Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
IgG contains one asparagine-linked biantennary carbohydrate in the Fc region of the heavy chain (1). Even though the heterogeneous biantennary structure of different molecules can be characterized by the variability of the presence of terminal galactose, the overall oligosaccharide pattern is constant for healthy individuals (2).
Changes in the extent of galactosylation in the N-glycans of IgG are related to several diseases such as cancer (3), arthritis (4), Castleman’s disease (5), periodontitis (6), and coeliac disease (7). Other emerging developments are in the use of recombinantly produced glycoproteins for therapeutic applications. Functional consequences can result from improper glycosylation. In the production of monoclonal antibodies it has been shown that varying dissolved oxygen concentrations in murine hybridoma cell lines during growth can influence the extent of galactosylation (8).
Specific glycosidases can be used to characterize the diversity of the carbohydrate structures in IgG. This application note describes the use of a β-N-Acetylglucosaminidase (recombinant expressed in E. coli) to remove terminal N-acetylglucosamine from the biantennary N-glycoprotein IgG. The amount of N-acetylglucosamine that is released is a direct result of the extent of galactosylation.
β-N-Acetylglucosaminidase (NEB μl @ -20°C).
Glycoprotein Substrate 10 mg/μl: 85 μl
GlycoBuffer 1 10x: 10 μl
β-N-Acetylglucosaminidase: 5 μl (20 units)
Total volume: 100 μl
), N-acetylglucosamine standard (Sigma #A8625), IgG from human serum (Sigma; #4506), 10X G1 buffer (supplied with enzyme).
- Prepare 10 mg/ml solution of IgG in water (store in aliquots of 100 "_blank" href="~/media/Catalog/All-Products/F63409F329664923AC045F699112208B/Application%20Notes/appNoteP0732.pdf">Download PDF Brochure
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- Fleming, S. C. et al. (1998) Journal of Clinical Pathology, 51, 825-830.
- Watson, M. et al. (2001) Arthritis and Rheumatism, 42, 1682-1690.
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- Gordana, S. et al. (2006) Journal of Periodontology, 77, 1887-1893.
- Cremata, J. A. et al. (2003) Clinical and Experimental Immunology, 133, 422-429.
- Kunkel, J.P. et al. (1998) Journal of Biotechnology, 62, 55-71.