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
FAQs for Endoglycosidases
- Do the Remove-iT glycosidases have the same specificity as enzymes without a tag?
- Can endoglycosidases be removed from the reaction?
- What is a good endoglycosidase substrate?
- What is the difference between Endo H and Endo Hf?
- Are NEB's endoglycosidases compatible with protease inhibitor cocktails?
- What is the difference between PNGase F and Endo H?
- Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
- Can glycosidases be used on whole cells?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- Can glycosidases be used in combination for extensive digestion?
- What are glycosidases and their uses?
Protocols for Endoglycosidases
- PNGase F Protocol
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Rapid PNGase F Protocols (P0710)
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- Reaction Conditions for PNGase A (P0707)
- Removal of Endo F2 by Magnetic Beads (P0772)
- Endo F2 Reaction Protocol (P0772)
- Loading a Sample (P7708)
- O-Glycosidase (P0733)
- O-Glycosidase Application Note 1 (P0733)
- Endo H/Endo Hf Protocol
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- Reaction Conditions for Endo D (P0742)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Reaction Conditions for Endo S (P0741)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Suggested Protocol for Loading the Unstained Protein Ladder, Broad Range (P7703)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Preparing and Loading Protein Ladders
- Enzymatic removal of N- and O-glycans using PNGase F or the Protein Deglycosylation Mix P0704
- Glycan Analysis of Murine IgG by Enzymatic Digestion
- O-Glycosidase Application Note 1
The Glycobiology brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
N-Glycan Composition Profiling for Quality Testing of Biotherapeutics
The Structure, Function and Importance of Carbohydrates
- Detailed Characterization of Several Glycosidase Enzymes
- Glycobiology Unit Conversion Chart
Other Tools & Resources
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