loetscher M. Moser B. Baggiolini M. J. Biol. Chem. 1994; 269: 16075-16081Abstract Full Text PDF PubMed Google Scholar). For MGSA, Val-26 was substituted with N-methylvaline, and for NAP-2, Leu-22 was substituted with N-methylleucine. In addition to disrupting hydrogen bonding across the dimer interface, the modification also introduces a bulky methyl group, which prevents monomers from coming close to each other. We show here that the monomeric analogs of NAP-2 and MGSA have an activity similar to that of the corresponding native proteins for neutrophil elastase release and induce a similar rise in free Ca2+ in Jurkat cells transfected with either IL8-R1 or IL8-R2, indicating that dimerization is not essential for receptor binding and functional activation. Furthermore, calculations of Kd by sedimentation equilibrium for both NAP-2 and MGSA indicate that they are predominantly monomeric at functional nanomolar concentrations. INTRODUCTIONRecruitment and accumulation of circulating leukocytes at the site of inflammation are mediated in part by a family of small molecular weight proteins called chemokines (1Miller M.D. Krangel M.S. Crit. Rev. Immunol. 1992; 12: 17-46PubMed Google Scholar, 2Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2258) Google Scholar, 3Furie M.B. Randolph G.J. Am. J. Pathol. 1995; 146: 1287-1301PubMed Google Scholar). They have four conserved cysteines and are called either CXC or CC chemokines, depending on whether the first two cysteines are separated by one amino acid (CXC) or are adjacent (CC). Functionally, the CXC chemokines predominantly attract neutrophils, whereas CC chemokines attract monocytes, lymphocytes, and eosinophils.Three well studied proteins of the CXC chemokine family are interleukin-8 (IL-8) 1The abbreviations used are: IL-8interleukin-8MGSAmelanoma growth-stimulatory activityNAP-2neutrophil-activating peptide-2RANTESregulated upon activation, normal T-cell expressed, and presumably secreted. , melanoma growth-stimulatory activity (MGSA), and neutrophil-activating peptide-2 (NAP-2). The structure of IL-8 has been solved by both NMR and x-ray methods, and both approaches indicated it to be a dimer (4Clore G.M. Appella E. Yamada M. Matsushima K. Gronenborn A.M. Biochemistry. 1990; 29: 1689-1696Crossref PubMed Scopus (413) Google Scholar, 5Baldwin E.T. Weber I.T. Charles R. Xuan J.-C. Appella E. Yamada M. Matsushima K. Edwards B.F.P. Clore G.M. Gronenborn A.M. Wlodawer A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 502-506Crossref PubMed Scopus (334) Google Scholar). NMR studies of MGSA (6Kim K.-S. Clark-Lewis I. Sykes B.D. J. Biol. Chem. 1994; 269: 32909-32915Abstract Full Text PDF PubMed Google Scholar, 7Fairbrother W.J. Reilly D. Colby T.J. Hesselgesser J. Horuk R. J. Mol. Biol. 1994; 242: 252-270Crossref PubMed Scopus (81) Google Scholar) and x-ray studies of a variant of NAP-2 (M6L) (8Malkowski M.G. Wu J.Y. Lazar J.B. Johnson P.H. Edwards B.F.P. J. Biol. Chem. 1995; 270: 7077-7087Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) indicated them to be a dimer and tetramer, respectively. The tertiary and quaternary structures of IL-8 and MGSA are similar, and NAP-2 adopts a similar structure while showing an additional level of complexity by forming a tetramer. The tertiary structure consists of a series of turns and loops in the N-terminal region followed by three β-strands and a C-terminal α-helix. In the dimeric structure, the three β-strands from each monomer form a six-stranded β-sheet with the helices transversing the β-sheet. On the basis of the dimeric structure of IL-8, it was previously thought that dimerization is essential for function. However, we subsequently demonstrated that an analog in which Leu-25 was substituted with N-methylleucine (L25NMe) was monomeric and had similar functional properties as the native IL-8 (19Clark-Lewis I. Dewald B. Loetscher M. Moser B. Baggiolini M. J. Biol. Chem. 1994; 269: 16075-16081Abstract Full Text PDF PubMed Google Scholar). The structure of this synthetic IL-8 monomer was solved by 1H NMR spectroscopy and was shown to be largely similar to that of the monomeric unit in the NMR and x-ray structures of the native dimer (16Clark-Lewis I. Schumacher C. Baggiolini M. Moser B. J. Biol. Chem. 1991; 266: 23128-23134Abstract Full Text PDF PubMed Google Scholar). Taken together, these observations suggested that dimerization of IL-8 is essential neither for structural integrity nor for functional activation.Two receptors (IL-8R1 and IL-8R2) that bind CXC chemokines have been identified and characterized in neutrophils, and they belong to the superfamily of seven transmembrane domain-containing proteins that bind to G-proteins. IL-8R2 binds IL-8, MGSA, and NAP-2 with equal affinity. In contrast, IL-8R1 binds only IL-8 with high affinity, but it does bind MGSA and NAP-2 with low affinity (9Rajarathnam K. Sykes B.D. Kay C.M. Dewald B. Geiser T. Baggiolini M. Clark-Lewis I. Science. 1994; 264: 90-92Crossref PubMed Scopus (282) Google Scholar, 10Rajarathnam K Clark-Lewis I. Sykes B.D. Biochemistry. 1995; 34: 12983-12990Crossref PubMed Scopus (87) Google Scholar, 11Gayle III, R.B. Sleath P.R. Srinivason S. Birks C.W. Weerawarna K.S. Cerretti D.P. Kozlosky C.J. Nelson N. Bos T.V. Beckmann M.P. J. Biol. Chem. 1993; 268: 7283-7289Abstract Full Text PDF PubMed Google Scholar). The N-terminal residues Glu-4, Leu-5, and Arg-6 2The numbering corresponds to the sequence in IL-8. The corresponding positions are 2, 3 and 4 in NAP-2 and 6, 7 and 8 in MGSA. (the "ELR" motif) (Fig. 1) are conserved and are absolutely essential for receptor binding in MGSA, NAP-2, and IL-8 (12LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar, 13Schumacher C. Clark-Lewis I. Baggiolini M. Moser B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10542-10546Crossref PubMed Scopus (148) Google Scholar, 14Hesselgesser J. Chitnis C.E. Miller L.H. Yansura D.G. Simmons L.C. Fairbrother W.J. Kotts C. Wirth C. Gillece-Castro B.L. Horuk R. J. Biol. Chem. 1995; 270: 11472-11476Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Differential binding to the two receptors has been attributed to a region of about 10 residues immediately preceding the first β-strand, and these residues are similarly located away from the interface (17Hébert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar, 18Schraufstätter I.U. Ma M. Oades Z.G. Barritt D.S. Cochrane C.G. J. Biol. Chem. 1995; 270: 10428-10431Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Mutation of charged residues at the dimer interface in IL-8 had no affect on activity (18Schraufstätter I.U. Ma M. Oades Z.G. Barritt D.S. Cochrane C.G. J. Biol. Chem. 1995; 270: 10428-10431Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Thus, functional data indicate that the residues at the dimer interface are not directly involved in receptor binding.In order to address the relevance of higher order states observed in structures of NAP-2 and MGSA, monomeric analogs of NAP-2 and MGSA were synthesized using a similar strategy employed in the synthesis of the monomeric IL-8 (19Clark-Lewis I. Dewald B. Loetscher M. Moser B. Baggiolini M. J. Biol. Chem. 1994; 269: 16075-16081Abstract Full Text PDF PubMed Google Scholar). For MGSA, Val-26 was substituted with N-methylvaline, and for NAP-2, Leu-22 was substituted with N-methylleucine. In addition to disrupting hydrogen bonding across the dimer interface, the modification also introduces a bulky methyl group, which prevents monomers from coming close to each other. We show here that the monomeric analogs of NAP-2 and MGSA have an activity similar to that of the corresponding native proteins for neutrophil elastase release and induce a similar rise in free Ca2+ in Jurkat cells transfected with either IL8-R1 or IL8-R2, indicating that dimerization is not essential for receptor binding and functional activation. Furthermore, calculations of Kd by sedimentation equilibrium for both NAP-2 and MGSA indicate that they are predominantly monomeric at functional nanomolar concentrations.