Different affinities of glycosaminoglycan oligosaccharides for monomeric and dimeric interleukin-8: a model for chemokine regulation at inflammatory sites.
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.
Goger
Birgit
B
Institute of Pharmaceutical Chemistry, Protein Chemistry and Biophysics Group, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria.
Halden
Yvonne
Y
Rek
Angelika
A
Mösl
Roland
R
Pye
David
D
Gallagher
John
J
Kungl
Andreas J
AJ
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D019281:Dimerization; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D016209:Interleukin-8 / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D013552:Swine
D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D016209:Interleukin-8; D009844:Oligosaccharides