The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity

The chemokine RANTES (regulated on activation normal T cell expressed and s ecreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as hepar in, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANT ES contains two clusters of basic residues, (RKNR47)-R-44 and (KKWVR59)-K-5 5. The first is a BBXB motif common in heparin-binding proteins, and the se cond is located in the loop directly preceding the C-terminal helix. We hav e mutated these residues to alanine, both as point mutations as well as tri ple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mu tant retained full binding capacity. Mutation of the (RKNR47)-R-44 Site red uced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as w ell as their ability to induce chemotaxis in vitro. In all assays the singl e point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at m icromolar concentrations. The triple 40s mutant was also able to inhibit HI V-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.