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
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
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DOI：
10.1074/jbc.274.23.16077
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发表时间：
1999-06-04
影响因子：
4.8
通讯作者：
Hunter, MG
中科院分区：
文献类型：
--
作者：
Czaplewski, LG;McKeating, J;Hunter, MG
人CC趋化因子巨噬细胞炎性蛋白（MIP）-1 α、MIP-1 β和RANTES（受正常T细胞表达的活化调节）自缔合以形成高分子量聚集体。为了探索趋化因子聚集的生物学意义，寻找非聚集变体。通过系统诱变和酵母表达获得的105个hMIP-1 α变体的表型进行了测定，hMIP-1 α残基Asp（26）和Glu（66）是自缔合过程的关键。在任一残基处的取代导致在0.5mg/ml下形成基本上均匀的四聚体。在hMIP-1 β和RANTES的同源位置上取代相同或类似的残基表明它们对聚集也是关键的。我们的分析表明，在位置26或66处的单个带电残基不足以支持广泛的聚集，并且必须存在两个带电残基。hMIP-1 α的三维NMR结构的解决方案使得能够比较hMIP-1 β和RANTES中的这些残基。hMIP-1 α、hMIP-1 β和RANTES的聚集和解聚形式通常具有等效的G蛋白偶联受体介导的生物学效力。因此，我们已经产生了新的试剂来评估hMIP-1 α，hMIP-1 β和RANTES聚集在体外和体内的作用。分解的趋化因子保留了它们的人类免疫缺陷病毒（HIV）抑制活性。令人惊讶的是，高浓度的RANTES，而不是解聚的RANTES变体，增强了M-和T-嗜性HIV分离株/毒株对细胞的感染。这一观察结果对趋化因子的潜在治疗用途具有重要意义，这意味着解聚形式可能是安全临床研究所必需的。
Human CC chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, 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, nonaggregating variants were sought. The phenotypes of 105 hMIP-1 alpha variants generated by systematic mutagenesis and expression in yeast were determined, hMIP-1 alpha residues Asp(26) and Glu(66) 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-1 beta 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-1 alpha has enabled comparison of these residues in hMIP-1 beta and RANTES. Aggregated and disaggregated forms of hMIP-1 alpha, hMIP-1 beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1 alpha, hMIP-1 beta, 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.