Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
复制标题
使用真核表达系统对糖蛋白进行 NMR 表征的稳定同位素标记方法
DOI：
10.1007/s10858-018-0169-2
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发表时间：
2018
影响因子：
2.7
通讯作者：
Kato Koichi
中科院分区：
文献类型：
--
作者：
Yanaka Saeko;Yagi Hirokazu;Yogo Rina;Yagi-Utsumi Maho;Kato Koichi
糖蛋白的特征在于其聚糖部分的异质性和动态性质，这妨碍了晶体学分析。NMR光谱学提供了潜在的优势，在处理这样的复杂系统，考虑到目标分子可以同位素标记。重组糖蛋白的代谢性同位素标记方法近年来得到了发展，这些方法使用了多种真核生产载体，包括哺乳动物、酵母、昆虫和植物细胞，每种载体都具有不同的N-聚糖多样化途径。酵母基因工程已经使同质高甘露糖型寡糖的过量表达与13 C标记的NMR表征其构象动力学。稳定的同位素辅助NMR光谱的效用也已被证明使用的Fc片段的免疫球蛋白G（IgG）作为模型糖蛋白，提供有用的信息，分子内的碳水化合物-蛋白质相互作用。通过使用哺乳动物表达系统对选定的氨基酸残基进行定制的氘化，已经实现了分子量为150 kDa的完整IgG的横向弛豫优化。这为表征以血清为代表的多分子拥挤系统中的分子相互作用网络提供了有用的探针。展望有关技术的发展，为定制糖型设计和同位素标记的重组糖蛋白进行了讨论。
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinctN-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate–protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150 kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.