O-Glycosidase

Description


 
O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.

Substrate Specificity:

Substrate Specificity:

Product Source

Cloned from Enterococcus faecalis and expressed in E. coli (1).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Glycoprotein Denaturing Buffer-2010X
GlycoBuffer 2-2010X
NP-40-2010%

Advantages and Features

Applications

  • Removal of Core 1 and Core 3 O-linked disaccharide glycans from glycoproteins

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of neuraminidase digested, non-denatured fetuin (2) in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

Non-Denaturing Unit Definition Assay
Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction mix is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by the Morgan and Elson Assay (2).

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

1X NP-40
1% NP-40 in MilliQ-H2O

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

10X GlycoBuffer 2:
50 mM sodium phosphate
pH 7.5 @ 25°C

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

65°C for 10 min

Molecular Weight

Apparent: 147000 daltons

Notes

  1. Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  3. It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides. A general Neuraminidase (#P0720) or the O-Glycosidase and Neuraminidase bundle (#E0540S)  is recommended.
  4. Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

References

  1. Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
  2. Morgan, W.T.J. and Elson, L.A. (1994). Biochem. J.. 28, 988-995.

FAQs

  1. What are the typical reaction conditions for O-Glycosidase?
  2. I tried using O-Glycosidase on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
  3. How much O-Glycosidase should I use to remove my carbohydrate under native conditions?
  4. How do I inhibit O-Glycosidase?

  5. Do detergents inhibit O-Glycosidase?
  6. Can Neuraminidase be used together in a digest with PNGase F and O-Glycosidase?
  7. Can I double digest PNGase F and O-Glycosidase?
  8. What is the difference between PNGase F, Endo H and O-Glycosidase?
  9. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
  10. What are glycosidases and their uses?
  11. Is it necessary to treat my glycoprotein concomitantly with Neuraminidase and O-Glycosidase?

Tech Tips

Don’t forget to include Neuraminidase (P0720) in your reaction! It is necessary to treat glycoproteins concomitantly with Neuraminidase and O-Glycosidase. Neuraminic Acid residues must be removed in order to allow O-Glycosidase to cleave the O-linked disaccharides.

NEB’s O-Glycosidase is the only available O-Glycosidase that catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins

You can use this enzyme under native or denaturing conditions

Under native conditions we recommend adding more enzyme and using longer incubation times

Under denaturing conditions the enzyme activity is increased two-fold. However, this observation may be substrate dependent.

Protocols

  1. O-Glycosidase Application Note 1 (P0733)
  2. O-Glycosidase (P0733)
  3. Endo-α-N-Acetylgalactosaminidase Application Note 1

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.

Feature Articles

Interactive Tools

Application Notes

Quality Control

Quality Assurance Statement

  • No contaminating exoglycosidase or proteolytic activity could be detected.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Glycosidase Activity (TLC):
    The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
  • Protease Activity (SDS-PAGE):
    The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

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]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.