Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution
Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution
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DOI：
10.1189/jlb.1009649
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发表时间：
2010-06-01
影响因子：
5.5
通讯作者：
Yamamoto, Tetsuro
中科院分区：
文献类型：
--
作者：
Jia, Nan;Semba, Umeko;Yamamoto, Tetsuro
来自大肠杆菌的SKP作为C5a受体的纯趋化配体吸引白细胞[1]。我们利用人工合成的多肽片段和Skp的定点突变体，鉴定了与C5a受体结合并激活C5a受体的亚分子区域-Gln103-Asp104-Arg105。由于Skp的Gln103的C5a氨基酸残基为Leu72，因此我们制备了Gln103 Leu-Skp突变体作为重组蛋白。通过这个突变，Skp获得了促分泌功能，包括诱导呼吸爆发和颗粒释放反应，以及白三烯的产生，以及C5a所显示的化学吸引。然而，当我们将C5a中的Leu72替换为Gln时，L72Q-C5a突变体大部分失去了促分泌功能。这些功能转换是使用模拟重组蛋白的受体结合/激活区域的合成肽来复制的。使用模拟多肽进行的受体结合分析表明，Leu72-C5a和Gln72-C5a多肽之间只有很小的差异。L72Q-C5a明显拮抗C5a的促分泌作用。这些结果表明，趋化反应和联合趋化/分泌反应之间的差异不是归因于受体的性质，而是配体的指导，至少在C5a受体介导的白细胞反应的情况下是这样。J.Leukoc。比奥尔。87：965-975；2010。
Skp derived from Escherichia coli attracts leukocytes as a pure chemotactic ligand of the C5a receptor [1]. We identified the submolecular region of Skp that binds and activates the C5a receptor to be -Gln103-Asp104-Arg105-using synthetic peptide fragments and site-directed mutants of Skp. As the C5a amino acid residue equivalent to Gln103 of Skp is Leu72, we prepared a Gln103Leu-Skp mutant as a recombinant protein. With this mutation, Skp gained secretagogue functions including induction of the respiratory burst and granule release reactions and leukotriene generation, in addition to the chemoattraction displayed by C5a. However, when we substituted Leu72 with Gln in C5a, the L72Q-C5a mutant largely lost its secretagogue function. These functional conversions were reproduced using synthetic peptides mimicking the receptor-binding/-activating regions of the recombinant proteins. Receptor-binding assays using the mimicking peptides demonstrated only a small difference between the Leu72-C5a and Gln72-C5a peptides. Consistently, L72Q-C5a apparently antagonized C5a secretagogue function. These results indicate that the difference between a chemotactic response and a combined chemotactic/secretory response can be attributed not to the nature of the receptor but to guidance by the ligand, at least in the case of C5a receptor-mediated leukocyte responses. J. Leukoc. Biol. 87: 965-975; 2010.