Structural glycomics by NMR

我们开发了一种系统的利用核磁共振（NMR）光谱分析结构糖组学的方法。在这种方法中，在核磁共振分析之前，目标糖蛋白的共价结构是在多维高效液相色谱数据的基础上确定的，这使得我们现在能够在分子、细胞和组织水平上进行定量糖基化分析，处理硫酸和葡萄糖醛基低聚糖。此外，通过多维高效液相色谱法构建的低聚糖标准库可以为蛋白质潜在的糖结合活性的综合分析提供有用的工具。我们已经成功地阐明了糖蛋白命运决定的分子机制，通过与多种细胞内凝集素作为分子伴侣、货物受体和泛素连接酶的相互作用。为了在原子水平上分析糖缀合物的结构、动力学和相互作用，我们采用了920 MHz超高场核磁共振波谱结合糖链稳定同位素标记技术。通过这种方法，我们揭示了人免疫球蛋白G1的铁区糖型依赖性构象改变，这影响了该糖蛋白的效应功能。我们的超高场核磁共振研究还揭示了β淀粉样蛋白和神经节苷脂簇之间的相互作用模式，这与阿尔茨海默病的发病有关。这些研究将为合理设计治疗性抗体以及靶向糖-蛋白相互作用的药物提供新的机会。

We have developed a systematic methods for structural glycomics by using nuclear magnetic resonance (NMR) spectroscopy. In this methodology, prior to NMR analyses, the covalent structures of target glycoproteins are determined on the basis of the multi-dimensional HPLC data, which now enable us to perform quantitative glycosylation profiling at molecular, cellular, and tissue levels dealing with sulfated and glucuronyl oligosaccharides. In addition, the library of the oligosaccharide standards constructed by the multi-dimensional HPLC can be a useful tool for comprehensive analyses of potential sugar-binding activities of proteins. We have successfully elucidated the molecular mechanisms of the glycoprotein-fate determination in cells through the interactions with a variety of intracellular lectins operating as molecular chaperones, cargo receptors, and ubiquitin ligases.To analyze structures, dynamics, and interactions of glycoconjugates at atomic level, we employed a 920 MHz ultra-high field NMR spectroscopy combined with stable isotope labeling techniques of sugar chains. By this approach, we have revealed glycoform-dependent conformational alteration of the Fe region of human immunoglobulin G1, which affects the effector functions of this glycoprotein. Our ultra-high field NMR study also revealed the interaction modes between amyloid β and ganglioside clusters, which are associated with the onset of Alzheimer's disease. These studies will offer a new opportunity for the rational design of therapeutic antibodies as well as drugs targeting sugar-protein interactions.