Different Affinities of Glycosaminoglycan Oligosaccharides for Monomeric and Dimeric Interleukin-8:  A Model for Chemokine Regulation at Inflammatory Sites

Abstract

The binding of interleukin-8 (IL-8) to heparan sulfate (HS) proteoglycans on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Fluorescence anisotropy measurements yielded an IL-8 dimerization constant of 120 nM. The binding affinities, obtained by isothermal fluorescence titration, of size-defined heparin and HS oligosaccharides to the chemokine were found to depend on the oligomerization state of IL-8: high affinity was detected for monomeric and low affinity was detected for dimeric IL-8, referring to a self-regulatory mechanism for its chemoattractant effect. The highest affinity for monomeric IL-8 was detected for the HS octamer with a K(d) < 5 nM whereas the dissociation constants of dimeric IL-8 were found in the medium micromolar range. No indication for increasing affinities for monomeric IL-8 with increasing oligosaccharide chain length was found. Instead, a periodic pattern was obtained for the dissociation constants of the GAG oligosaccharides with respect to chain length, referring to optimum and least optimum chain lengths for IL-8 binding. GAG disaccharides were identified to be the minimum length for chemokine binding. Conformational changes of the dimeric chemokine, determined using CD spectroscopy, were detected only for the IL-8/HS complexes and not for heparin, pointing to an HS-induced activation of the chemokine with respect to receptor binding. Thermal unfolding of IL-8 yielded a single transition at 56 degrees C which was completely prevented by the presence of undigested HS or heparin, indicating structural stabilization, thereby prolonging the biological effect of the chemokine.

Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma

“…HSPG probably increase the effective concentration at the cell membrane and then facilitate ErbB activation. An additional explanation might be that HSPGs induce a conformational change in EGF-ligands which would stabilize them in an active conformation, as it was shown for the chemokine IL-8 (Goger et al, 2002). As the non HSbinding EGF-ligands have no effect on myeloma cells, one can speculate that they are unable to activate their Figure 7 Binding of EGF-family members to HSPG is required for their myeloma cell growth factor activity.…”

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

“…De-N-sulfated, De-2-O-sulfated, and De-6-O-sulfated heparins were prepared as described previously (27). Different sized heparin fragments from 12 to 2 disaccharides in length were prepared by high resolution gel chromatography of low molecular weight heparin as described previously (28). For all of the experiments, the heparins were used at equal weight/volume ( g/ml) concentrations.…”

Induction of a CXCL8 binding site on endothelial syndecan‐3 in rheumatoid synovium

“…The binding affinities have been variously reported in the nanomolar or micromolar range for CXCL8, CCL2, CCL3, and CCL5 (14)(15)(16). Chemokines display differences in their strength of interactions with glycosaminoglycans.…”

“…The functional importance of chemokine-HS interactions has been shown in vivo, because administration of modified chemokines that exhibit reduced affinity to HS or heparin resulted in diminished leukocyte recruitment (31,32). Other functions of glycosaminoglycan-chemokine interactions have been proposed, including the formation of immobilized chemokine gradients in the extracellular matrix and the protection of these mediators from degradation, resulting in the prolongation of their activities (15,35,36).…”