Characterization of the chemokine CXCL11-heparin interaction suggests two different affinities for glycosaminoglycans.
Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.
Severin
India C
IC
Merck Serono Geneva Research Centre, 9 Chemin des Mines, 1202 Geneva, Switzerland.
Gaudry
Jean-Philippe
JP
Johnson
Zoë
Z
Kungl
Andreas
A
Jansma
Ariane
A
Gesslbauer
Bernd
B
Mulloy
Barbara
B
Power
Christine
C
Proudfoot
Amanda E I
AE
Handel
Tracy
T
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002465:Cell Movement; D054371:Chemokine CXCL11 / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D005260:Female; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054367:Receptors, CXCR3 / Q000737:chemistry; D018417:Th1 Cells / Q000378:metabolism
D054371:Chemokine CXCL11; D000939:Epitopes; D006025:Glycosaminoglycans; D054367:Receptors, CXCR3; D006493:Heparin; D000409:Alanine