在H-CDR3循环中具有二硫化键的亲和成熟抗体
Mutsumi Yoshida1, Yuya Hanazono2, Nobutaka Numoto2
1Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto, 606-8522, Japan.
Archives of biochemistry and biophysics
|June 23, 2024
概括
抗体中的二硫化键增强了抗原的结合和稳定性. 这项研究表明,抗NP抗体中的特定二硫化键对于高亲和抗原识别和复杂形成至关重要.
科学领域:
- 免疫学 免疫学 免疫学
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 抗体亲和力成熟通过体质突变增强了抗原结合.
- 像E11这样的高度成熟的抗NP抗体在重链的CDR3中具有Cys96H和Cys100H,可能形成二硫化键.
研究的目的:
- 通过评估E11 scFv及其Cys突变体,研究二硫化键在抗原结合中的作用.
- 确定这些抗体抗原结合的热力学和动力学参数.
主要方法:
- 产生单链Fv (scFv) 抗体,包括E11及其氨酸突变体 (E11_C96KH/C100EH,E11_C96KH/C100QH).
- 确定抗原结合热力学和动力学.
- 高分辨率的晶体结构确定NP-free和NP-bound E11 scFvs.
- 小角度X射线散射 (SAXS) 分析.
主要成果:
- 与野生型E11 scFv相比,氨酸突变体表现出较低的结合亲缘关系,这表明二硫化键对抗原结合的贡献.
- 在还原剂的存在下,E11 scFv亲和力下降,进一步支持二硫化键的重要性.
- 晶体结构揭示了二硫化键,阐明了抗原识别机制,允许与其他抗NP抗体进行比较.
- 萨克斯数据显示,抗原结合会诱导溶液中E11 scFv的局部形状变化.
结论:
- Cys96H和Cys100H之间的二硫化键显著促进了E11 scFv的抗原结合亲和力和稳定复合物的形成.
- 结构洞察力解释了改变抗原结合亲和力和热稳定性的分子基础.
- 抗原结合会诱导抗体的结构灵活性,影响结合动态.
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