Efficient O-glycan Hydrolysis

The degree and composition of the glycosylation of proteins are critical for a wide range of biological processes, and alterations of the glycan structures may impact the function of the glycoprotein. In the development of biopharmaceuticals, the N- and O-linked glycans may obstruct the analysis and characterization of other post-translational modifications (PTMs) or the amino acid sequence. For this reason, Genovis has developed OglyZOR™ and SialEXO™ to rapidly remove O-glycans from native glycoproteins.

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OglyZOR and SialEXO are available for ordering. Buy online or visit 'How to order'. 

Antibody Characterization

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OglyZOR

OglyZOR™ Product Page

The OglyZOR enzyme specifically hydrolyzes core 1 and core 3 type O-glycan disaccharides on native glycoproteins

OglyZOR Activity

The OglyZOR enzyme acts on O-linked disaccharides and efficiently hydrolyzes the glycoprotein in a native state. This makes the enzymes suitable for preparation of samples prior to LC-MS analysis for identification of other PTMs or confirmation of amino acid sequences. A schematic presentation of the OglyZOR activity is presented in Fig. 1 below.

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Figure 1. Schematic overview of the OpeRATOR activity

OglyZOR Activity Requires Removal of Sialic Acids

To investigate the OglyZOR activity on native glycoproteins, the enzyme was incubated with etanercept, TNFR and abatacept for 1 h at 37°C with or without SialEXO. When the sialic acids are removed, the OglyZOR enzyme efficiently hydrolyzes the O-glycans from all three substrates (Fig. 2)

 

Figure 2. Efficient removal of O-glycans from native glycoproteins using OglyZOR and SialEXO.

 

OglyZOR Deglycosylation of O-glycosylated Proteins

The OglyZOR O-glycosidase was benchmarked to other O-glycosidases present on the market. All enzymatic reactions were carried out according to the manufacturers instructions. When incubating OglyZOR and a competing enzyme with TNFR, a clear difference in the migration pattern on SDS-PAGE was observed (Fig. 3). Using OglyZOR, a single band is obtained indicating complete removal of O-glycans, whereas the competing enzyme leaves a smear of TNFR indicative of an incomplete hydrolysis of O-glycans. Interestingly, using fetuin as a substrate, a similar result was obtained using OglyZOR and the competing enzyme. In both experiments, a visible band was observed in the competing enzyme lane, indicating that more enzyme is present in the reaction.

 

Figure 3. Comparison of the enzymatic activity of OglyZOR and SialEXO to commercially available endoglycosidases and sialidases. All incubations (4 h) were performed according to the manufacturers instructions, and the samples were separated on SDS-PAGE.

SialeXO Activity on Sialic Acid Linkages

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 (Fig. 4).

 

Figure 4. SialEXO activity on sialic acid linkages from the substrates 3’-sialyllactose (α2-3 bonds), 6’-sialyllactose (α2-6 bonds), and colominic acid (α2-8 bonds).

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O-glycosidase for Native Glycoproteins

The OglyZOR enzyme hydrolyzes core 1 and core 3 type O-glycans on native glycoproteins.

  • Hydrolyzes O-glycans on glycoproteins
  • 2-4 h reaction
  • Requires removal of sialic acids using SialEXO™ (included)
  • Core 1 and core 3 type O-glycan disaccharides

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Removal of Sialic Acids from Native Glycoproteins

SialEXO efficiently hydrolyzes all sialic acids from native glycoproteins.

  • Hydrolyzes sialic acids on N- and O-linked glycans
  • 2 h reaction
  • Requires no co-factors
  • α2-3, α2-6 and α2-8-linked sialic acids

Patent and Disclaimer

OglyZOR™ and SialEXO™

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

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

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

©2018 Genovis AB.