Enzymes for O-glycans

Enzymes for O-glycans

Enzymes for O-glycan Analysis

The Genovis product line of enzymes for O-glycans consists of:

OpeRATOR™ - O-protease for O-glycan-specific digestion of glycoproteins

OglyZOR™ - Endoglycosidase for O-glycans (disaccharides) of core 1 and core 3

SialEXO™ - Exoglycosidase acting on sialic acids

An overview of the enzymes are presented on this page. Please visit the individual product pages for full details on the different enzymes for O-glycans.

 

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OpeRATOR is an O-protease digesting O-glycosylated proteins N-terminally of the S/T glycosylation site.

The OpeRATOR enzyme is a novel tool for analysis of O-glycans on glycoproteins. The endoprotease binds to the O-glycans and digests the amino acid backbone N-terminally of the S/T residues. O-glycans are required for OpeRATOR activity and the enzyme will not digest glycoproteins with only N-linked glycans. The digestion generates glycopeptides decorated with O-glycans and allows for O-glycan profiling and site-occupancy determination using mass spectrometry.

O-glycopeptide Mapping of Etanercept

The OpeRATOR enzyme can be used to map O-glycans on therapeutic proteins. In the example below, etanercept was stripped from N-glycans and incubated with OpeRATOR and SialEXO to generate O-glycopeptides. The graph illustrates the peptides generated with OpeRATOR and show the O-glycans in the hinge region, but also an O-glycan in the TNF alpha receptor.

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Figure 1. Representation of glycopeptides of etanercept generated using OpeRATOR. N-glycans of etanercept were removed using PNGaseF, and SialEXO was used to remove sialic acids, then OpeRATOR was incubated with etanercept and the resulting glycopeptides analyzed using LC/MS.

Key Characteristics

  • O-glycan specific endoprotease
  • Digests N-terminally of O-glycan-occupied serine or threonine
  • Requires O-glycan for activity
  • No activity on glycoproteins with only N-glycans
  • Pretreatment with SialEXO™ increases performance

 

OpeRATOR Product Page

 

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OglyZOR is an endoglycosidase that specifically hydrolyzes core 1- and 3-linked O-glycans (disaccharides) on native glycoproteins.

The enzyme acts on native glycoproteins, and does not require denaturation of the substrate. The sialic acids of the glycans should be removed for OglyZOR activity. For this reason, the OglyZOR enzyme is provided together with SialEXO.

OglyZOR Deglycosylation of O-glycosylated Proteins

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Figure 2. Comparison of the enzymatic activity of OglyZOR and SialEXO to the commercially available endoglycosidase and sialidase from E. faecalis and C. perfringens. All incubations were performed according to the manufacturers' instructions. The enzymatic reactions with TNFα receptor and etanercept were separated on SDS-PAGE.

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Key Characteristics

  • Endoglycosidase acting on O-glycans
  • Active on native glycoproteins
  • Specific for core 1 and core 3 O-glycans (disaccharides)
  • Requires pretreatment with SialEXO

OglyZOR Product Page

 

 

  1. TNFα receptor
  2. + SialEXO and OglyZOR
  3. + Endoglycosidase (E. faecalis) and sialidase (C. perfringens)
  4. Etanercept
  5. + SialEXO and OglyZOR
  6. + Endoglycosidase (E. faecalis) and sialidase (C. perfringens)

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SialEXO is a sialidase mix for complete removal of sialic acids from 2 mg native glycoprotein

SialEXO is used for removal of sialic acids on native glycoproteins, and it works on both O- and N-linked glycans. It is a combination of two sialidases, acting on α2-3, α2-6 and α2-8 linkages and it will remove sialic acids from 2 mg glycoprotein in 2 h at 37°C.

SialEXO acts on α2-3, α2-6 and α2-8 Linked Sialic Acids

SialEXO was incubated with substrates representing α2-3, α2-6 and α2-8 linked sialic acids and the graph below shows the hydrolysis of the sialic acids and demonstrates the ability of SialEXO to remove sialic acids with the indicated linkages.

Figure 1
Figure 3. The substrate specificity of SialEXO was investigated through quantification of liberated free sialic acids from the substrates 3’-sialyllactose (α2-3 bonds), 6’-sialyllactose (α2-6 bonds), and colominic acid (α2-8 bonds).

 

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Key Characteristics

  • Acts on native glycoproteins
  • Cleaves α2-3, α2-6 and α2-8 linked sialic acids
  • Hydrolyzes sialic acids within 2 h
  • Acts on both N- and O-linked glycans
  • Compatible with OpeRATOR and OglyZOR

SialEXO Product Page

 

 

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Patent and Disclaimer

OglyZOR™, OpeRATOR™ and SialEXO™

All rights reserved. Aspects of OglyZOR™, OpeRATOR™ and SialEXO™ technologies are encompassed by pending patent applications in the name of Genovis AB.

The trademarks OglyZOR™, OpeRATOR™ and SialEXO™ are the property of Genovis AB.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

©2017 Genovis AB.