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 extracell ular matrix, The binding site on the chemokine interleukin-8 (IL-8) fo r the glycosaminoglycan heparin has been characterized using a systema tic series of site-directed mutants of IL-8 in which the basic residue s of the protein have been replaced by alanine. Mutation of K64 and R6 8 caused the largest decrease in affinity for a heparin Sepharose matr ix, with smaller effects seen with mutations of K20, R60, and K67. Hep arin-derived disaccharides that could disrupt the IL-8-heparin Sepharo se interaction were identified by a competitive binding assay. Heteron uclear NMR spectroscopic titration of N-15-labeled IL-8 with a trisulf ated disaccharide revealed a cluster of residues on IL-8 which were pe rturbed by disaccharide binding. These data identify a heparin-binding surface on IL-8 that includes the C-terminal ct-helix and the proxima l loop around residues 18-23, The heparin-binding site is spatially di stinct from the residues involved in receptor binding.