Glycosylation is an extremely diverse, enzymatic process resulting in the covalent attachment of sugars (glycans and monosaccharides) to proteins. About half of all cellular proteins are glycosylated. Most, such as transmembrane receptors, secreted proteins, organelle residents and surface ligands become glycosylated either coupled to or shortly after synthesis of the protein in the rough endoplasmic reticulum (ER). In addition to proteins, lipids and proteoglycans can also be glycosylated.
Glycosylation has many functions. Glycosylation aids in protein folding and stability and serves as a quality-control checkpoint for properly folded proteins. Glycosylation alters protein: protein interactions. Glycans can serve as recognition domains to aid in proper trafficking of proteins within the cell as well as facilitate ligand-receptor interactions resulting in signal transduction pathways. Glycans help mediate cell-cell adhesion and immune responses. In addition, glycosylation can alter the solubility of a protein. Changes in glycosylation patterns are linked to diseases, highlighting the importance of glycosylation to protein function.
Glycosylation is a dynamic event that requires the action of multiple enzymes. Glycosyltransferases link sugars to proteins while glycosidases remove sugars from proteins. Glycosylation enzymes are segregated to different cellular organelles and act on proteins as they traverse the secretory pathway. These step-wise interactions add, remove and trim sugar moieties resulting in a diverse range of glycosylated products. Different cell types contain different glycosylation enzymes altering the types of glycans that can be added.
There are five types of glycosylation: N-linked, O-linked, glypiation, C-linked and phosphoglycosylation. This article focuses on the two most frequent types of glycosylation: N-linked and O-linked.
N-linked glycosylation is the most frequent type of glycosylation and occurs in eukaryotes and Achaea. Approximately 90% of glycoproteins contain N-linked glycosylation. In N-linked glycosylation, sugars are attached to the amino group of an asparagine (N) side chain. N-linked glycans tend to be large, bulky and diverse, requiring the action of many independent enzymes.
All N-linked attachments to proteins are initially identical. The enzymes that transfer N-linked glycans are located in the lumen of the ER. Upon recognition of the appropriate consensus site, a glycan containing 14 sugar residues is attached en bloc to the protein as it is translated. This glycan is then modified by trimming enzymes. When the protein proceeds into the Golgi apparatus, the glycan modifications become diversified by the action of multiple resident glycosyltransferases and glycosidases located in the different stacks. Fully glycosylated proteins can contain either complex oligosaccharides containing multiple sugar types, high-mannose oligosaccharides (multiple mannose residues) or a mixture of both.
O-linked glycosylation is most frequently found on mucins (proteins that form mucous secretions), extracellular matrix components and on antibodies. In O-linked glycosylation, a monosaccharide is attached to the hydroxyl group of serine or threonine posttranslationally. O-linked glycosylation can also occur on oxidized forms of lysine and proline within certain proteins. Sugars are added one at a time by different enzymes and the overall structure of an O-linked glycan is less diverse and complex than an N-linked glycan. Although O-linked glycosylation typically occurs in the Golgi apparatus, it has also been reported in the cytosol and nucleus.
Glycosylation enzymes recognize a consensus sequence when initiating glycosylation. The consensus sequence for N-linked glycosylation is Asn-X-Ser/Thr (where X is any amino acid except Pro) and more rarely Asn-X-Cys. O-linked glycosylation merely requires a serine or threonine without a consensus sequence. Protein prediction software can be used to predict potential glycosylation sites on a protein.
Changes in molecular weight
Glycosylation is usually suspected if a protein contains potential glycosylation sites and migrates slower in a gel than expected based on its predicted molecular weight. Often, multiple forms of the protein are observed, with indiscrete higher molecular weight bands due to the presence of multiple glycosylated forms.
Enzymatic and chemical treatment
Deglycosylating enzymes and chemicals can be used in initial studies to study glycosylation of a protein. In general, a sample is either left untreated or treated with the enzyme/chemical. The protein can then be analyzed by SDS-PAGE and protein staining or Western blotting to detect the protein of interest. A shift in molecular weight upon treatment is indicative of removal of sugar residues. Alternatively, treated samples may be analyzed by mass spectrometry.
Several enzymes can be used for analysis of glycosylation. Endonuclease H removes simple N-linked glycosylations occurring in the ER while PNGase F removes almost all N-linked glycans. No single enzyme can cleave O-linked glycans, however the sugars can be trimmed by exoglycosidases, which then makes the glycan susceptible to removal by O-Glycosidase.
Exposing glycoproteins to an alkaline environment results in sugar release, termed β-elimination. Treatment with sodium hydroxide can result in complete degradation of the glycan. Alternatively, anhydrous hydrazine can be added to a lyophilized glycoprotein to release both N- and O-linked glycans. Temperature variation can be used to determine which type of glycan is released: O-linked glycans are removed at 60°C while N-linked glycans are released at 95°C. While the glycan is released intact, the protein may be degraded.
Although glycan sugars are usually not reactive to stains, glycoproteins can be detected using the periodic acid-Schiff (PAS) reaction. Reaction of sugars with periodic acid oxidizes sugars into aldehydes or ketones, which can be detected using multiple dyes and detected in gels or on membranes. The reaction can also be used to couple glycosylated proteins to labels, such as biotin or horseradish peroxidase, for other detection methods. Although this method can be used to determine if a protein is glycosylated it does not discriminate between the different types of glycosylation.
Lectins are proteins found in animals, plants and microorganisms that specifically bind to sugar molecules. More than 2000 lectins have been described and many are commercially available. Different lectins have various specificities and can be used to detect different types of glycosylation. However, lectins have different affinities for various sugars and recognition can overlap. Lectins can be conjugated to probes for use in blotting or immunohistochemistry or immobilized to solid supports for affinity purification.
Anti-glycan antibodies are used to study glycosylation of proteins. Antibodies can have greater specificity than lectins and target particular linkages. Commercial antibodies are available that recognize all 5 types of glycosylation linkages. The antibodies can be used in any immunodetection assay (e.g. Western blot, FACS, etc).
Mass spectrometry can be used to characterize and quantitate the glycans associated with proteins. However, glycans are extremely variable and often have similar mass, making analysis by mass spec challenging. Often, multiple types of analyses are required. To study the linkage site, the protein and associated glycans is often left intact. In other studies, the glycans are removed sequentially by enzymatic and chemical treatments and the glycans are analyzed separately from the protein.
Photo courtesy of Jun.