Removal of glycan groups from proteins via enzymatic methods is preferable to chemical removal because it is gentler and can provide complete sugar removal with no protein degradation.
Endoglycosidases cleave entire glycan groups from glycoproteins. The most commonly used endoglycosidase, Peptide-N-Glycosidase F (PNGase F), is actually an amidase, which catalyzes the deglycosylation of most N-linked glycoproteins. The enzyme catalyzes the cleavage between the innermost N-Acetylglucosamine (GlcNAc) of the chitobiose core and the amine group of the asparagine residue (1).There is a variety of endoglycosidases that are active on N-linked glycans. They all have identical cleavage sites between the two GlcNAc residues of the chitobiose core, but they recognize and cleave different types of N-linked glycans. Endoglycosidase H (Endo H) (NEB #P0702, NEB #P0703) cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins (1). Endoglycosidase S (Endo S) (NEB #P0741) is highly specific for removing N-linked glycans from the heavy chain of native IgG (2). While Endoglycosidase D (Endo D) (NEB #P0742) cleaves paucimannose N-linked glycans(3).
There are fewer endoglycosidases also called α-N-Acetylgalactosaminidases active on O-linked glycans. The majority of O-glycosidases found to date are active only on a disaccharide Galβ1,3GalNAcα-O-Ser/Thr requiring the removal of Sialic acid and any other attached sugar residues before the enzyme can catalyze the removal of this Core 1 disaccharide from the glycoprotein. However there is one enzyme with a slightly broader O-Glycosidase specificity (NEB #P0733, NEB #E0540), and it is able to catalyze the removal of Core 1 (Galβ1,3GalNAc) or Core 3 (GlcNAcβ1,3GalNAc) O-linked disaccharides from glycoproteins (4).
- Maley, F. et al. (1989) Anal. Biochem., 180, 195-204.
- Collin, M. and Olsén, A. (2001). The EMBO Journal. 20, 3046-3055. PMID: 11406581
- Mizuochi, T. et al. (1984) J. Biochem. 95, 1209-1213.
- Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008) Glycobiology, 18, 799-805. PMID: 18635885
- Do detergents inhibit exoglycosidases/endoglycosidases?
- Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
- What are Glycosidases and their uses?
- What is the difference between PNGase F and Endo H?
- What is a good endoglycosidase substrate?
- Are NEB's endoglycosidases compatible with protease inhibitor cocktails?
- What is the difference between Endo H and Endo Hf?
- Can endoglycosidases be removed from the reaction?
- Can glycosidases be used in combination for extensive digestion?
- Can glycosidases be used on whole cells?
- Do the Remove-iT glycosidases have the same specificity as enzymes without a tag?
- Loading a Sample (P7708)
- O-Glycosidase Application Note 1 (P0733)
- O-Glycosidase (P0733)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Suggested Protocol for Loading the Unstained Protein Ladder, Broad Range (P7703)
- Endo H/Endo Hf Protocol
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Reaction Conditions for Endo S (P0741)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Reaction Conditions for Endo D (P0742)
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- PNGase F Protocol
- Rapid PNGase F Protocols (P0710)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- Reaction Conditions for PNGase A (P0707)
- Endo F2 Reaction Protocol (P0772)
- Removal of Endo F2 by Magnetic Beads (P0772)
The Structure, Function and Importance of Carbohydrates
Read about the structure, function, and importance of Carbohydrates from biology experts at NEB.
O-glycan analysis of therapeutic proteins enabled by O-glycoprotease
This article from European Pharmaceutical Review describes tools that can be used for O-glycan analysis of therapeutic proteins.
- Glycoproteomics Brochure
- Detailed Characterization of Several Glycosidase Enzymes
- Glycobiology Unit Conversion Chart
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Learn about glycobiology and its importance in clinical and diagnostic applications in this episode of NEB TV. Also, hear more about how NEB is setting the bar for product quality in this rapidly growing field.
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