Identification of a Glycosaminoglycan Binding Surface on Human Interleukin-8

Abstract

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using a systematic series of site-directed mutants of IL-8 in which the basic residues of the protein have been replaced by alanine.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.

IL-8 Released Constitutively by Primary Bronchial Epithelial Cells in Culture Forms an Inactive Complex with Secretory Component

“…These observations have since been confirmed and extended to the inhibitory effect of heparin on IL-8 bound to endothelial cell surface heparan sulfate proteoglycans (44). The interaction of IL-8 with heparin involves highly sulfated residues on the glycosaminoglycan (45). However, the interaction of IL-8 with the carbohydrate residues, fucose, mannose, galactose, N-acetyl glucosamine, and neuraminic acid, on SC (8) is likely to be different and is the subject of our further investigations.…”

Tissue-Specific Mechanisms Control the Retention of IL-8 in Lungs and Skin

“…Proteoglycans are found on epithelial cells, in the extracellular matrix, and on endothelial cells in the lungs (41). IL-8 has been shown to bind to endothelial cells in skin and isolated GAGs such as heparin and heparan sulfate in vitro (15,18,20). The molecular interaction between IL-8 and GAG is specific and requires a GAG binding domain on IL-8 and complementary sequences on the GAG heparan sulfate (15,42).…”

“…IL-8 has been shown to bind to endothelial cells in skin and isolated GAGs such as heparin and heparan sulfate in vitro (15,18,20). The molecular interaction between IL-8 and GAG is specific and requires a GAG binding domain on IL-8 and complementary sequences on the GAG heparan sulfate (15,42). The observation that IL-8 selectively binds to different GAG families with binding to heparin Ͼ heparan sulfate Ͼ dermatan sulfate Ͼ chondroitin sulfate is further evidence of specificity for this molecular interaction (19).…”

“…The three-dimensional structures of CXC and CC chemokines are highly conserved and contain two spatially separate binding domains, a high-affinity binding domain and a glycosaminoglycan (GAG)-binding domain (12)(13)(14)(15)(16)(17). The high-affinity binding domain of IL-8 is responsible for activation and chemotaxis of PMN, while the GAG-binding domain binds to the GAGs heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate in vitro (13,15,18,19).…”

“…The high-affinity binding domain of IL-8 is responsible for activation and chemotaxis of PMN, while the GAG-binding domain binds to the GAGs heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate in vitro (13,15,18,19). The presence of a GAG binding domain on IL-8 has led several investigators to propose that, in tissue, chemokines are immobilized on the surface of endothelial cells and extracellular matrix through interactions with GAGs and that leukocytes migrate over fixed gradients (e.g., haptotactic gradients) rather than along soluble gradients (20 -27).…”