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
- What is the difference between PNGase F and Endo H?
- Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
- 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?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- Can glycosidases be used on whole cells?
- What are glycosidases and their uses?
- Can glycosidases be used in combination for extensive digestion?
Protocols for Endoglycosidases
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- Reaction Conditions for Endo D (P0742)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Reaction Conditions for Endo S (P0741)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- 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)
- Reaction Conditions for PNGase A (P0707)
- 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)
- Removal of Endo F2 by Magnetic Beads (P0772)
- Endo F2 Reaction Protocol (P0772)
- Endo H/Endo Hf Protocol
- Suggested Protocol for Loading the Unstained Protein Ladder, Broad Range (P7703)
- Loading a Sample (P7708)
- O-Glycosidase (P0733)
- O-Glycosidase Application Note 1 (P0733)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
Application Notes for Endoglycosidases
N-Glycan Composition Profiling for Quality Testing of Biotherapeutics
The Structure, Function and Importance of Carbohydrates
The Glycobiology brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
The Glycoproteomics brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
- Detailed Characterization of Several Glycosidase Enzymes
- Glycobiology Unit Conversion Chart
Other Tools & Resources
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.
Learn how glycosidases are used to analyze multiple glycoprotein samples. Here, the model glycoprotein, hCGβ, which carries both N- and O-glycans, is demonstrated.
Learn more about the structural elucidation of complex and diverse heparin oligosaccharides using Bacteroides Heparinase I, II and III in combination with downstream mass spec analysis.