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Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES: Characterization of active disaggregated chemokine variants

Lookup NU author(s): Dr Steven Evans

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Abstract

Human CC chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, non-aggregating variants were sought. The phenotypes of 105 hMIP-1α variants generated by systematic mutagenesis and expression in yeast were determined, hMIP-1α residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1β and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three- dimensional NMR structure of hMIP-1α has enabled comparison of these residues in hMIP-1β and RANTES. Aggregated and disaggregated forms of hMIP- 1α, hMIP-1β, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1α, hMIP-1β, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.


Publication metadata

Author(s): Czaplewski LG, McKeating J, Craven CJ, Higgins LD, Appay V, Brown A, Dudgeon T, Howard LA, Meyers T, Owen J, Palan SR, Tan P, Wilson G, Woods NR, Heyworth CM, Lord BI, Brotherton D, Christison R, Craig S, Cribbes S, Edwards RM, Evans SJ, Gilbert R, Morgan P, Randle E, Schofield N, Varley PG, Fisher J, Waltho JP, Hunter MG

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 1999

Volume: 274

Issue: 23

Pages: 16077-16084

Print publication date: 04/06/1999

ISSN (print): 0021-9258

ISSN (electronic):

Publisher: American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.274.23.16077

DOI: 10.1074/jbc.274.23.16077

PubMed id: 10347159


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