Structural and Functional Analysis of the RANTES-Glycosaminoglycans Interactions

Abstract

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.

Autocrine CCL5 Signaling Promotes Invasion and Migration of CD133 + Ovarian Cancer Stem‐Like Cells via NF‐κB‐Mediated MMP‐9 Upregulation

“…It is well-established that CCL5 mediates its biological activities through activation of the G-protein-coupled receptors CCR1, CCR3, and CCR5 [49]. We observed expression of all the CCL5 receptors (CCR1, CCR3, and CCR5) in ovarian CSLCs.…”

The C-terminal Nonapeptide of Mature Chemerin Activates the Chemerin Receptor with Low Nanomolar Potency

“…A dual role of chemerin cannot therefore be excluded, and specific actions of the cystatin-like module are presently being analyzed. The N-terminal domain of chemerin might also interact with proteoglycans, similarly to the core domain of chemokines, and this property could be essential for promoting leukocyte migration in vivo (32,33).…”

The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH

“…Residues Arg-47 and Arg-17 of CCL5 have been shown to play a crucial role in the CCL5-CCR1 binding event (29,30), and the N terminus of CCL5 is known to be crucial for CCR1 signaling (30,31). Although it has previously been shown that pools of heparin-derived oligosaccharides can inhibit the binding of CCL5 to its receptor, CCR1 (29), no specific sulfation pattern or motif is known to be optimal for binding to CCL5. GAG heterogeneity makes such evaluations particularly challenging and presents a role for computational methods to provide theoretical insights.…”

“…Residues Arg-47 and -17 of CCL5 are known to play a crucial role in the CCL5-CCR1 binding event (29,30), whereas the N terminus of CCL5 is crucial for CCR1 signaling (30,31). Based on the per-residue MM-GBSA data, each ligand interacts with these residues to varying degrees.…”