Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Molecules. 2017 Sep 27;22(10):1619. doi: 10.3390/molecules22101619.
Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.
尽管抗体的功能是在分子拥挤和混杂的分子间相互作用的异质血流中执行的，但到目前为止，对抗体相互作用的详细结构特征的研究都是在同质的体外条件下进行的。NMR 光谱学有可能在异质环境中研究蛋白质结构，假设目标蛋白质可以用 NMR 活性同位素标记。基于我们成功开发的抗体糖蛋白的同位素标记，在这里我们应用 NMR 光谱学来表征异质细胞外环境中的抗体相互作用，以小鼠 IgG-Fc 作为测试分子。在人血清中，许多源自 Fc 骨架的 HSQC 峰的强度都有不同程度的衰减。来自血清中的多克隆 IgG 的 Fab 片段也会引起类似的光谱变化，但血清白蛋白不会，这表明在没有预先免疫的情况下，人血清中存在一组与小鼠 IgG-Fc 反应的抗体。血清多克隆 IgG 识别的区域覆盖了 Fc 的整个分子表面，包括与 Fc 受体和 C1q 的结合位点。NMR 观察将为在生理相关的异质环境中对生物制药（包括治疗性抗体）进行详细表征提供有用的工具，也将深入了解免疫系统中多克隆抗体的分子识别。
