Aggregation of the chemokine MIP-1 alpha is a dynamic and reversible phenomenon. Biochemical and biological analyses.

Abstract

Macrophage inhibitory protein (MIP)-1 alpha is a potent inhibitor of hemopoietic stem cell proliferation and is a member of a family of pro-inflammatory mediators, the chemokine family. This molecule along with other members of the chemokine family exists as a peptide of 8 kDa but has a strong tendency for noncovalent extensive self-aggregation. As this aggregation may interfere with biological activity, we have produced nonaggregating variants of MIP-1 alpha which display a range of molecular sizes. The mutants, produced by sequential neutralization of carboxyl-terminal acidic residues, display native molecular masses representative of tetramers, dimers, and monomers. Intriguingly when these mutants are assessed in comparison with native MIP-1 alpha for bioactivity in vitro, they are seen to be equipotent in both stem cell assays and in monocyte shape-change assays, suggesting that there is no requirement for aggregation in either of these biological contexts. This indicates that the aggregated MIP-1 alpha and the aggregated mutants spontaneously disaggregate under assay conditions and ultimately function as monomers. We have further demonstrated the ability of MIP-1 alpha to disaggregate spontaneously in dilute solution by enzyme-linked immunosorbent assay analysis of fractions obtained from gel filtration of varying concentrations of MIP-1 alpha. The aggregation of MIP-1 alpha is therefore a dynamic and reversible phenomenon which has little, if any, impact on bioactivity in vitro.