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 #P0732), N-acetylglucosamine standard (Sigma #A8625), IgG from human serum (Sigma; #4506), 10X G1 buffer (supplied with enzyme).
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- Prepare 10 mg/ml solution of IgG in water (store in aliquots of 100 μl @ -20°C).
- Set up the exo-glycosidase digestion reaction:
Glycoprotein Substrate 10 mg/μl: 85 μl
GlycoBuffer 1 10x: 10 μl
β-N-Acetylglucosaminidase: 5 μl (20 units)
Total volume: 100 μl
- Incubate at 37°C for 4 hours. Add 200 μl water followed by 600 μl methanol. Chill overnight at 4°C to precipitate proteins. After the overnight precipitation, spin the sample at 14K rpm for 30 minutes, and reserve the supernatant.
- Concentrate supernatant to dryness with a Speed Vac set at medium heat (Savant; equipped with a high vacuum pump and finger trap immersed in a Dewar containing isopropanol and dry ice). Reconstitute with 400 μl Milli-Q water.
- De-ionize the sample from step 4 by gently rocking in 200 μl of prepared mixed bed ion exchange resin AGAG 501-X8 for 5 minutes (Bio-Rad; #142-6424). Collect the supernatant with a 1ml syringe using a 23 gauge needle. Note: before use, the resin must be converted to the acetate form by soaking in an equal volume of 1 M acetic acid followed washing ten times with equal volumes of water.
- Remove the needle and load the entire sample (400 μl) from Step 5 to an activated Sep-Pak cartridge (Waters; #WAT051910). Collect the entire flow through (400 μl). Wash the Sep-Pak 2 times with 400 μl of Milli-Q water and pool the washes with the flow through. Concentrate to 70 μl using a Speed Vac. Note: before use, the Sep-Paks were activated by washing two times with 400 μl methanol followed by 4 times with 400 μl Milli-Q water.
- Detect free N-acetylglucosamine (GlcNAc) by HPAEC-PAD Chromatography using the following conditions: Column: CarboPac 20 with Amino Guard. Elution: 20mM NaOH isocratic for 12 minutes, 150mM regeneration for 10 minutes, flow rate: 0.5 μl/min. Detection: Pulse electrochemical, Au electrode, quadruple potential. Injection sample: 30 μl, with or without internal GLcNAc standard (30 nanograms).
- Kornfeld, R. et al. (1971) Journal of Biological Chemistry, 246, 3259-3268.
- Parekh, R. B. et al. (1988) Lancet, 1, 966-969.
- Fleming, S. C. et al. (1998) Journal of Clinical Pathology, 51, 825-830.
- Watson, M. et al. (2001) Arthritis and Rheumatism, 42, 1682-1690.
- Kanayam, Y. et al. (1991) Clinica Chimica Acta, 197, 00221-00228.
- 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.