Glycosylation is one of the most common post-translational modifications of proteins, as shown in Figure 1.
N-linked glycosylation occurs when glycans are attached to asparagine residues on the core protein. O-linked glycosylation occurs when glycans are attached to serine or threonine residues. Both chemical and enzymatic methods exist for removing oligosaccharides from glycoproteins. However, chemical methods such as β-elimination with mild alkali or mild hydrazinolysis can be harsh and may result in incomplete sugar removal and degradation of the protein; whereas, enzymatic methods are much gentler and can provide complete sugar removal with no protein degradation.
PNGase F is the most effective enzymatic method for removing almost all
N-linked oligosaccharides from glycoproteins. PNGase F digestion deaminates the aspargine residue to aspartic acid, and leaves the oligosaccharide intact, keeping it suitable for further analysis. Oligosaccharides containing a fucose α(1-3)-linked to the glycan core are, however, resistant to PNGase F which can occur on some plant and insect glycoproteins. Steric hindrance slows or inhibits the action of PNGase F on certain residues of glycoproteins; therefore denaturation of the glycoprotein by heating with SDS and DTT greatly increases the rate of deglycosylation. Other commonly used endoglycosidases such as Endoglycosidase H are not suitable for general deglycosylation of N-linked sugars because of their limited specificities and because they leave one N-acetylglucosamine residue attached to the asparagine.
O-linked glycans, monosaccharides must be removed by a series of exoglycosidases until only the Galβ1-3GalNAc (core 1) and/or the GlcNAcβ1-3GalNAc (core 3) cores remain attached to the serine or threonine. The Enterococcus faecalis O-Glycosidase, also called O-Glycosidase, also called Endo-α- N-Acetylgalactosaminidase, can then remove these core structures with no modification of the serine or threonine residues. Any modification of the core structures, including sialyation, will block the action of the O-Glycosidase. Sialic acid residues are easily removed by a general α2-3,6,8 Neuraminidase. In addition, exoglycosidases such as β(1-4)Galactosidase and β-N-Acetylglucosaminidase can be included in deglycosylation reactions to remove other complex modifications often known to be present on the core structures. This combination of enzymes will not remove all O-linked oligosaccharides but should remove many common oligosaccharide structures.
Figure 1: A glycoprotein modified with O-linked and N-linked glycosylation
Figure 2: Enzymatic Deglycosylation of Bovine Fetuin
100 µg Bovine Fetuin Control was deglycosylated using the denaturing reaction conditions. 25 µg of the reaction was loaded onto a 10/20 SDS-PAGE gel. Lane 1: Protein Ladder (10-250 kDa) ( NEB #P7703 ), Lane 2: 25 µg untreated Fetuin control, Lane 3: 25 µg denatured Fetuin control, Lane 4: 25 µg deglycosylated denatured Fetuin, Lane 5: 5 µl Deglycosylation Mix
Deglycosylation Enzyme Mix:
20 µl PNGase F Glycerol
20 µl O-Glycosidase:
20 µl Neuraminidase:
20 µl β1-4
Fetuin, 0.5 mg (Fetuin contains sialylated N-linked and
Deglycosylation Enzyme Mix supplied in:
50 mM NaCl, 20 mM
Tris-HCl (pH 7.5 @ 25°C) and 2.6 mM EDTA.
Description of Enzymes Included in the
Deglycosylation Enzyme Mix: ,
also known as Endo-α- N-Acetylgalactosaminidase, is a
recombinant enzyme cloned from Enterococcus faecalis (1). It catalyzes
the removal of core 1 and core 3 O-linked disaccharides from
glycoproteins. The molecular weight is approximately 147
PNGase F, also known as Peptide:
N-glycosidase F, is an enzyme purified from Flavobacterium
meningosepticum (2). PNGase F is an amidase which cleaves between the
innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex
oligosaccharides from N-linked glycoproteins unless α(1-3) core
fucosylated. The molecular weight is approximately 36
Neuraminidase, also known as Sialidase, is a
recombinant enzyme cloned from Clostridium perfringens (3) and
overexpressed in E. coli (4). It catalyzes the hydrolysis of terminal, non-reducing α2,3,
α2,6, and α2,8 linked N-acetylneuraminic acid residues from
glycoproteins and oligosaccharides. The molecular weight is approximately 43
β1-4 Galactosidase, is a recombinant enzyme cloned
from Bacteroides fragilis (5). It is a highly specific exoglycosidase
that catalyzes the hydrolysis of terminal, non-reducing β1-4 linked D-galactopyranosyl residues from
oligosaccharides and glycoproteins. The molecular weight is approximately 94
β-, is a recombinant enzyme
cloned from N-Acetylglucosaminidase Xanthomonas manihotis (6). It is a highly specific
exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing
β- N-Acetylglucosamine residues from oligosaccharides and glycoproteins. The molecular
weight is approximately 71 kDa.
The following reagents are supplied with this product:
Store at (°C) Concentration
Glycoprotein Denaturing Buffer -20 10X
NP-40 -20 10%
GlycoBuffer 2 -20 10X
Fetuin 4 10 mg/ml