Identification and characterization of a glycosaminoglycan recognition element of the C chemokine lymphotactin.
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 (approximately 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.
Peterson
Francis C
FC
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Elgin
E Sonay
ES
Nelson
Timothy J
TJ
Zhang
Fuming
F
Hoeger
Theresa J
TJ
Linhardt
Robert J
RJ
Volkman
Brian F
BF
D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D018925:Chemokines / Q000378:metabolism; D019744:Chemokines, C; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008222:Lymphokines / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012795:Sialoglycoproteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013997:Time Factors
D018925:Chemokines; D019744:Chemokines, C; D003062:Codon; D006025:Glycosaminoglycans; D008222:Lymphokines; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C092390:XCL1 protein, human; C018997:lymphotactin; D006493:Heparin; D001120:Arginine