Structural and functional analysis of the RANTES-glycosaminoglycans interactions

Chemokines mediate their biological activity through activation of G protei n coupled receptors, but most chemokines, including RANTES, are also able t o bind glycosaminoglycans (GAGs). Here, we have investigated, by site-direc ted mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analy sis. Our results indicate that (i) RANTES exhibited selectivity in GAGs bin ding with highest affinity (K-d = 32.1 nM) for heparin, (ii) RANTES uses th e side chains of residues R44, K45, and R47 for heparin binding, and blocki ng these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expre ssing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our result s indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps th e CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzym atic removal of cell surface GAGs by glycosidases did not affect CCR5 bindi ng and Ca2+ response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to tha t found in surface plasmon resonance experiments. Thus, cell surface GAGs i s not a prerequisite for receptor binding or signaling, but soluble GAGs ca n inhibit the binding and the functional response of RANTES to CCR5 express ing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inf lammatory situations and locations.