Identification of amino acid residues critical for aggregation of human CCchemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES - Characterization of active disaggregated chemokine variants
Lg. Czaplewski et al., Identification of amino acid residues critical for aggregation of human CCchemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES - Characterization of active disaggregated chemokine variants, J BIOL CHEM, 274(23), 1999, pp. 16077-16084
Human CC chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 be
ta, and RANTES (regulated on activation normal T cell expressed) self-assoc
iate to form high-molecular mass aggregates. To explore the biological sign
ificance of chemokine aggregation, nonaggregating variants were sought. The
phenotypes of 105 hMIP-1 alpha variants generated by systematic mutagenesi
s and expression in yeast were determined, hMIP-1 alpha residues Asp(26) an
d Glu(66) were critical to the self-association process. Substitution at ei
ther 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-1 beta and RANTES demonstrated that they were also
critical to aggregation. Our analysis suggests that a single charged residu
e at either position 26 or 66 is insufficient to support extensive aggregat
ion and that two charged residues must be present. Solution of the three-di
mensional NMR structure of hMIP-1 alpha has enabled comparison of these res
idues in hMIP-1 beta and RANTES. Aggregated and disaggregated forms of hMIP
-1 alpha, hMIP-1 beta, and RANTES generally have equivalent G-protein-coupl
ed receptor-mediated biological potencies. We have therefore generated nove
l reagents to evaluate the role of hMIP-1 alpha, hMIP-1 beta, and RANTES ag
gregation in vitro and in vivo. The disaggregated chemokines retained their
human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, hi
gh concentrations of RANTES, but not disaggregated RANTES variants, enhance
d infection of cells by both M- and T-tropic HIV isolates/strains. This obs
ervation has important implications for potential therapeutic uses of chemo
kines implying that disaggregated forms may be necessary for safe clinical
investigation.