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Lookup NU author(s): Sarah Fritchley
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The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, 44RKNR47 and 55KKWVR59. The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the 44AANA47 mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the 55AAWVA59 mutant retained full binding capacity. Mutation of the 44RKNR47 site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single 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 micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.
Author(s): Proudfoot AEI, Fritchley S, Borlat F, Shaw JP, Vilbois F, Zwahlen C, Trkola A, Marchant D, Clapham PR, Wells TNC
Publication type: Article
Publication status: Published
Journal: Journal of Biological Chemistry
Year: 2001
Volume: 276
Issue: 14
Pages: 10620-10626
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology, Inc.
URL: http://dx.doi.org/10.1074/jbc.M010867200
DOI: 10.1074/jbc.M010867200
PubMed id: 11116158
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