Glycoproteins are composed of oligosaccharide chains and proteins and have attracted great attention in proteomics and glycomics research. They exist in a variety of organisms in various forms such as enzymes, lectins, immunoglobulins, and membrane proteins. Glycoproteins have been reported to have multiple functions, including anti-tumor, antioxidant, lowering blood glycans, immune enhancement, and anti-radiation effects. Abnormal glycosylation is closely related to the occurrence and development of some diseases, thus the in-depth study of deglycosylation has become a research hotspot. A variety of methods have been developed to remove glycan chains in glycoproteins, including enzymatic methods, chemical methods, recombinant DNA technology, glycosylation inhibitors, etc. These methods are useful in simplifying the analysis of peptide moieties and glycan components of glycoproteins, removing glycoprotein heterogeneity, removing carbohydrate epitopes from antigens, studying ligand binding, and for the quality control of glycoprotein pharmaceuticals.
Figure 1. Enzymatic deglycosylation of proteins bearing N-linked glycans.
(Source: Fitchette, A. C. et al., 2007)
Deglycosylation Strategies
There are several methods available for the deglycosylation of glycoproteins. These methods can broadly be classified into enzymatic methods, chemical methods, and hybrid methods that combine enzymatic and chemical approaches.
Enzymatic Methods:
Endoglycosidase deglycosylation Endoglycosidases are a group of enzymes commonly used in the deglycosylation processes. These enzymes have different specificities for glycoproteins.
PNGase F
PNGase F (peptide N-glycosidase F) is a commonly used enzyme for the removal of N-linked glycans from glycoproteins. It cleaves the glycosidic bond between the asparagine residue and the attached N-glycan.
PNGase A
PNGase A is specifically useful for removing fucose-type glycans.
Endo F
Endo F1 can cleave high mannose and hybrid-type N-glycans. Endo F2 and F3 are capable of cleaving two-antennary and three-antennary N-glycans. However, Endo F3 does not work on hybrid and oligomannose glycans.
Endo H
Endo H targets the chitobiose core structure of oligomannose N-glycans and some hybrid oligosaccharides.
O-glycosidase
O-glycosidases, such as O-glycanase, are used for the removal of O-linked glycans. These enzymes cleave the glycosidic bond between the serine or threonine residue and the attached O-glycan.
Exoglycosidase deglycosylation Exoglycosidases are a type of enzyme that acts on the glycan chain of glycoproteins. Examples of exoglycosidases include sialidase, β-N-acetylglucosaminidase, β-galactosidase, β-mannosidase, α-mannosidase, α-L-fructosidase, α-N-acetylgalactosidase, and more. These enzymes work together to specifically release glycan groups from glycoproteins.
Sialidase
Sialidase can slowly hydrolyze sialic acid in glycoproteins.
β-galactosidase can slowly hydrolyze galactose residues in glycoproteins.
α-Mannosidase
α1-2,3mannosidase catalyzes the hydrolysis of specific α1-2 and α1-3-D-mannose residues in oligosaccharides; α1-6 mannosidase can catalyze the unbranched α1-6 glycosidic bonds in oligosaccharides.
By employing a combination of exoglycosidases, researchers can achieve specific and controlled deglycosylation of glycoproteins, leading to different functional outcomes.
Mixed enzymes to remove glycosyls Due to the heterogeneity of glycans, deglycosylation with a single enzyme cannot achieve complete removal of glycan groups, so a mixture of multiple enzymes is usually required.
Chemical Methods:
Hydrazinolysis: Hydrazinolysis involves the treatment of glycoproteins with hydrazine, which breaks the glycosidic bonds between the protein and the attached glycans. This method is effective for releasing intact N-linked glycans, but it is less specific and can cause partial degradation of the protein backbone.
β-Elimination: β-Elimination employs strong alkaline conditions to cleave the O-glycosidic linkage between the protein and the O-linked glycan. This method is often used in combination with subsequent chemical or enzymatic steps for complete deglycosylation.
Trifluoromethanesulfonic acid method: It has a rapid reaction, mild conditions, and does not affect protein conformation. This method is suitable for analyzing all types of glycan chains.
Hybrid Methods:
Combination of enzymatic and chemical approaches: In some cases, a combination of enzymatic and chemical methods is employed for deglycosylation. For example, treatment with PNGase F can be followed by chemical methods like hydrazinolysis or β-elimination to achieve complete deglycosylation.
Structure-Function Relationship Research Glycosylation can drastically influence the structure, stability, and function of glycoproteins. By removing the glycans through deglycosylation, researchers can obtain a clearer understanding of the relationship between the protein structure and its biological activity. This information is crucial for deciphering the functional implications of glycoproteins in health and disease.
Protein Characterization Removing carbohydrate groups from glycoproteins is crucial for effective protein identification using mass spectrometry (MS) analysis. The microheterogeneity of attached glycans in glycopeptides results in lower detection sensitivity and signal suppression. Moreover, the presence of bulky oligosaccharides in native glycoproteins hinders complete proteolytic digestion, which is necessary for peptide fragment elution from gels in MS identification. Deglycosylation of glycopeptides prior to tryptic digestion improves protein identification and facilitates the identification of glycosylation sites on the protein core.
Vaccine Development Glycoproteins are often key components of vaccines, as they can elicit strong immune responses. However, glycan structures on these proteins can mask or modulate immune recognition sites. Deglycosylation allows for the exposure of epitopes, enhancing the immune response and aiding in the development of more effective vaccines.
Therapeutic Protein Development Protein deglycosylation plays a pivotal role in the development of therapeutic proteins, such as monoclonal antibodies. Glycosylation patterns can significantly impact the pharmacokinetics, immunogenicity, and effector functions of therapeutic proteins. By precisely controlling the glycan profile through deglycosylation, researchers can optimize the therapeutic properties of these proteins, improving their efficacy and safety.
References
Fitchette A C, et al. Plant proteomics and glycosylation. Methods in Molecular Biology. 2007: 317-342.
Ma B, et al. Protein glycoengineering: An approach for improving protein properties. Frontiers in Chemistry. 2020, 8: 622.
Chen W, et al. Comprehensive analysis of protein N-glycosylation sites by combining chemical deglycosylation with LC-MS. Journal of Proteome Research. 2014, 13(3): 1466-1473.
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Figure 1. Enzymatic deglycosylation of proteins bearing N-linked glycans. 