Kato Koichi, Yanaka Saeko, Yagi Hirokazu
Graduate School of Pharmaceutical Sciences, Nagoya City University.
Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences.
Yakugaku Zasshi. 2018;138(12):1495-1502. doi: 10.1248/yakushi.18-00020-3.
Detailed structural characterization of protein biopharmaceuticals is a critical step in research and development; however, this step is often hampered by the structural complexities associated with glycosylation. Most protein biopharmaceuticals are modified with structurally heterogeneous and dynamic oligosaccharides which govern the physicochemical properties, functionality, pharmacokinetics, and potential pathogenicity of these glycoproteins. Considering this, we have developed a structural biological approach to describe the dynamic three-dimensional structures and interactions of glycoproteins as biopharmaceuticals. We developed an NMR technique assisted by metabolic stable-isotope labeling that can provide useful atomic-level probes for detecting and characterizing structural perturbations of glycoproteins caused by alterations in solution conditions and production protocols, as well as by mutagenesis. We have applied this method in conjunction with X-ray crystallography to investigate the structural impacts of varying glycoforms of the Fc region of immunoglobulin G (IgG), thereby elucidating the functional roles of the Fc glycans. In particular, we have successfully elucidated the structural mechanisms by which defucosylation of the IgG-Fc region increases its affinity for Fcγ receptor IIIa, leading to an improvement in ameliorating antibody-dependent cell-mediated cytotoxicity. In addition, we applied our stable-isotope-assisted NMR method to analyzing biomolecular interactions in serum environments, which are characterized by molecular crowding and promiscuous intermolecular interactions. An integrative structural biological approach combining NMR spectroscopy, X-ray crystallography, neutron scattering, atomic force microscopy, and molecular dynamics simulation will provide new research tools that will enable the visualization of dynamic structures and interactions of glycoproteins of pharmaceutical interest, thereby providing valuable insights for the development of biopharmaceuticals.
蛋白质生物药物的详细结构表征是研发过程中的关键步骤；然而，这一步骤常常受到与糖基化相关的结构复杂性的阻碍。大多数蛋白质生物药物都被结构异质且动态的寡糖修饰，这些寡糖决定了这些糖蛋白的物理化学性质、功能、药代动力学和潜在致病性。考虑到这一点，我们开发了一种结构生物学方法来描述作为生物药物的糖蛋白的动态三维结构和相互作用。我们开发了一种由代谢稳定同位素标记辅助的核磁共振技术，该技术可以提供有用的原子水平探针，用于检测和表征由溶液条件和生产方案的改变以及诱变引起的糖蛋白结构扰动。我们已将此方法与X射线晶体学结合应用，以研究免疫球蛋白G（IgG）Fc区域不同糖型的结构影响，从而阐明Fc聚糖的功能作用。特别是，我们成功阐明了IgG-Fc区域去岩藻糖基化增加其对Fcγ受体IIIa亲和力的结构机制，从而改善了抗体依赖性细胞介导的细胞毒性。此外，我们将稳定同位素辅助的核磁共振方法应用于分析血清环境中的生物分子相互作用，血清环境的特点是分子拥挤和混杂的分子间相互作用。结合核磁共振光谱、X射线晶体学、中子散射、原子力显微镜和分子动力学模拟的综合结构生物学方法将提供新的研究工具，能够可视化具有药物意义的糖蛋白的动态结构和相互作用，从而为生物药物的开发提供有价值的见解。
