Overview of Glycobiology

Learn about the core sequences and common modifications of N-linked and O-linked glycans in this video. Analysis of these glycans and/or peptide portions of the glycoprotein can be accomplished with the use of deglycosylation enzymes, which are explained in detail. Unlike other chemical deglycosylation methods, enzymatic treatment is much gentler and can provide complete sugar removal with no protein degradation.


Secretory and membrane proteins are often post-translationally modified with sugar chains called, glycans. Glycans are essential for the stability and function of the mature protein. They effect enzymatic activity, half-life, and receptor binding among other biological processes. Proteins can be modified with different kinds of carbohydrates. N-glycans and O-glycans being the most common types in eukaryotic systems.

N-glycans are attached to asparagine residues of the protein, shown with an N. N-glycans have a common biosynthesis pathway, which is reflected in the common structure of their core. N-glycans can be high mannose, which means, all the component monosaccharides are mannose residues, with the exception of the two-core N-actyleglucosamine, or GlcNAc, residues. N-glycans can also be complex, which means the mannose residues are only present at the core, and the branches are extended with other monosaccharides, such as N-actyleglucosamine, or GlcNAc, galactose, sialic acid, and fucose.

O-glycans start with the addition of an N-actyleglucosamine, or GalNAc, residue to a serine, shown with an S, or threonine, shown with a T. O-glycans are often short chains consisting of only a few sugars, or they can be extended by the addition of different monosaccharides, resulting in a longer chain.

Deglycosylation refers to the removal of glycans from proteins. Enzymatic deglycosylation is the method of choice, because it preserves the integrity of the protein, and sometimes the glycan chains for further analysis. Because enzymes are specific, using several enzymes at once, or in tandem, provides valuable information about the structure of the glycoprotein.

PNGase F is able to remove most N-glycans as an intact glycan molecule. If present, the O-glycans, remain attached to the protein. The released glycans are intact and are ready for further characterization using analytical methods, such as mass spectrometry.

The protein deglycosylation mix is a combination of PnGase F, O-glycosidase, neuraminidase, also called, sialidase, beta GlcNAcase and beta 1-4galactosidase. As shown before, PNGase F removes most N-glycans. O-glycosidase removes core one and core three O-glycans. The exoglycosidases, sialidase, beta GlcNAcase, and beta 1-4galactosidase, can be used to trim down O-glycans to their core structures.

The protein deglycosylation mix can remove short O-glycans, and longer chain O-glycans, in a step-by-step reaction. The mix also removes N-glycans, however, the complex N-glycans are degraded by some of the enzymes. Only high mannose N-glycans remain as a block. Although some glycans are reduced to individual components, and are not suitable for analysis, the protein remains intact. This is the method of choice, if one wants to study the properties of the naked polypeptide.

Additional exoglycosidases, for instance, beta 1-3galactosidase, alpha galactosidase, and fucosidase can be used along with the protein deglycosylation mix. Some glycoproteins have O-glycans that are resistant to the protein deglycosylation mix. However, additional glycosidases can be used along with the protein deglycosylation mix to facilitate the removal of the glycan. In this example, fucosidase and alpha GalNAcase are used and the resistant glycan is removed.

One application for PNGase F is to remove all N-glycans, leaving the protein ready for O-glycan isolation. Alkaline chemical treatments, like beta elimination, release O-glycans as a block for further analysis. Note that the protein is destroyed in the process. Enzymatic release of glycans is often the better alternative, if one desires to analyze the properties of the deglycosylated, or naked, protein.

Some asparagine consensus sites carry a glycan molecule, and others are free of glycans. This property of glycoproteins is called, occupancy. Protein occupancy is a dynamic feature and can change to modulate protein function. Using glycosidases, like EndoH, that cleave between the two GlcNAc residues can help to determine occupancy in glycoproteins. Trypsin is used to digest the protein. The peptides are analyzed by mass spectrometry. It can be concluded that a glycan was present in any peptide that now carries the additional mass of a GlnNAc monosaccharide.

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