Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 71, Issue 3, Pages 193-202Publisher
SPRINGER
DOI: 10.1007/s10858-018-0169-2
Keywords
Stable isotope labeling; Oligosaccharide; Glycoprotein; Eukaryotic expression system; Antibody; Serum
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Funding
- Taiyo Nippon Sanso Co.
- Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT, MEXT/JSPS Grant [JP25102008, JP15K07935, JP17H06414, JP17H05893]
- Grants-in-Aid for Scientific Research [15K07935, 17K15441, 25102001, 25102008] Funding Source: KAKEN
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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 C-13 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.
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