O-Linked Glycan Strategies

Glycobiology Analysis Manual, 2nd Edition

• 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.

Figure 1. Cleavage site and structural requirements for O-Glycosidase. Any modification of the core structure will prevent cleavage.

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

Figure 2. 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.

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.

Figure 3. 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 2.

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).

Figure 4. O-linked hexasaccharide structures containing β(1→4)-linked galactose (Gal) and β(1→6)-linked N-acetylglucosamine (GlcNAc) as well as di and trisialated O-linked glycans.

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

References

1. Iwase H, Hotta K. Release of O-Linked Glycoprotein Glycans by Endo-?-N-Acetylgalactosaminidase.151-160. https://doi.org/10.1385/0-89603-226-4:151

2. Fukuda M, Lauffenburger M, Sasaki H, Rogers ME, Dell A. 1987. Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins.. Journal of Biological Chemistry. 262(25):11952-11957. https://doi.org/10.1016/s0021-9258(18)45301-x

3. P?hlsson P, Blackall DP, Ugorski M, Czerwinski M, Spitalnik SL. 1994. Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells. Glycoconjugate J. 11(1):43-50. https://doi.org/10.1007/bf00732431

4. Saito S, Levery S, Salyan M, Goldberg R, Hakomori S. 1994. Common tetrasaccharide epitope NeuAc alpha 2?>3Gal beta 1?>3(Neu-Ac alpha 2?>6)GalNAc, presented by different carrier glycosylceramides or O-linked peptides, is recognized by different antibodies and ligands having distinct specificities.. Journal of Biological Chemistry. 269(8):5644-5652. https://doi.org/10.1016/s0021-9258(17)37509-9

5. Spiro RG, Bhoyroo VD. 1974. Structure of the O-Glycosidically Linked Carbohydrate Units of Fetuin. Journal of Biological Chemistry. 249(18):5704-5717. https://doi.org/10.1016/s0021-9258(20)79875-3

6. UCHIDA Y, TSUKADA Y, SUGIMORI T. 1979. Enzymatic Properties of Neuraminidases from Arthrobacter ureafaciens. 86(5):1573-1585. https://doi.org/10.1093/oxfordjournals.jbchem.a132675

7. HARRD K, DAMM JBL, SPRUIJT MPN, BERGWERFF AA, KAMERLING JP, DEDEM GWK, VLIEGENTHART JFG. 1992. The carbohydrate chains of the beta subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates. Eur J Biochem. 205(2):785-798. https://doi.org/10.1111/j.1432-1033.1992.tb16843.x

8. Wilkins PP, McEver RP, Cummings RD. 1996. Structures of the O-Glycans on P-selectin Glycoprotein Ligand-1 from HL-60 Cells. Journal of Biological Chemistry. 271(31):18732-18742. https://doi.org/10.1074/jbc.271.31.18732

9. Glasgow L, Paulson J, Hill R. 1977. Systematic purification of five glycosidases from Streptococcus (Diplococcus) pneumoniae. Journal of Biological Chemistry. 252(23):8615-8623. https://doi.org/10.1016/s0021-9258(19)75265-x

10. Jin C, Cherian RM, Liu J, Playà-Albinyana H, Galli C, Karlsson NG, Breimer ME, Holgersson J. 2020. Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues. Glycoconj J. 37(4):485-498. https://doi.org/10.1007/s10719-020-09931-1

11. Jiang M, Hart GW. 1997. A Subpopulation of Estrogen Receptors Are Modified by O-Linked N-Acetylglucosamine. Journal of Biological Chemistry. 272(4):2421-2428. https://doi.org/10.1074/jbc.272.4.2421

12. Stults NL, Cummings RD. 1993. O-Linked fucose in glycoproteins from Chinese hamster ovary cells. Glycobiology. 3(6):589-596. https://doi.org/10.1093/glycob/3.6.589

13. Chiba A, Matsumura K, Yamada H, Inazu T, Shimizu T, Kusunoki S, Kanazawa I, Kobata A, Endo T. 1997. Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve ?-Dystroglycan. Journal of Biological Chemistry. 272(4):2156-2162. https://doi.org/10.1074/jbc.272.4.2156