Chung Il Yup, Kim Yong Hyun, Choi Moon Kyung, Noh Yoon Jung, Park Choon-Sik, Kwon Do Yoon, Lee Duck Yeon, Lee Young Seek, Chang Hun Soo, Kim Key-Sun
Department of Biochemistry and Molecular Biology, Hanyang University, Ansan, Kyunggi-do, Republic of Korea.
Biochem Biophys Res Commun. 2004 Feb 6;314(2):646-53. doi: 10.1016/j.bbrc.2003.12.134.
Eotaxin selectively binds CC chemokine receptor (CCR) 3, whereas monocyte chemotactic protein (MCP)-3 binds CCR1, CCR2, and CCR3. To identify the functional determinants of the chemokines, we generated four reciprocal chimeric chemokines-M10E9, M22E21, E8M11, and E20M23-by shuffling the N-terminus and N-loop of eotaxin and MCP-3. M22E21 and E8M11, which shared the N-loop from MCP-3, bound to monocytes with high affinity, and activated monocytes. In contrast, M10E9 and E20M23, which lacked the N-loop, failed to bind and transduce monocyte responses, identifying the N-loop of MCP-3 as the selectivity determinant for CCR1/CCR2. A BIAcore assay with an N-terminal peptide of CCR3 (residues 1-35) revealed that all chimeras except E20M23 exhibited varying degrees of binding affinity with commensurate chemotaxis activity of eosinophils. Surprisingly, E20M23 could neither bind the CCR3 peptide nor activate eosinophils, despite having both N-terminal motifs from eotaxin. These results suggest that the two N-terminal motifs of eotaxin must cooperate with other regions to successfully bind and activate CCR3.
嗜酸性粒细胞趋化因子选择性结合CC趋化因子受体（CCR）3，而单核细胞趋化蛋白（MCP）-3结合CCR1、CCR2和CCR3。为了确定趋化因子的功能决定因素，我们通过交换嗜酸性粒细胞趋化因子和MCP-3的N端和N环，生成了四种相互嵌合的趋化因子——M10E9、M22E21、E8M11和E20M23。共享MCP-3 N环的M22E21和E8M11以高亲和力结合单核细胞，并激活单核细胞。相比之下，缺乏N环的M10E9和E20M23无法结合并传导单核细胞反应，从而确定MCP-3的N环是CCR1/CCR2的选择性决定因素。一项使用CCR3 N端肽（第1至35位氨基酸残基）的生物传感器分析表明，除E20M23外，所有嵌合体都表现出不同程度的结合亲和力以及相应的嗜酸性粒细胞趋化活性。令人惊讶的是，尽管E20M23具有嗜酸性粒细胞趋化因子的两个N端基序，但它既不能结合CCR3肽，也不能激活嗜酸性粒细胞。这些结果表明，嗜酸性粒细胞趋化因子的两个N端基序必须与其他区域协同作用，才能成功结合并激活CCR3。
