Identification of a glycosaminoglycan binding surface on human interleukin-8.
The activation of leukocytes by chemokines is believed to be mediated via binding of chemokines to glycosaminoglycan chains of the extracellular matrix. The binding site on the chemokine interleukin-8 (IL-8) for the glycosaminoglycan heparin has been characterized using a systematic series of site-directed mutants of IL-8 in which the basic residues of the protein have been replaced by alanine. Mutation of K64 and R68 caused the largest decrease in affinity for a heparin Sepharose matrix, with smaller effects seen with mutations of K20, R60, and K67. Heparin-derived disaccharides that could disrupt the IL-8-heparin Sepharose interaction were identified by a competitive binding assay. Heteronuclear NMR spectroscopic titration of 15N-labeled IL-8 with a trisulfated disaccharide revealed a cluster of residues on IL-8 which were perturbed by disaccharide binding. These data identify a heparin-binding surface on IL-8 that includes the C-terminal alpha-helix and the proximal loop around residues 18-23. The heparin-binding site is spatially distinct from the residues involved in receptor binding.
Department of Chemistry, University of York, Heslington, United Kingdom.
G S
GS
Kuschert
A J
AJ
Hoogewerf
A E
AE
Proudfoot
C W
CW
Chung
R M
RM
Cooke
R E
RE
Hubbard
T N
TN
Wells
P N
PN
Sanderson
D000345:Affinity Labels; D001667:Binding, Competitive / Q000235:genetics; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004187:Disaccharides / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives
D000345:Affinity Labels; D004187:Disaccharides; D006025:Glycosaminoglycans; D016209:Interleukin-8; C037405:heparin-sepharose; D006493:Heparin; D012685:Sepharose