Abstract
Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans. It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes. By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate. We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate. These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of </=14 monosaccharide residues that may be fully N-acetylated. Binding to interleukin-8 correlates with the occurrence of the di-O-sulfated disaccharide unit -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. We suggest that the heparan sulfate sequence binds in horseshoe fashion over two antiparallel-oriented helical regions on the dimeric protein.
Highlights
Glycosaminoglycans (GAGs),1 i.e. the linear, sulfate-substituted carbohydrate constituents of proteoglycans, are ubiquitous components of cell surfaces
Minimal Size of Heparin Oligosaccharide Binding to IL-8 — The smallest oligosaccharide fragment able to bind to IL-8 was identified by in-solution binding studies using radiolabeled size-defined heparin fragments
The interaction between heparin and IL-8 was previously shown to be of relatively low affinity, with a Kd of ϳ6 ϫ 10Ϫ6 M [26], and we considered the possibility of a charge-dependent interaction involving a shorter saccharide sequence that had escaped detection due to the ionic conditions of the assay
Summary
Materials—Recombinant human IL-8 was expressed in Escherichia coli and purified as described previously [15], except that a final step of affinity chromatography on heparin-agarose was added. All O-desulfated heparin fragments were re-N-sulfated as described [21] before final separation by gel chromatography on Bio-Gel P-10. 1 nmol each of the indicated 3H-labeled modified heparin 18-mers was incubated with 10 nmol of protein in a final volume of 1 ml of phosphate-buffered saline. To ensure complete N-substitution following chemical N-sulfation, saccharide samples were tested for N-unsubstituted glucosamine residues by treatment with nitrous acid at pH 4 [24], followed by gel chromatography on a column (1 ϫ 150 cm) of Sephadex G-50 in 1 M NaCl. Size separation of selectively desulfated heparin preparations on a Superose 6 column gave an average molecular mass for all preparations of 10 –11 kDa. no appreciable cleavage had occurred during chemical processing. The proportions of nonsulfated disaccharides were determined by high-voltage paper electrophoresis on Whatman No 3MM paper in pyridine acetate buffer, pH 5.3
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