Identification of the Glycosaminoglycan Binding Site of the CC Chemokine, MCP-1

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1β/CCL4 (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (654) Google Scholar). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production. In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1β/CCL4 (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (654) Google Scholar). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production. Chemokines are small 8-10-kDa proteins that control the migration of specific leukocyte populations during inflammatory responses, hematopoeisis, and routine immune surveillance. They exert their biological effects by binding to seven transmembrane G protein-coupled receptors on leukocytes, triggering changes in the cytoskeleton and in adhesive interactions with the extracellular matrix and cell surfaces to produce locomotion (2Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 3Baggiolini M. Nature. 1998; 392: 565-568Crossref PubMed Scopus (2407) Google Scholar, 4Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2109) Google Scholar). Inflammatory chemokines can also stimulate further cellular activation, resulting in destructive processes such as lysosomal enzyme release, generation of toxic products from the respiratory burst, and apoptosis (5Baggiolini M. Kernen P. Deranleau D.A. Dewald B. Biochem. Soc. Trans. 1991; 19: 55-59Crossref PubMed Scopus (15) Google Scholar). Control over cell populations that are mobilized from the vasculature is determined in large part by the specific chemokines that are secreted and the corresponding receptors that are expressed on the migrating cells. To date, 45 human chemokines and 18 receptors have been discovered. They segregate into four families (CXC, CC, CX3C, and C) based on the pattern of cysteine residues in the ligands (6Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1130) Google Scholar). They can be further classified as either inducible chemokines, which are expressed as a consequence of physiological stress and inflammation (7D'Ambrosio D. Panina-Bordignon P. Sinigaglia F. J. Immunol. Methods. 2003; 273: 3-13Crossref PubMed Scopus (148) Google Scholar), or as constitutive chemokines which are the homeostatic ligands responsible for routine leukocyte trafficking and developmental processes (8Kunkel E. Butcher E. Adv. Exp. Med. Biol. 2002; 512: 65-72Crossref PubMed Scopus (14) Google Scholar, 9Butcher E.C. Picker L.J. Science. 1996; 272: 60-66Crossref PubMed Scopus (2519) Google Scholar). In general, many chemokines bind multiple receptors and many receptors bind multiple chemokines, creating the potential for combinatorial diversity in their functions. Traditionally, the overlapping receptor-binding capacity of chemokines has been perceived as redundant (10Mantovani A. Immunol. Today. 1999; 20: 254-257Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). However, there is mounting evidence that different chemokines interacting with the same receptor may activate different sets of signaling pathways, leading to finely tuned cellular responses (11Devalaraja M.N. Richmond A. Trends Pharmacol. Sci. 1999; 20: 151-156Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Moreover, some chemokines act as agonists of some receptors and antagonists of others (12Dairaghi D.J. Fan R.A. McMaster B.E. Hanley M.R. Schall T.J. J. Biol. Chem. 1999; 274: 21569-21574Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 13Rosenkilde M.M. Kledal T.N. Brauner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Clark-Lewis I. M. M. PubMed Scopus Google Scholar, P. S. Clark-Lewis I. M. 2003; PubMed Scopus Google Scholar). 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In to the which are is thought to the binding capacity of a The by concentrations of with in for at The to with and with the addition of of the at in for to a B. A. D. J. 2002; PubMed Scopus Google Scholar). heparin molecular into a of in and the in of the on a and of and and a The binding by to a the resulting as of the in the to a changes in to be in vivo cell as (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (654) Google Scholar). with of or with of in the same 18 the the and the with a on a at of at proteins at a of that in and with specific of heparin and heparin from to as well as in the in the concentrations determined from the Biochem. PubMed Scopus Google and molecular specific to at of the at equilibrium the in and in with of the based on the from to the the at is the at is the is the specific is the of the is the molecular of the and is the to and the molecular and for the to to the the to the are by the to or is the for the of is the for the of the at the of during the of a The for the the of the and the by and The specific of determined from to the of and and as and Google Scholar). The specific of the as a of the specific of and heparin P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), The to be The of the by the and by for of the of and heparin with of the A. A. H. 2002; PubMed Scopus Google Scholar, D.A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. J. Biochem. PubMed Scopus Google Scholar). The to is that and in with be there is heparin with and in to some of the into The to at for at which with of in the same to to with and of the and the to for at and to and on a products by of the GAG Binding on alanine mutants to the GAG binding site of To that the residues with surface mutants that receptor binding we to S. D. I. A. M. S. D. B. Handel T.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. S. D. J. S. D. M. B. J. I. Handel T.M. 1999; PubMed Scopus Google Scholar). In the present study, we also of the as for the changes are localized to the of the of the mutants in for of the and that of The mutants in a of in GAG binding heparin as a Although heparin has been as a cell surface M. A. M. J. Biol. 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J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Using we to for the of the mutants with heparin The for in with a S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The also that and are in the to that for a to the addition of the the of some of the basic residues in or which the residues as The mutants and In to the heparin the that and has a in with from the assays. a physiological we several of the mutants in in vivo (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (654) Google Scholar). with the small effects observed for mutants in the in binding mutants on in vivo with The to which to as demonstrated (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. 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A. H. 2002; PubMed Scopus Google Scholar, D.A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. J. Biochem. PubMed Scopus Google Scholar). the products of cross-linking and heparin of different from to of In with sedimentation equilibrium we a corresponding to the tetramer in the of and heparin in the of or and that the is the to tetramer formation also observed the formation of a small of in the of octasaccharide and also with sedimentation the cross-linking of in the of heparin a of large molecular with a at the of the of of and on the GAG on a In study, we identified residues that to the of with by several in heparin binding assays. In of the residues are The of and residues is with and heparin observed in the of and the of from with heparin or I. Chem. 2002; PubMed Scopus Google Scholar). the heparin binding and the in vivo identified and as to GAG the and that to the the and the GAG binding residues mapped onto the surface of the in with the residues identified as for receptor binding and signaling S. A. S. D. J. S. D. M. B. J. I. Handel T.M. 1999; PubMed Scopus Google Scholar, D. M. B. S. A. S. F. D. I. A. A. Handel T.M. 1999; PubMed Scopus Google Scholar). is the residues that the receptor binding and the GAG binding of and to alanine have a small on receptor binding and S. A. S. D. J. S. D. M. B. J. I. Handel T.M. 1999; PubMed Scopus Google Scholar). possible to a with GAG receptor binding with for we that the in to stimulate cell migration in the essential of interactions (1Proudfoot A.E.I. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N.C. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (654) Google Scholar). 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Z. A. A. F. and A. E. I. for may the tetramer, which the for heparin by the of GAG-induced tetramer octasaccharide to the of the by of the small of the we the that the tetramer is of to the GAG the of the of the to the GAG binding site in of and tetramer formation at the of tetramer we the of the is can be in of the structure of the tetramer and GAG binding octasaccharide be to the site and to of the by the four is also with the that are of tetramer However, on the of interactions for chemokines and in a GAG the surface of the tetramer, as has been for J. Biol. 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