O-Linked Glycan Strategies

• O-Glycosidase
• Removal of Sialic Acid Residues
• Removal of β-Linked Galactose and N-Acetylglucosamine
• Other O-Linked Oligosaccharide Modifications
• Materials

Releasing O-linked glycans, or O-glycans, can involve various steps including removing different non-reducing end residues with exoglycosidases. Read on to learn about O-linked glycan strategies, such as the actions of O-glycosidase and how to remove di and trisialylation, β-linked galactose, and N-acetylglucosamine.

O-Glycosidase

While most N-linked oligosaccharides can be removed using PNGase F, there is no enzyme comparable to PNGase F for removing intact O-linked sugars. O-Glycosidase (also known as Endo-α-N-acetylgalactosaminidase or O-Glycanase) hydrolyzes the serine or threonine-linked unsubstituted O-glycan core [Gal-β(1→3)-GalNAc] (Figure 1). Any modification of the core structure can block the action of O-glycosidase. To avoid this issue, exoglycosidases like sialidases can be used to reduce the glycoform down to a core structure before or concurrently with the O-glycosidase treatment.

Monosaccharides must be sequentially hydrolyzed by a series of exoglycosidases until only the Gal-β(1→3)-GalNAc core remains. O-Glycosidase (ex. G1163 or 324716) can then remove the intact core structure with no modification of the serine or threonine residues.¹ Denaturation of the glycoprotein does not appear to significantly enhance O-deglycosylation.

Removal of Sialic Acid Residues

The most common modification of the core Gal-β(1→3)-GalNAc is a mono, di, or trisialylation.^2,3,4,5 These sialic acid residues are easily removed by α-(2→3,6,8,9)-neuraminidase (ex. N8271) since only this enzyme is capable of efficient cleavage of the NeuAc-α(2→8)-NeuAc bond (Figure 2).⁶

Removal of β-Linked Galactose and N-Acetylglucosamine

Another commonly occurring O-linked hexasaccharide structure contains β(1→4)-linked galactose (Gal) and β(1→6)-linked N-acetylglucosamine (GlcNAc) as well as sialic acid.^7,8 Hydrolysis of this glycan will require, in addition to neuraminidase, a β(1→4)-specific galactosidase (ex. G0413) and an N-acetylglucosaminidase (ex. A6805) (Figure 3). A non-specific galactosidase (ex. 345788 or G5635) will hydrolyze β(1→3)-galactose from the core glycan and leave an O-linked GalNAc residue that cannot be removed by O-glycosidase.

α-Neuraminidase (ex. N8271), β(1-4)-Galactosidase (ex. G0413), and β-N-Acetylglucosaminidase (ex. A6805) can be used for the hydrolysis of these and any other O-linked structures containing α-sialic acid, β(1-4)-linked galactose (Gal), or β-linked N-acetylglucosamine (GlcNAc) such as polylactosamine (Figure 4).

Other O-Linked Oligosaccharide Modifications

Less common modifications that have been found on O-linked oligosaccharides include α-linked galactose (Gal) and α-linked fucose.^8.9 α-Gal has found to be a common allergen found in animal tissue, and can lead to meat allergies or xenotransplant immunogenicity. ¹⁰ A non-specific α-Galactosidase (ex. G8507) can be used to hydrolyze this type of linkage. N-acetylglucosamine attached directly to the peptide backbone (found on nuclear proteins) and α-linked N-acetylgalactosamine (found in mucins) have also been reported.¹¹ Additional exoglycosidases are necessary for complete O-deglycosylation when these residues are present. Fucose and mannose directly O-linked to proteins cannot presently be removed enzymatically. ^12,13