Glycosylation

O Linked Glycan Strategies

O-Linked Glycan Strategies

There is no enzyme comparable to PNGase F for removing intact O-linked sugars. Monosaccharides must be sequentially hydrolyzed by a series of exoglycosidases until only the Gal-b(1-3)-GalNAc core remains. O-Glycosidase6 can then remove the core structure intact 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 modification of the core Gal-b(1-3)-GalNAc is a mono, di, or trisialylation.7,8,9,10 These residues are easily removed by a2-(3,6,8.9)-Neuraminidase11 since only this enzyme is capable of efficient cleavage of the NeuAc-a(2-8)-NeuNAc bond. Another commonly occuring O-linked hexasaccharide structure contains b(1-4)-linked galactose and b(1-6)-linked N-acetylglucosamine as well as sialic acid.12,13b(1-4)-specific galactosidaseN-acetylglucosaminidase. A non-specific galactosidase will hydrolyze b(1-3)-galactose from the core glycan and leave O-linked GalNAc that can not be removed by O-Glycosidase. b(1-4)-Galactosidase and b?N-Acetylglucosaminidase can be used for the hydrolysis of these and any other O-linked structures containing b(1-4)-linked galactose or b-linked N-acetylglucosamine such as polylactosamine. Less common modifications that have been found on O-linked oligosaccharides include a-linked galactose and a-linked fucose.13,14 Directly O-linked N-acetylglucosamine (found on nuclear proteins)15 and a-linked N-acetylgalactosamine (found in mucins) have also been reported. Additional exoglycosidases are necessary for complete O-deglycosylation when these residues are present. Fucose16 and mannose17 directly O-linked to proteins can not presently be removed enzymatically. Hydrolysis of this glycan will require, in addition to neuraminidase, a and an


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Endoglycosidase Specificities

PNGase F is available as a solution and a lyophilized powder. PNGase F cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless a(1-3) core fucosylated. The asparagine must be peptide bonded at both termini. The asparagine residue from which the glycan is removed is deaminated to aspartic acid.

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Peptide-N-Glycosidase A hydrolyzes oligosaccharides containing a fucose a(1-3)-linked to the asparagine-linked N-acetylglucosamine, commonly found in glycoproteins from plants or parasitic worms. These types of glycans are resistant to PNGase F. Like PNGase F the asparagine asparagine residue from which the glycan is removed is deaminated to aspartic acid. However, it is ineffective when sialic acid is present on the N-linked oligosaccharide.

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O-Glycosidase hydrolyzes the serine or threonine-linked unsubstituted O-Glycan core [Gal-b(1-3)-GalNAc]. Any modification of the core structure can block the action of O-Glycosidase.


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Endoglycosidase F1 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. High mannose (oligomannose) and hybrid structures can be removed by Endoglycosidase F1, but not complex, oligosaccharides. Useful under native or non-denaturing deglycosylation conditions.

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Endoglycosidase F2 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. Endo F2 cleaves biantennary complex and to a lesser extent high mannose oligosaccharides. Fucosylation has little effect on Endo F2 cleavage of biantennary structures. Endo F2 will not cleave hybrid structures. Useful under native or non-denaturing deglycosylation conditions.

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Endoglycosidase F3 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. Endo F3 cleaves non-fucosylated biantennary and triantennary structures at a slow rate, but only if peptide-linked. Core fucosylation of biantennary structures increases activity up to 400-fold. Endo F3 has no activity on oligomannose and hybrid molecules. Endo F3 will also cleave fucosylated trimannosyl core structures on free and protein-linked oligosaccharides. Native deglycosylation of complex tetrantennary glycans requires sequential hydrolysis down to the trimannosyldiacetylchitobiose core.

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Endoglycosidase H is available as a native lyophilized powder, recombinant solution, or a recombinant solution with reaction buffer. Endo H cleaves between the N-acetylglucosamine residues of the diacetylchitobiose core of N-linked glycans. Oligomannose and most hybrid type glycans, including core fucosylated are hydrolyzed by Endo H. However complex type oligosaccharides are not.

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Kits for Enzymatic Deglycosylation

E-DEGLY Kit for General Enzymatic Deglycosylation
Deglycosylation of most N- and O-linked glycoproteins can be accomplished with Sigma’s E-DEGLY Kit. This kit contains the enzymes and reagents required for sequential and intact hydrolysis of glycan components under denaturing and some non-denaturing conditions.

N-DEGLY Kit for Native Deglycosylation of N-Linked Glycans
Many N-linked glycoproteins are resistant to deglycosylation using PNGase F under non-denaturing conditions. The N-DEGLY kit utilizes the three Endoglycosidase enzymes (F1, F2, F3) with reaction buffers to overcome this problem.

PP0200 ProteoProfile Enzymatic In-Gel N-Deglycosylation Kit
This kit has been optimized to provide a convenient and reproducible method to N-deglycosylate and trypsin-digest protein samples from 1D or 2D polyacrylamide gel pieces for subsequent MS or HPLC analysis.

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References

  1. Szkudinski, M.W., et al., Asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin. Endocrinology, 136, 3325-3330 (1995).
  2. Tarentino, A.L., and Plummer, T.H., Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificitly of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymol., 230, 44-57 (1994).
  3. Taga, E. M., et al., Structural and Chemical characterization of a homogeneous peptide N-glycosidase from almond. Biochemistry, 23, 815-22 (1984).
  4. Kobata, A., Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal. Biochem., 100, 1-14 (1979).
  5. Trimble, R.B., and Tarentino, A.L., Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J. Biochem., 266, 1646-1651 (1991).
  6. Iwase, H., and Hotta, K., Release of O-linked glycoprotein glycans by endo-alpha-N-acetyl-D-galactosaminidase. Methods Mol. Biol., 14, 151-159 (1993).
  7. Fukuda, M., et al., Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins. J. Biol. Chem., 262, 11952-11957 (1987).
  8. Pahlsson, P., et al., Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells. Glycoconj. J., 11, 43-50 (1994).
  9. Saito, S., et al., Common tetrasaccharide epitope NeuAc a 2-->3 Gal b1 -->3(Neu-Aca2-->6)GalNAc, presented by different carrier glycosylceramides or O-linked peptides, is recognized by different antibodies and ligands having distinct specificities. J. Biol. Chem., 269, 5644-5652 (1994).
  10. Spiro, R. G., and Bhoyroo, V. D., Structure of the O-glycosidically linked carbohydrate units of fetuin. J. Biol. Chem., 249, 5704-5717 (1974).
  11. Uchida, Y., et al., Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J. Biochem. (Tokyo), 86, 573-585 (1979).
  12. Hard, K., et al., 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, 785-798 (1992).
  13. Wilkins, P.P., et al., Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells. J. Biol. Chem., 271, 18732-18742 (1996).
  14. Glasgow, L.R., et al., Systematic purification of five glycosidases from Streptococcus pneumonia. J. Biol. Chem., 252, 8615-8623 (1977).
  15. Jiang, M.S., and Hart, G. W., A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem., 272, 2421-2428 (1977).
  16. Stults, N.L., and Cummings, R. D., O-linked fucose in glycoprotein from Chinese hamster ovary cells. Glycobiology, 3, 589-596 (1993).
  17. Chiba, A., et al., Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve a-dystroglycan. The role of a novel O-Mannosyl-type oligosaccharide in the binding of a-dystroglycan with laminin. J. Biol. Chem., 272, 2156-2162 (1977).

 

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