Structural and functional analysis of the RANTES-glycosaminoglycans interactions.
Chemokines mediate their biological activity through activation of G protein coupled receptors, but most chemokines, including RANTES, are also able to bind glycosaminoglycans (GAGs). Here, we have investigated, by site-directed mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analysis. Our results indicate that (i) RANTES exhibited selectivity in GAGs binding with highest affinity (K(d) = 32.1 nM) for heparin, (ii) RANTES uses the side chains of residues R44, K45, and R47 for heparin binding, and blocking these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expressing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our results indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps the CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzymatic removal of cell surface GAGs by glycosidases did not affect CCR5 binding and Ca(2+) response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to that found in surface plasmon resonance experiments. Thus, cell surface GAGs is not a prerequisite for receptor binding or signaling, but soluble GAGs can inhibit the binding and the functional response of RANTES to CCR5 expressing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inflammatory situations and locations.
Martin
L
L
CEA (Commissariat à l'Energie Atomique) Saclay, Département d'Ingénierie et d'Etudes des Protéines, F-91191 Gif-sur-Yvette, France.
Blanpain
C
C
Garnier
P
P
Wittamer
V
V
Parmentier
M
M
Vita
C
C
D000107:Acetylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D018946:Chemokine CCL5 / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D006023:Glycoproteins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D026563:LDL-Receptor Related Protein-Associated Protein; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D054389:Receptors, CCR1; D054397:Receptors, CCR3; D019713:Receptors, CCR5 / Q000378:metabolism / Q000502:physiology; D019707:Receptors, Chemokine / Q000378:metabolism / Q000502:physiology; D012995:Solubility; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
D002352:Carrier Proteins; D018946:Chemokine CCL5; D006023:Glycoproteins; D006025:Glycosaminoglycans; D026563:LDL-Receptor Related Protein-Associated Protein; D054389:Receptors, CCR1; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D006493:Heparin; D001120:Arginine; D006026:Glycoside Hydrolases; D008239:Lysine