Chemical Deglycosylation
Overview of Chemical Deglycosylation
The chemical method is to use chemical reagents to effectively release the glycan chain without destroying the integrity of the glycan chain and protein structure, such as hydrazinolysis, periodate oxidation-β elimination method, trifluoroacetic acid method, trifluoromethanesulfonic acid method, etc. For N-linked glycopeptides, hydrazinolysis can effectively release N-glycan chains. For O-linked glycopeptides, the periodate oxidation-β elimination method and trifluoroacetic acid method can effectively release O-linked glycan chains. O-linked glycosidic bonds are sensitive to alkalis. Under alkaline conditions, the O-glycosylated groups of Ser/Thr undergo β- elimination to form unsaturated bonds, and the double bonds are easily added by nucleophile attack. Trifluoroacetic acid breaks the peptide chain by transamination, thereby releasing the glycan chain. The trifluoromethanesulfonic acid method is suitable for all glycan chains, but the glycan chain removal is not thorough enough.
Fig. 1 Representative O-linked and N-linked glycans on the surface of a mammalian cell. (Munkley, 2017)
Chemical Deglycosylation Strategies
The hydrazinolysis method can deglycosylate N-glycan chains. Glycoproteins are deacetylated after hydrazinolysis and then release glycan chains. To avoid the hydrolysis and deacetylation of glycan chains by hydrazine, the glycan chains need to be reduced and acetylated after hydrazinolysis. The disadvantages of this method are: firstly, the acetyl group of N-acetylamino glycan, the acetyl group of sialic acid, and glycolyl group will be hydrolyzed under the conditions of hydrazinolysis; secondly, the remaining hydrazine or amino acids will bind to the ends of reducing glycan; thirdly, the high reaction temperature will cause the loss of GlcNAc; finally, this process must strictly maintain anhydrous conditions, and the hydrazinolysis conditions are severe, which will cause protein denaturation, and it is only used for obtaining glycan chains for structural analysis. However, if used with caution, hydrazinolysis can be applied commercially in large quantities.
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Periodate oxidation-β elimination
The key to the sodium periodate oxidation-β elimination method is that the C3 position of the side chain glycan group should be no substituents. If there is a substituent on the C3 position, the sulfonic trifluoromethane (TFMSA) treatment is required. The first step of the periodate oxidation-β elimination method is to form dialdehydes at the C3 and C4 positions of N-acetylgalactosamine, and then under alkaline conditions, the dialdehyde derivatives undergo β-elimination reaction. O-linked glycosidic bonds are sensitive to alkali. This method can completely remove O-linked glycans under alkaline conditions. For N-linked glycans, GlcNAc residues will remain on Asn after removal.
The trifluoroacetic acid method (TFA) breaks the peptide chain by transamination, and the N-trifluoroacetate group replaces the N-acetyl group in the amino glycan. The disadvantage of this reaction is that the O-glycosidic bond between serine and threonine as well as the N-acetylgalactosamine bond will be broken, and the galactosamine residue will be partially cleaved. Thus the oligosaccharides released by this reaction cannot be recovered quantitatively and the reducing end of the chain will be partially degraded.
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Trifluoromethanesulfonic acid
The trifluoromethanesulfonic acid (TFMS) method can remove all glycan groups except the monosaccharide directly connected to the peptide chain. The disadvantage of this method is that if sialic acid exists at the end of the glycan chain, the efficiency of the reaction will be affected, and the structure of the glycan chain will be destroyed, resulting in incomplete and inaccurate information. In addition, the process of this method must be kept strictly anhydrous.
Fig.2 The scheme of TFMS deglycosylation. (Edge, 2003)
Services at Creative Biolabs
The importance of chemical deglycosylation is partly reflected in the potential to realize commercial deglycosylation in large quantities. As an industry-leading glycoprotein-based services provider, Creative Biolabs has established a comprehensive technology platform providing reliable glycoprotein analysis services:
If you are interested in our services or you have any other questions, please feel free to contact us for more information.
References
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Munkley, J. Glycosylation is a global target for androgen control in prostate cancer cells. Endocr Relat Cancer. 2017, 24(3): R49-R64.
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Edge, A.S. Deglycosylation of glycoproteins with trifluoromethane sulphonic acid: elucidation of molecular structure and function. Biochem J. 2003, 376(Pt 2): 339-50.
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