Glycosylation (see also chemical glycosylation) is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). In biology, glycosylation mainly refers in particular to the enzymatic process that attaches glycans to proteins, or other organic molecules. This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins). Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesized in the rough endoplasmic reticulum undergo glycosylation. It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification. Aglycosylation is a feature of engineered antibodies to bypass glycosylation. Five classes of glycans are produced:
- N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate.
- O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide
- phosphoglycans linked through the phosphate of a phosphoserine;
- C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain
- glypiation, which is the addition of a GPI anchor that links proteins to lipids through glycan linkages.
Glycosylation is the process by which a carbohydrate is covalently attached to a target macromolecule, typically proteins and lipids. This modification serves various functions. For instance, some proteins do not fold correctly unless they are glycosylated. In other cases, proteins are not stable unless they contain oligosaccharides linked at the amide nitrogen of certain asparagine residues. The influence of glycosylation on the folding and stability of glycoprotein is twofold. Firstly, the highly soluble glycans may have a direct physicochemical stabilisation effect. Secondly, N-linked glycans mediate a critical quality control check point in glycoprotein folding in the endoplasmic reticulum. Glycosylation also plays a role in cell-to-cell adhesion (a mechanism employed by cells of the immune system) via sugar-binding proteins called lectins, which recognize specific carbohydrate moieties. Glycosylation is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. Glycosylation also underpins the ABO blood group system. It is the presence or absence of glycosyltransferases which dictates which blood group antigens are presented and hence what antibody specificities are exhibited. This immunological role may well have driven the diversification of glycan heterogeneity and creates a barrier to zoonotic transmission of viruses. In addition, glycosylation is often used by viruses to shield the underlying viral protein from immune recognition. A significant example is the dense glycan shield of the envelope spike of the human immunodeficiency virus.
Overall, glycosylation needs to be understood by the likely evolutionary selection pressures that have shaped it. In one model, diversification can be considered purely as a result of endogenous functionality (such as cell trafficking). However, it is more likely that diversification is driven by evasion of pathogen infection mechanism (e.g. Helicobacter attachment to terminal saccharide residues) and that diversity within the multicellular organism is then exploited endogenously.
Glycosylation increases diversity in the proteome, because almost every aspect of glycosylation can be modified, including:
- Glycosidic bond—the site of glycan linkage
- Glycan composition—the types of sugars that are linked to a given protein
- Glycan structure—can be unbranched or branched chains of sugars
- Glycan length—can be short- or long-chain oligosaccharides
There are various mechanisms for glycosylation, although most share several common features:
- Glycosylation, unlike glycation, is an enzymatic process. Indeed, glycosylation is thought to be the most complex post-translational modification, because of the large number of enzymatic steps involved.
- The donor molecule is often an activated nucleotide sugar.
- The process is non-templated (unlike DNA transcription or protein translation); instead, the cell relies on segregating enzymes into different cellular compartments (e.g., endoplasmic reticulum, cisternae in Golgi apparatus). Therefore, glycosylation is a site-specific modification.
N-linked glycosylation is a very prevalent form of glycosylation and is important for the folding of many eukaryotic glycoproteins and for cell–cell and cell–extracellular matrix attachment. The N-linked glycosylation process occurs in eukaryotes in the lumen of the endoplasmic reticulum and widely in archaea, but very rarely in bacteria. In addition to their function in protein folding and cellular attachment, the N-linked glycans of a protein can modulate a protein's function, in some cases acting as an on/off switch.
Xylose, fucose, mannose, and GlcNAc phosphoserine glycans have been reported in the literature. Fucose and GlcNAc have been found only in Dictyostelium discoideum, mannose in Leishmania mexicana, and xylose in Trypanosoma cruzi. Mannose has recently been reported in a vertebrate, the mouse, Mus musculus, on the cell-surface laminin receptor alpha dystroglycan4. It has been suggested this rare finding may be linked to the fact that alpha dystroglycan is highly conserved from lower vertebrates to mammals.
A mannose sugar is added to the first tryptophan residue in the sequence W–X–X–W (W indicates tryptophan; X is any amino acid). Thrombospondins are one of the most commonly C-mannosylation is unusual because the sugar is linked to a carbon rather than a reactive atom such as nitrogen or oxygen. Recently,[when?] the first crystal structure of a protein containing this type of glycosylation has been determined—that of human complement component 8, .
Formation of GPI anchors (glypiation)Edit
Glypiation is a special form of glycosylation that features the formation of a GPI anchor. In this kind of glycosylation a protein is attached to a lipid anchor, via a glycan chain. (See also prenylation.)
Glycosylation can also be effected using the tools of synthetic organic chemistry. Unlike the biochemical processes, synthetic glycochemistry relies heavily on protecting groups (e.g. the 4,6-O-benzylidene) in order to achieve desired regioselectivity. The other challenge of chemical glycosylation is the stereoselectivity that each glycosidic linkage has two stereo-outcomes, alpha-/beta or cis-/trans. Generally, the alpha- or cis- glycoside is more challenging to synthesis. New methods have been developed based on solvent participation or the formation of bicyclic sulfonium ions as chiral-auxiliary groups.
Over 40 disorders of glycosylation have been reported in humans. These can be divided into four groups: disorders of protein N-glycosylation, disorders of protein O-glycosylation, disorders of lipid glycosylation and disorders of other glycosylation pathways and of multiple glycosylation pathways. No effective treatment is known for any of these disorders. 80% of these affect the nervous system.
Effects on therapeutic efficacyEdit
It has been reported that mammalian glycosylation can improve the therapeutic efficacy of biotherapeutics. For example, therapeutic efficacy of recombinant human interferon gamma, expressed in HEK 293 platform, was improved against drug-resistant ovarian cancer cell lines.
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- GlycoEP : In silico Platform for Prediction of N-, O- and C-Glycosites in Eukaryotic Protein Sequences PLoS ONE 8(6): e67008
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- NetNGlyc: The NetNglyc server predicts N-glycosylation sites in human proteins using artificial neural networks that examine the sequence context of Asn-Xaa-Ser/Thr sequons.
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