Structural and functional analysis of the RANTES-glycosaminoglycans interactions

Area of Science:

• Immunology
• Biochemistry
• Cell Biology

Background:

• Chemokines are crucial signaling molecules that activate G protein-coupled receptors.
• Many chemokines, including RANTES, also interact with glycosaminoglycans (GAGs).
• The role of these GAG interactions in chemokine function is not fully understood.

Purpose of the Study:

• To investigate the specific RANTES basic residues involved in GAG binding.
• To determine the biological relevance of RANTES-GAG interactions for receptor binding and activation.
• To elucidate the role of cell-surface and soluble GAGs in RANTES-mediated signaling.

Main Methods:

• Site-directed mutagenesis and chemical acetylation of RANTES.
• Surface plasmon resonance kinetic analysis for GAG binding affinity.
• Functional assays using CHO-K1 cells expressing chemokine receptors (CCR5, CCR1, CCR3).
• Enzymatic removal of cell surface GAGs and addition of soluble GAGs.

Main Results:

• RANTES demonstrated selective GAG binding, with highest affinity for heparin (K(d) = 32.1 nM).
• Residues R44, K45, and R47 are critical for RANTES heparin binding.
• The heparin-binding site (40s loop) is crucial for CCR1 and CCR3 binding/activation, but minimally involved in CCR5 interactions.
• Cell-surface GAGs are not essential for CCR5 binding or signaling, but soluble GAGs inhibit RANTES-CCR5 interactions.

Conclusions:

• RANTES exhibits specific binding to GAGs, particularly heparin, mediated by key basic residues.
• GAG binding influences RANTES interaction with specific chemokine receptors (CCR1, CCR3) but not CCR5.
• While not essential for receptor engagement, GAGs can modulate chemokine activity, suggesting a role in regulating inflammatory responses in vivo.