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  • Protein Deglycosylation Mix

    Description

    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

    20 µl O-Glycosidase:
    40,000,000 units/ml

    20 µl Neuraminidase:
    50,000 units/ml

    20 µl β1-4 Galactosidase:
    8,000 units/ml

    20 µl β-N-acetylglucosaminidase:
    4,000 units/ml

    Substrate Control:
    Fetuin, 0.5 mg (Fetuin contains sialylated N-linked and O-linked glycans)

    Deglycosylation Enzyme Mix supplied in:
    50 mM NaCl, 20 mM Tris-HCl (pH 7.5 @ 25°C) and 0.1 mM Na2EDTA.

    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.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Glycoprotein Denaturing Buffer10X
    NP-4010%
    G7 Reaction Buffer10X
    Fetuin410 mg/ml

    Advantages and Features

    Applications

    • 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.

    Properties and Usage

    Unit Definition


    1X Glycoprotein Denaturing Buffer
    0.5% SDS
    40 mM DTT

    1% NP-40
    1% NP-40

    Reaction Conditions

    1X G7 Reaction Buffer

    1X G7 Reaction Buffer:
    50 mM sodium phosphate
    pH 7.5 @ 25°C

    Storage Temperature

    4°C

    Storage Conditions

    50 mM NaCl
    20 mM Tris-HCl
    0.1 mM EDTA
    pH 7.5 @ 25°C

    Notes

    1. Storage: It is recommended to store this kit at 4°C. All components of the kit will be stable for at least one year if stored correctly.
    2. Deglycosylation Mix is not recommended for use on Mucin-like substrates.
    3. PMSF may inhibit PNGase F activity and should not be used with the Protein Deglycosylation Mix

    References

    1. Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
    2. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.
    3. Roggentin, P. et al. (1988). FEBS Lett. 238 (1), 31-34.
    4. Guan, C. New England Biolabs, Inc. Unpublished observation
    5. McLeod, E. New England Biolabs, Inc. Unpublished observation
    6. Guthrie, E.P., Shimer, E.P. New England Biolabs, Inc. Unpublished observation

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    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 info@neb.com or fill out the Technical Support Form for appropriate document.
    1. I tried the Protein Deglycosylation Mix on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
    2. Do detergents inhibit the Protein Deglycosylation Mix?
    3. Are Protease Inhibitors acceptable for use with the Protein Deglycosylation Mix reaction?
    4. How much Protein Deglycosylation Mix should I use to remove my carbohydrate under native conditions?
    5. What is a good endoglycosidase substrate?
    1. Reaction Protocols for Protein Deglycosylation Mix (P6039)

    Application Notes

    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