AGGREGATION OF THE CHEMOKINE MIP-1-ALPHA IS A DYNAMIC AND REVERSIBLE PHENOMENON - BIOCHEMICAL AND BIOLOGICAL ANALYSES

Macrophage inhibitory protein (MIP)-1 alpha is a potent inhibitor of h emopoietic 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 bu t has a strong tendency for noncovalent extensive self-aggregation. As this aggregation may interfere with biological activity, we have prod uced nonaggregating variants of MIP-1 alpha which display a range of m olecular sizes. The mutants, produced by sequential neutralization of carboxyl-terminal acidic residues, display native molecular masses rep resentative of tetramers, dimers, and monomers. Intriguingly when thes e mutants are assessed in comparison with native MIP-1 alpha for bioac tivity in vitro, they are seen to be equipotent in both stem cell assa ys and in monocyte shape-change assays, suggesting that there is no re quirement 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 funct ion as monomers. We have further demonstrated the ability of MIP-1 alp ha to disaggregate spontaneously in dilute solution by enzyme-linked i mmunosorbent assay analysis of fractions obtained from gel filtration of varying concentrations of MIP-1 alpha. The aggregation of MIP-1 alp ha is therefore a dynamic and reversible phenomenon which has little, if any impact on bioactivity in vitro.