Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
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 distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C 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.
KeywordsStable isotope labeling Oligosaccharide Glycoprotein Eukaryotic expression system Antibody Serum
Stable isotope labeling was supported by Taiyo Nippon Sanso Co. We particularly thank Dr. Tsutomu Terauchi for useful discussion. We also thank Dr. Hajime Sato (Bruker BioSpin) for his help in the NMR measurements. We thank Ms. Kiyomi Senda and Ms. Kumiko Hattori (Nagoya City University) for their help in protein purification. This work was partly supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT, MEXT/JSPS Grants in Aid for Scientific Research (JP25102008, JP15K07935, JP17H06414 and JP17H05893).
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