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.
To remove 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
Free:
500,000 units/ml
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:
O-Glycosidase,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
kDa.
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
kDa.
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
kDa.
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
kDa.
β-N-Acetylglucosaminidase, is a recombinant enzyme
cloned from 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.
This product is related to the following categories:
This kit contains all of the enzymes, reagents, and controls needed to remove almost all N-linked and simple O-linked glycans as well as some complex O-linked glycans. This kit contains enzyme sufficient for 20 reactions or the cleavage of as much as 2 mg of glycoprotein.
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
This is a single cocktail that combines all five enzymes
Can be used under native or denaturing conditions
Under native conditions we recommend adding more enzyme and using longer incubation times
Enzyme activity in this mix is inhibited by SDS, therefore under denaturing conditions it is essential to have NP-40 present in the reaction mixture in a 1:1 ratio.
A good positive control substrate is Fetuin
Magnelli P, Bielik A, Guthrie E. (2012) Identification and characterization of protein glycosylation using specific endo- and exoglycosidases Methods Mol Biol; 801 , 189-211 . PubMedID: 21987255
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please email us at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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