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 β-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

O-Linked Glycan Strategies

Figure 1.O-Linked Glycan Strategies

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.

PNGase F

Figure 2.PNGase F

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.

Peptide-N-Glycosidase A

Figure 3.Peptide-N-Glycosidase A

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.

O-Glycosidase

Figure 4.O-Glycosidase

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.

Endoglycosidase F1

Figure 5.Endoglycosidase F1

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.

Endoglycosidase F2

Figure 6.Endoglycosidase F2

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.

Endoglycosidase F3

Figure 7.Endoglycosidase F3

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.

Endoglycosidase H

Figure 8.Endoglycosidase H

Kits for Enzymatic Deglycosylation

E-DEGLY Kit for General Enzymatic Deglycosylation
Deglycosylation of most N- and O-linked glycoproteins can be accomplished with our 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.

Materials
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Reference

1.
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Tarentino AL, Plummer TH. 1994. [4] Enzymatic deglycosylation of asparagine-linked glycans: Purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum.44-57. http://dx.doi.org/10.1016/0076-6879(94)30006-2
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Taga EM, Waheed A, Van Etten RL. 1984. Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond. Biochemistry. 23(5):815-822. http://dx.doi.org/10.1021/bi00300a006
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Kobata A. 1979. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Analytical Biochemistry. 100(1):1-14. http://dx.doi.org/10.1016/0003-2697(79)90102-7
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Trimble R, Tarentino A. 1991. Identification of Distinct Endoglycosidase (Endo) Activities in Flavobacterium meningosepticum: Endo F1, Endo F2 and Endo F3. J Biochem. 266 1646-1651.
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Iwase H, Hotta K. Release of O-Linked Glycoprotein Glycans by Endo-?-N-Acetylgalactosaminidase.151-160. http://dx.doi.org/10.1385/0-89603-226-4:151
7.
Fukuda M. 1987. Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins.. J Biol Chem. 26211952-11957.
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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. http://dx.doi.org/10.1007/bf00732431
9.
Saito 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.. j biol chem. 269(8):5644-5652.
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Spiro R, Bhoyroo V. 1974. Structure of the O-glycosidically linked carbohydrate units of fetuin. 249. 185704-5717.
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UCHIDA Y, TSUKADA Y, SUGIMORI T. 1979. Enzymatic Properties of Neuraminidases from Arthrobacter ureafaciens. 86(5):1573-1585. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a132675
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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. http://dx.doi.org/10.1111/j.1432-1033.1992.tb16843.x
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Wilkins PP, McEver RP, Cummings RD. 1996. Structures of theO-Glycans on P-selectin Glycoprotein Ligand-1 from HL-60 Cells. J. Biol. Chem.. 271(31):18732-18742. http://dx.doi.org/10.1074/jbc.271.31.18732
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Glasgow L, Paulson J, Hill R. 1977. Systematic purification of five glycosidases from Streptococcus (Diplococcus) pneumoniae. J biol chem. 252(23):8615-8623.
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Jiang M, Hart GW. 1997. A Subpopulation of Estrogen Receptors Are Modified byO-LinkedN-Acetylglucosamine. J. Biol. Chem.. 272(4):2421-2428. http://dx.doi.org/10.1074/jbc.272.4.2421
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Stults NL, Cummings RD. 1993. O-Linked fucose in glycoproteins from Chinese hamster ovary cells. Glycobiology. 3(6):589-596. http://dx.doi.org/10.1093/glycob/3.6.589
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Chiba A, Matsumura K, Yamada H, Inazu T, Shimizu T, Kusunoki S, Kanazawa I, Kobata A, Endo T. 1997. Structures of SialylatedO-Linked Oligosaccharides of Bovine Peripheral Nerve ?-Dystroglycan. J. Biol. Chem.. 272(4):2156-2162. http://dx.doi.org/10.1074/jbc.272.4.2156

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