Graham G J, MacKenzie J, Lowe S, Tsang M L, Weatherbee J A, Issacson A, Medicherla J, Fang F, Wilkinson P C, Pragnell I B
Beatson Institute for Cancer Research, Cancer Research Campaign, Beatson Laboratories, Bearsden, Glasgow, United Kingdom.
J Biol Chem. 1994 Feb 18;269(7):4974-8.
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.
巨噬细胞抑制蛋白（MIP）-1α是造血干细胞增殖的强效抑制剂，属于促炎介质家族——趋化因子家族的一员。该分子与趋化因子家族的其他成员一样，以8 kDa的肽形式存在，但具有强烈的非共价广泛自我聚集倾向。由于这种聚集可能会干扰生物活性，我们制备了一系列具有不同分子大小的非聚集性MIP-1α变体。通过对羧基末端酸性残基进行逐步中和产生的这些突变体，呈现出代表四聚体、二聚体和单体的天然分子量。有趣的是，当将这些突变体与天然MIP-1α在体外进行生物活性评估比较时，发现在干细胞测定和单核细胞形态变化测定中它们具有同等效力，这表明在这两种生物学环境中都不需要聚集。这表明聚集的MIP-1α和聚集的突变体在测定条件下会自发解聚，最终以单体形式发挥作用。我们通过对不同浓度MIP-1α凝胶过滤得到的级分进行酶联免疫吸附测定分析，进一步证明了MIP-1α在稀溶液中自发解聚的能力。因此，MIP-1α的聚集是一种动态且可逆的现象，对体外生物活性几乎没有影响（如果有影响的话也很小）。
