IL-1β epitope mapping using site-directed mutagenesis and hydrogen -: Deuterium exchange mass spectrometry analysis
IL-1β epitope mapping using site-directed mutagenesis and hydrogen -: Deuterium exchange mass spectrometry analysis
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DOI：
10.1021/bi0505464
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发表时间：
2005-08-23
期刊：
影响因子：
2.9
通讯作者：
Kuhstoss, S
中科院分区：
文献类型：
--
作者：
Lu, JR;Witcher, DR;Kuhstoss, S
Hu 007是一种人源化IgG 1单克隆抗体，可结合并中和人、食蟹猴和兔IL-1 β，但仅与小鼠和大鼠IL-1 β弱结合。Biacore实验证明Hu 007和I型IL-1受体竞争结合IL-1 β。增加盐的浓度降低的缔合速率，只有适度的影响的解离速率，这表明远程静电的初始复合物的形成是至关重要的。为了理解Hu 007的配体结合特异性，我们绘制了参与识别的关键残基。将食蟹猴IL-1 β中的选定残基突变为小鼠IL-1 β中的相应残基，并使用Biacore通过表面等离子体共振测量来评估变化对结合的影响。具体地，F150 S的取代使结合亲和力降低100倍，表明疏水相互作用在稳定抗体/抗原复合物中的重要性。将cIL-1 β N端和C端区域附近的三个氨基酸替换为小鼠IL-1 β中发现的氨基酸（V3 I/S5 Q/F150 S），使Hu 007与IL-1 β的结合亲和力降低约1000倍。相反，将小鼠IL-1 β中的相应残基突变为人序列导致结合亲和力增加约1000倍。氢-氘交换/质谱分析证实，由于抗体结合，IL-1 β的这些区域被保护免于交换。该研究的结果表明，Hu 007与位于IL-1 β的β-桶结构的开放端的区域结合，并阻断IL-1 β与其受体的结合。
Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus, and rabbit IL-1 beta but only weakly binds to mouse and rat IL-1 beta. Biacore experiments demonstrated that Hu007 and the type-I IL-1 receptor competed for binding to IL-I beta. Increasing salt concentrations decrease the association rate with only moderate effects on the dissociation rate, suggesting that long-range electrostatics are critical for formation of the initial complex. To understand the ligand-binding specificity of Hu007, we have mapped the critical residues involved in the recognition of Selected residues in cynomolgus IL-1 beta were mutated to the corresponding residues in mouse IL-1 beta, and the effects of the changes on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically, substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobic interactions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the Nand C-terminal regions of cIL-1 beta with those found in mouse IL-1 beta (V3I/S5Q/F150S) decreased the binding affinity of Hu007 to IL-1 beta by about 1000-fold. Conversely, mutating the corresponding residues in mouse IL-1 beta to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1 beta were protected from exchange because of antibody binding. The results from this study demonstrate that Hu007 binds to a region located in the open end of the beta-barrel structure of IL-1 beta and blocks binding of IL-1 beta to its receptor.