O-Linked Glycan Strategies

Glycobiology Analysis Manual, 2nd Edition



While most N-linked oligosaccharides can be removed using PNGase F, a comparable enzyme for removing intact O-linked sugars has not been identified. Monosaccharides must be sequentially hydrolyzed by a series of exoglycosidases until only the Gal-β(1→3)-GalNAc core remains. At that point, O-glycosidase can remove the intact core structure with no modification of the serine or threonine residue. Denaturation of the glycoprotein does not appear to significantly enhance O-deglycosylation.

Any modification of the core structure can block the action of O-glycosidase. The most common modifications of the core Gal-β(1→3)-GalNAc are mono-, di-, and trisialylation. These residues are easily removed by α-(2→3,6,8,9)-neuraminidase. Only this enzyme is capable of efficient cleavage of the NeuAc-α(2→8)-NeuAc bond (see Figure 1). Another commonly occuring O-linked hexasaccharide structure contains β(1→4)-linked galactose (Gal) and β(1→6)-linked N-acetylglucosamine (GlcNAc) as well as sialic acid. Hydrolysis of this glycan requires, in addition to neuraminidase, a β(1→4)- specific galactosidase and an N-acetylglucosaminidase. A galactosidase that is not linkage specific will hydrolyze β(1→3)-galactose from the core glycan, leaving an O-linked GalNAc residue that cannot be removed by O-glycosidase.

Sequential glycolytic cleavage

Figure 1. In sequential glycolytic cleavage, disialylated and trisialylated O-linked glycans have the sialic acid residues (NeuNAc) removed by α(2→3,6,8,9) neuraminidase. The Core 1 type glycan is then cleaved from the O-linkage by O-glycosidase.

β(1→4)-Galactosidase and β-N-acetylglucosaminidase can be used for the hydrolysis of these and any other O-linked structures containing β(1→4)-linked galactose (Gal) or β-linked N-acetylglucosamine (GlcNAc) such as polylactosamine (see Figure 2). Less common modifications that have been found on O-linked oligosaccharides include α-linked galactose (Gal) and α-linked fucose. N-Acetylglucosamine attached directly to the peptide backbone (found on nuclear proteins) and α- linked N-acetylgalactosamine (found in mucins) have also been reported. 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.

Disialylated O-linked Core 2 hexasaccharide

Figure 2. Disialylated O-linked Core 2 hexasaccharide is sequentially degraded by (1) removal of sialic acid residues (NeuNAc) using α(2→3,6,8,9) neuraminidase, (2) removal of β(1→4)-galactose (Gal) residues using β(1→4)-galactosidase, and (3) removal of N-acetylglucosamine (GlcNAc) using N-acetylglucosaminidase. The remaining Core 1 type glycan can then be cleaved from O-linkage using O-glycosidase as shown in Figure 1.


Synonyms: Endo-α-N-acetylgalactosaminidase; O-Glycanase O-Glycosidase hydrolyzes the serine or threonine-linked unsubstituted O-glycan core [Gal-β(1→3)-GalNAc] (see Figure 3). Any modification of the core structure can block the action of O-glycosidase.

Cleavage site and structural requirements for O-Glycosidase

Figure 3. Cleavage site and structural requirements for O-Glycosidase. Any modification of the core structure will prevent cleavage.


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