Identification and Characterization of a Glycosaminoglycan Recognition Element of the C Chemokine Lymphotactin

Abstract

Chemokine-mediated recruitment of leukocytes in vivo depends on interactions with cell surface glycosaminoglycans. Lymphotactin, the unique member of the “C” chemokine subclass, is a highly basic protein that binds heparin, a glycosaminoglycan, with high affinity (∼10 nm). We detected lymphotactin-heparin binding by NMR and mapped this interaction to a narrow surface that wraps around the protein. Substitutions in and around this binding site and surface plasmon resonance analysis of heparin binding affinity identified two arginine residues of lymphotactin as critical for glycosaminoglycan binding. Both arginine mutant proteins and the combined double mutant had dramatically diminished in vivo activity in a leukocyte recruitment assay, suggesting that the lymphotactin-glycosaminoglycan interactions detected in vitro are important for the function of this chemokine. Our results demonstrate that like other chemokines, lymphotactin utilizes highly specific glycosaminoglycan-binding sites that represent potential targets for drug development. Chemokine-mediated recruitment of leukocytes in vivo depends on interactions with cell surface glycosaminoglycans. Lymphotactin, the unique member of the “C” chemokine subclass, is a highly basic protein that binds heparin, a glycosaminoglycan, with high affinity (∼10 nm). We detected lymphotactin-heparin binding by NMR and mapped this interaction to a narrow surface that wraps around the protein. Substitutions in and around this binding site and surface plasmon resonance analysis of heparin binding affinity identified two arginine residues of lymphotactin as critical for glycosaminoglycan binding. Both arginine mutant proteins and the combined double mutant had dramatically diminished in vivo activity in a leukocyte recruitment assay, suggesting that the lymphotactin-glycosaminoglycan interactions detected in vitro are important for the function of this chemokine. Our results demonstrate that like other chemokines, lymphotactin utilizes highly specific glycosaminoglycan-binding sites that represent potential targets for drug development. A family of small secreted proteins, chemokines recruit leukocytes from the circulatory system to mediate inflammatory responses in host defense and wound healing (1Keane M.P. Strieter R.M. Crit. Care Med. 2000; 28: N13-N26Crossref PubMed Scopus (81) Google Scholar), control angiogenesis, and regulate lymphoid development (2Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3334) Google Scholar, 3Gale L.M. McColl S.R. Bioessays. 1999; 21: 17-28Crossref PubMed Scopus (113) Google Scholar). Chemokine sequences are divided into four subfamilies, based on the number and spacing of conserved cysteine residues. Many chemokine structures are known, and members of all four subfamilies adopt a canonical tertiary fold, typically stabilized by a pair of disulfide bonds (4Kuloglu E.S. McCaslin D.R. Kitabwalla M. Pauza C.D. Markley J.L. Volkman B.F. Biochemistry. 2001; 40: 12486-12496Crossref PubMed Scopus (55) Google Scholar); some also form homodimeric structures (5Fernandez E.J. Lolis E. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 469-499Crossref PubMed Scopus (518) Google Scholar). Chemokines stimulate leukocyte migration by activating specific G protein-coupled receptors expressed in their target cell populations (3Gale L.M. McColl S.R. Bioessays. 1999; 21: 17-28Crossref PubMed Scopus (113) Google Scholar, 6Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2119) Google Scholar). This signaling network of roughly 50 chemokines and 20 receptors is both selective and redundant: specific leukocyte types express a particular receptor and are recruited only in response to their cognate chemokines, however, many receptors can be activated by more than one chemokine ligand (7Proudfoot A.E. Nat. Rev. Immunol. 2002; 2: 106-115Crossref PubMed Scopus (626) Google Scholar). Human lymphotactin (hLtn/XCL1) 1The abbreviations used are: hLtn, human lymphotactin; IL, interleukin; GAG, glycosaminoglycan; RANTES, regulated on activation normal T cell expressed and secreted; MIP, macrophage inflammatory protein; SDF-1, stromal cell-derived factor 1; MCP, monocyte chemoattractant protein; IP, interferon-γ-inducible protein. recruits T lymphocytes and natural killer cells through its specific receptor XCR1 in normal immune function and chronic inflammatory conditions (8Hedrick J.A. Zlotnik A. Clin. Immunol. Immunopathol. 1998; 87: 218-222Crossref PubMed Scopus (39) Google Scholar, 9Wang J.D. Nonomura N. Takahara S. Li B.S. Azuma H. Ichimaru N. Kokado Y. Matsumiya K. Miki T. Suzuki S. Okuyama A. Immunology. 1998; 95: 56-61Crossref PubMed Scopus (32) Google Scholar). For example, an inappropriate or uncontrolled T cell infiltration driven by hLtn expression is a factor in both Crohn's disease (10Middel P. Thelen P. Blaschke S. Polzien F. Reich K. Blaschke V. Wrede A. Hummel K.M. Gunawan B. Radzun H.J. Am. J. Pathol. 2001; 159: 1751-1761Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and rheumatoid arthritis (11Blaschke S. Middel P. Dorner B.G. Blaschke V. Hummel K.M. Kroczek R.A. Reich K. Benoehr P. Koziolek M. Muller G.A. Arthritis Rheum. 2003; 48: 1858-1872Crossref PubMed Scopus (52) Google Scholar). Interestingly, the unique ability of hLtn to specifically chemoattract lymphocytes has been exploited therapeutically in combination with the T cell-activating cytokine interleukin-2 (IL-2). T cells recruited by hLtn and activated by IL-2 generated potent antitumor immunity in human clinical trials for treatment of neuroblastoma, where studies with either hLtn or IL-2 alone failed to generate a significant response (12Rousseau R.F. Haight A.E. Hirschmann-Jax C. Yvon E.S. Rill D.R. Mei Z. Smith S.C. Inman S. Cooper K. Alcoser P. Grilley B. Gee A. Popek E. Davidoff A. Bowman L.C. Brenner M.K. Strother D. Blood. 2003; 101: 1718-1726Crossref PubMed Scopus (108) Google Scholar). Hence, hLtn and its analogs with activity toward XCR1 will be extremely valuable as agents for T cell recruitment in cancer vaccines (6Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2119) Google Scholar), whereas specific antagonists of hLtn activity may be useful for treating certain autoimmune or inflammatory diseases (7Proudfoot A.E. Nat. Rev. Immunol. 2002; 2: 106-115Crossref PubMed Scopus (626) Google Scholar). Protein-glycosaminoglycan (GAG) interactions are essential for in vivo signaling by the chemokine network of the immune system (13Kuschert G.S. Coulin F. Power C.A. Proudfoot A.E. Hubbard R.E. Hoogewerf A.J. Wells T.N. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (500) Google Scholar, 14McFadden G. Kelvin D. Biochem. Pharmacol. 1997; 54: 1271-1280Crossref PubMed Scopus (45) Google Scholar, 15Proudfoot A.E. Eur. J. Dermatol. 1998; 8: 147-157PubMed Google Scholar). High affinity chemokine binding to cell surface GAGs is thought to serve in the formation of a concentration gradient leading back to the site of chemokine production. These interactions may regulate the types of chemokines immobilized at inflammatory sites, because the content and composition of cell surface glycosaminoglycans vary with the type and location of endothelial tissues (13Kuschert G.S. Coulin F. Power C.A. Proudfoot A.E. Hubbard R.E. Hoogewerf A.J. Wells T.N. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (500) Google Scholar). Mutagenesis studies on a number of CC and CXC chemokines, including RANTES, MIP-1α, MIP-1β, MCP-1, IL-8, SDF-1, and IP-10 identified groups of basic residues responsible for GAG binding (16Koopman W. Krangel M.S. J. Biol. Chem. 1997; 272: 10103-10109Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 17Laurence J.S. Blanpain C. Burgner J.W. Parmentier M. LiWang P.J. Biochemistry. 2000; 39: 3401-3409Crossref PubMed Scopus (104) Google Scholar, 18Koopmann W. Ediriwickrema C. Krangel M.S. J. Immunol. 1999; 163: 2120-2127Crossref PubMed Google Scholar, 19Proudfoot A.E.I. Fritchley S. 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Lee E.M. Sun J. Luster A.D. J. Biol. Chem. 2003; 278: 17066-17074Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Whereas a cluster of basic residues commonly associated with heparin-binding proteins (the BBXB motif (25Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar)) appears in the 40's loop of the CC chemokines RANTES, MIP-1α, and MIP-1β, GAG binding sites identified in other chemokines are highly divergent (26Lortat-Jacob H. Grosdidier A. Imberty A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1229-1234Crossref PubMed Scopus (221) Google Scholar). Cell-based in vitro assays for chemotaxis (or Ca2+-flux) showed that GAG binding mutants of RANTES, MIP-1α, and MIP-1β remained fully functional, suggesting that G protein-coupled receptor binding and activation is independent of the interactions with extracellular matrix (20Chakravarty L. Rogers L. Quach T. Breckenridge S. Kolattukudy P.E. J. Biol. Chem. 1998; 273: 29641-29647Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 24Campanella G.S. Lee E.M. Sun J. Luster A.D. J. Biol. Chem. 2003; 278: 17066-17074Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 27Proudfoot A.E. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (661) Google Scholar). However, the same mutants with disrupted GAG binding sites were nonfunctional in vivo, demonstrating that the activity of chemokines depends on both types of interactions A.E. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (661) Google Scholar). structures are for many chemokines, been A is for heparin to the cytokine basic factor S. R.E. PubMed Scopus Google Scholar), the are of a chemokine signaling will of GAG binding that represent targets for development of specific chemokine antagonists G. Kelvin D. Biochem. Pharmacol. 1997; 54: 1271-1280Crossref PubMed Scopus (45) Google Scholar, A.E. Power C.A. C. Wells T.N. Immunol. 2003; PubMed Scopus (91) Google Scholar). chemokines, hLtn is highly basic and binds heparin with high affinity (∼10 through some combination of its and arginine residues (13Kuschert G.S. Coulin F. Power C.A. Proudfoot A.E. Hubbard R.E. Hoogewerf A.J. Wells T.N. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (500) Google Scholar). this the interaction hLtn and a heparin by NMR identified a GAG a of basic residues. this generated mutant proteins with affinity for heparin, and showed that in vivo hLtn Our results a specific GAG binding site for the chemokine lymphotactin and that the interaction is essential for in vivo and the of human in a into a protein with a site (4Kuloglu E.S. McCaslin D.R. Kitabwalla M. Pauza C.D. Markley J.L. Volkman B.F. Biochemistry. 2001; 40: 12486-12496Crossref PubMed Scopus (55) Google Scholar). A of this from at a site in the hLtn the of this an for expression of hLtn based on the used to generate a hLtn with and sites at the and to into a to the of the to a of residues and of a 1989; PubMed Scopus Google Scholar) site as a of the affinity Many chemokines are by however, lymphotactin an G. J. K. S. D. N. N. J. K. T. Zlotnik A. PubMed Scopus Google Scholar), and of the a hLtn protein B. S. F. P. R. P. Clark-Lewis I. Kroczek R.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the from the expression in a protein of residues of hLtn This hLtn protein is to as hLtn this of and the expression were by and hLtn expression into E. were at in and 50 the cell expression by the of to a concentration of cells were for and at For with cells were in as the hLtn in the of the and by the for and from a in of and by two through a were by at for at in of 50 by through a and by at for This protein with in for at into a and with of with of were and of to of the affinity by the conditions to 50 50 and of a of and for at of the hLtn disulfide by of the into of by of the of hLtn to 20 by in an a hLtn from or by high and of all hLtn were by and and were from heparin, and were from were on a were and all protein were to by high and used to because of a were the same and agents as the and a from in and surface by and all studies heparin immobilized on a failed because of binding of hLtn to the were at heparin of hLtn through its groups to a for NMR analysis of a also the interaction of immobilized hLtn with heparin from heparin A. C. PubMed Scopus Google Scholar, G. L. D. J. 2000; 100: Full Text Full Text PDF PubMed Scopus Google Scholar). were at in 20 for a of heparin with the where is is is is and or an of heparin binding for hLtn proteins, heparin of immobilized through protein groups on a as F. M. R. Biochem. 2002; PubMed Scopus Google Scholar). by the of a response control cell by a This surface and a of hLtn mutants were at in 20 to conditions used for NMR analysis of the hLtn (4Kuloglu E.S. McCaslin D.R. Kitabwalla M. Pauza C.D. Markley J.L. Volkman B.F. Biochemistry. 2001; 40: 12486-12496Crossref PubMed Scopus (55) Google Scholar) and heparin binding as NMR were on a with a NMR hLtn were in 20 S. C. Johnson J. B. Scopus Google Scholar) were at with of and in the and in the for were by from from heparin in of the or A. C. PubMed Scopus Google Scholar) or a heparin to the binding site of heparin G. L. D. J. 2000; 100: Full Text Full Text PDF PubMed Scopus Google Scholar). of heparin by and for the were by from the protein (4Kuloglu E.S. McCaslin D.R. Kitabwalla M. Pauza C.D. Markley J.L. Volkman B.F. Biochemistry. 2001; 40: 12486-12496Crossref PubMed Scopus (55) Google Scholar) because for be through the of the A used to of the arginine and other to were with and with of or 20 of mutants of hLtn in of the were by with of and the for number of cells from a of Human binds heparin, like chemokines (13Kuschert G.S. Coulin F. Power C.A. Proudfoot A.E. Hubbard R.E. Hoogewerf A.J. Wells T.N. Biochemistry. 1999; 38: 12959-12968Crossref PubMed Scopus (500) Google Scholar). We the of the interaction immobilized hLtn and heparin by and an of at in 20 interactions been to form a that be by of heparin to a of hLtn an used to the binding of a of heparin from to 20 residues. for the heparin from to leading to that one or more of the heparin sequences hLtn in a for NMR Our showed that hLtn the canonical chemokine as a at in (4Kuloglu E.S. McCaslin D.R. Kitabwalla M. Pauza C.D. Markley J.L. Volkman B.F. Biochemistry. 2001; 40: 12486-12496Crossref PubMed Scopus (55) Google Scholar), and that the protein is conditions E.S. McCaslin D.R. Markley J.L. Volkman B.F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). normal chemokine with a that at in however, the of this are conditions the two are in as detected by is to is more from analysis the is to of other chemokines with and the high has or whereas may a the of lymphotactin is to be important for in vivo the all NMR and at in 20 with at of used NMR to a of heparin and A. C. PubMed Scopus Google Scholar, G. L. D. J. 2000; 100: Full Text Full Text PDF PubMed Scopus Google Scholar) in the conditions used for of the hLtn 20 of of either the or to of hLtn of in and a in NMR with the formation of of the protein to the including protein and a of and were of a of the the same conditions suggesting that the GAG binding site of hLtn at residues to form a specific We this by a heparin G. L. D. J. 2000; 100: Full Text Full Text PDF PubMed Scopus Google Scholar). of and of the for a unique of residues of hLtn, including a of the of heparin were from residues in a demonstrating that the binding in the of the NMR appears that the binds hLtn with affinity to by a of heparin However, the were in the of at and binding a with NMR is formation of a high affinity a GAG of or more and heparin may hLtn as for other chemokines A.J. Proudfoot A.E. Borlat F. Clark-Lewis I. Power C.A. Wells Biochemistry. 1997; PubMed Scopus Google Scholar). However, the for with heparin for its binding the heparin is to target the GAG important for hLtn activity in We the NMR of this specific to residues of the chemokine that form the heparin binding structures for a of chemokines the same tertiary of a and are the of GAG because of interactions with the binding sites identified in other chemokines of basic residues the in the and in the A.E. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (661) Google Scholar), whereas residues and the loop been as in chemokine receptor P. Clark-Lewis I. J. Biol. 2001; PubMed Google Scholar). for and in hLtn four of the hLtn that are by the of including the the residues of the and residues Whereas are in cluster to a potential GAG interaction surface on the of the hLtn a number of and residues were identified in the of residues that and an that to an in the of the arginine in hLtn, and to the with heparin and residues are of the hLtn by We used results to a to binding. of of the basic residues identified from to by We of mutant by NMR of the and or mutant the hLtn tertiary with of a of and a protein based on NMR of the NMR of hLtn to that of the tertiary and that be to its in heparin binding by this and proteins also a of and Both proteins and were used in of binding to immobilized were by for of the hLtn at in conditions to NMR studies of the including and had or on the affinity for heparin, a factor of of hLtn 20 binding of both the and proteins were by a factor of more than with the in the on the double mutant its by NMR and its interaction with heparin by and combined to the affinity for heparin to hLtn an independent of heparin binding all mutant proteins from a a this assay, are with binding proteins in the by the in for interaction hLtn mutant protein and at in 20 in a hLtn of hLtn in the and of a heparin were at in 20 for and are from the arginine of of the double or from the of of the double mutant protein with heparin in that the heparin binding site had been residues to be to GAG as by NMR and on the hLtn and a potential heparin binding surface that a of the of this GAG binding is of two basic and that are in the other in the hLtn tertiary of hLtn of residues in GAG binding of other chemokines has proteins that fully in in vitro assays for chemotaxis (or Ca2+-flux) A.E. Handel T.M. Johnson Z. Lau E.K. LiWang P. Clark-Lewis I. Borlat F. Wells T.N. Kosco-Vilbois M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1885-1890Crossref PubMed Scopus (661) Google Scholar). A more is to the of the Human has in vivo activity in as by recruitment of leukocytes into the J.A. V. D. L. Y. S. J. Handel T. Zlotnik A. J. Immunol. 1997; Google Scholar). We this leukocyte recruitment to in vivo activity for a of hLtn including with diminished heparin binding hLtn to be a potent leukocyte chemoattractant in and this activity is on tertiary because the protein failed to recruit significant of cells proteins with disrupted heparin binding sites, and showed diminished activity in the in vivo recruitment with and heparin as as hLtn were in vivo, at Whereas this may a in formation of activity also be diminished either in binding or activation of the specific hLtn or stabilized an essential of the chemokine. However, and other are and on the hLtn surface and with heparin to the of the Interestingly, whereas heparin for the mutants and are roughly with hLtn, and the double mutant binds with proteins were to an in Hence, either of the at and disrupted interactions to a in heparin binding affinity had These results that of GAG binding can for the formation of chemokine signaling with analysis of interactions on to a NMR to the interaction of a heparin with specific residues of the protein. These residues and that are in the were for to their to the binding many CC chemokines, the 40's loop of hLtn a basic cluster that one of the identified as a heparin binding the of RANTES, and of all basic residues the BBXB motif to or GAG binding W. Ediriwickrema C. Krangel M.S. J. Immunol. 1999; 163: 2120-2127Crossref PubMed Google Scholar, 19Proudfoot A.E.I. Fritchley S. Borlat F. Shaw J.P. Vilbois F. Zwahlen C. Trkola A. Marchant D. Clapham P.R. Wells T.N. J. Biol. Chem. 2001; 276: 10620-10626Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 23Sadir R. Baleux F. Grosdidier A. Imberty A. Lortat-Jacob H. J. Biol. Chem. 2001; 276: 8288-8296Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, J.S. Blanpain C. A.D. Parmentier M. LiWang P.J. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). However, results showed of this cluster of basic residues in hLtn, only to the affinity for Whereas a GAG binding site has been for the CXC chemokines, studies a conserved basic in the of IP-10 and that in GAG binding. residues critical to GAG binding are in in For example, the of and and the 40's loop of IP-10 basic residues that in GAG binding G.S. Hoogewerf A.J. Proudfoot A.E. Chung C.W. Cooke R.M. Hubbard R.E. Wells T.N. Sanderson P.N. Biochemistry. 1998; 37: 11193-11201Crossref PubMed Scopus (170) Google Scholar, 24Campanella G.S. Lee E.M. Sun J. Luster A.D. J. Biol. Chem. 2003; 278: 17066-17074Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, E. V. M. J. T. Biochem. J. PubMed Scopus Google Scholar). hLtn, a a in GAG whereas of basic residues in the heparin hLtn GAG binding surface and a unique combination of basic residues that in many CXC and CC chemokines, and the of GAG binding sites in Whereas the chemokines are highly basic proteins, the of specific GAG binding residues family For example, basic residues in the of MCP-1, a CC are critical to GAG whereas the essential residues in other CC chemokines are in a BBXB motif in the 40's loop (20Chakravarty L. Rogers L. Quach T. Breckenridge S. Kolattukudy P.E. J. Biol. Chem. 1998; 273: 29641-29647Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). of a GAG binding motif the chemokine family that in the extracellular matrix through and highly protein However, results and studies demonstrate that chemokine GAG interactions are highly specific and by basic residues that are unique for chemokine. T cell chemoattractant lymphotactin and binds heparin with We used NMR to binding of a heparin to hLtn, and identified a surface a of the basic residues. Our analysis of binding by and in vivo activity showed that NMR as an and for the for high affinity GAG binding. We like other chemokines, hLtn function depends on interactions with of the extracellular and that high affinity of glycosaminoglycans by this chemokine is specifically by the of and that the be and NMR studies of binding used heparin E. V. M. J. T. Biochem. J. PubMed Scopus Google Scholar) for or heparin A.E.I. Fritchley S. Borlat F. Shaw J.P. Vilbois F. Zwahlen C. Trkola A. Marchant D. Clapham P.R. Wells T.N. J. Biol. Chem. 2001; 276: 10620-10626Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, LiWang P.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), are to a GAG binding site on the chemokine Whereas the heparin used in this binds hLtn with affinity the conditions that with an of the protein a that the heparin binding site This can be by and a lymphotactin that only the leukocyte recruitment activity in C. T. J. and B. F. These results to that specific to the are essential for lymphotactin Our studies will be at the specific for both of this chemokine.