Structural and functional analysis of the β-chemokine RANTES: Proposal of a three-site binding hypothesis

Abstract

Chemokines comprise a family of low molecular weight proteins involved in a variety of biological activities including the activation and regulation of immune responses. A subset of chemokines, including Regulated Upon Activation Normal T Cell Expressed and Secreted (RANTES), Macrophage Inflammatory Protein (MIP)-1alpha, MIP-1beta and Macrophage Derived Chemokine (MDC), block HIV-1 entry of susceptible cells. The anti-viral activity of chemokines is dependent upon binding to 7-transmembrane (TM) G protein-coupled receptors, which also serve as receptors for HIV-1 viral binding to cells. In addition to this dependence upon protein receptors we have demonstrated a reliance of chemokine function on cell surface glycosaminoglycans (GAGs). Through the use of an anti-RANTES monoclonal antibody developed in our laboratory, mAb 4A12, residues in the C-terminal region of RANTES have been identified as those residues critical for GAG binding. Preincubation of mAb 4A12 with RANTES blocks RANTES induced Ca2+ mobilization, inhibits RANTES binding to the cell surface and reverses the HIV-1 anti-viral activity of this chemokine, thus implicating the dependence of chemokine function on cell surface GAGs. Additionally, enzymatic removal of cell surface GAGs from PBMC inhibits the ability of RANTES to mobilize Ca2+ in these cells. Further implication of the importance of GAGs in chemokine function is provided by studies in which cells inherently expressing low levels of cell surface GAGs exhibit reduced ability to mobilize Ca2+ in response to RANTES. Finally, adding back GAGs as soluble complexes to chemokines does not restore chemokine function. Soluble RANTES/GAG complexes are unable to induce Ca2+ mobilization or chemotaxis in susceptible cells; yet they retain HIV-1 anti-viral properties. In conclusion, our data suggest that the current model describing chemokine interactions with protein receptors needs to be extended to include the GAG interaction necessary for functional chemokine binding. Additionally, our findings suggest soluble chemokine/GAG complexes represent 7-TM receptor ligands that no longer elicit the potentially deleterious effects of cell signaling through Ca2+ mobilization and chemotaxis, yet retain their anti-viral activity and therefore represent a potential therapeutic formulation to treat HIV-1 infection.