Glycosylation is the reaction in which acarbohydrate (or 'glycan'), i.e. aglycosyl donor, is attached to a hydroxyl or other functional group of another molecule (aglycosyl acceptor) in order to form aglycoconjugate. In biology (but not always in chemistry),glycosylation usually refers to an enzyme-catalysed reaction, whereasglycation (also 'non-enzymatic glycation' and 'non-enzymatic glycosylation') may refer to a non-enzymatic reaction.[1]
Glycosylation is a form of co-translational andpost-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins.[2] The majority of proteins synthesized in therough endoplasmic reticulum undergo glycosylation. Glycosylation is also present in thecytoplasm and nucleus as theO-GlcNAc modification. Aglycosylation is a feature of engineered antibodies to bypass glycosylation.[3][4] Five classes of glycans are produced:
Glycosylation is the process by which acarbohydrate iscovalently attached to a targetmacromolecule, typicallyproteins andlipids. This modification serves various functions.[5] For instance, some proteins do not fold correctly unless they are glycosylated.[2] In other cases, proteins are not stable unless they containoligosaccharides linked at theamidenitrogen of certainasparagine residues. The influence of glycosylation on the folding and stability ofglycoprotein 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.[6] Glycosylation also plays a role in cell-to-cell adhesion (a mechanism employed by cells of theimmune system) viasugar-binding proteins calledlectins, which recognize specific carbohydrate moieties.[2] Glycosylation is an important parameter in the optimization of many glycoprotein-based drugs such asmonoclonal antibodies.[6] Glycosylation also underpins theABO blood group system. It is the presence or absence ofglycosyltransferases which dictates which blood groupantigens 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 tozoonotic transmission of viruses.[7] 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 thehuman immunodeficiency virus.[8]
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 ascell 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.[citation needed]
Glycosylation can also modulate the thermodynamic and kinetic stability of the proteins.[9]
Glycosylation increases diversity in theproteome, because almost every aspect of glycosylation can be modified, including:[citation needed]
There are various mechanisms for glycosylation, although most share several common features:[2]
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. TheN-linked glycosylation process occurs ineukaryotes in the lumen of the endoplasmic reticulum and widely inarchaea, but very rarely inbacteria. In addition to their function in protein folding and cellular attachment, theN-linked glycans of a protein can modulate a protein's function, in some cases acting as an on/off switch.[citation needed]
O-linked glycosylation is a form of glycosylation that occurs ineukaryotes in theGolgi apparatus,[11] but also occurs inarchaea andbacteria.
Xylose,fucose,mannose, andGlcNAcphosphoserineglycans have been reported in the literature. Fucose and GlcNAc have been found only inDictyostelium discoideum, mannose inLeishmania mexicana, and xylose inTrypanosoma 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.[12]
Amannose sugar is added to the firsttryptophan residue in the sequence W–X–X–W (W indicates tryptophan; X is any amino acid). AC-C bond is formed between the first carbon of thealpha-mannose and the second carbon of the tryptophan.[13] However, not all the sequences that have this pattern are mannosylated. It has been established that, in fact, only two thirds are and that there is a clear preference for the secondamino acid to be one of the polar ones (Ser,Ala,Gly and Thr) in order for mannosylation to occur. Recently there has been a breakthrough in the technique of predicting whether or not the sequence will have a mannosylation site that provides an accuracy of 93% opposed to the 67% accuracy if we just consider the WXXW motif.[14]
Thrombospondins are one of the proteins most commonly modified in this way. However, there is another group of proteins that undergoC-mannosylation, type Icytokine receptors.[15]C-mannosylation is unusual because the sugar is linked to acarbon rather than a reactive atom such asnitrogen oroxygen. In 2011, the first crystal structure of a protein containing this type of glycosylation was determined—that of human complement component 8.[16] Currently it is established that 18% of humanproteins, secreted andtransmembrane undergo the process of C-mannosylation.[14] Numerous studies have shown that this process plays an important role in the secretion ofTrombospondin type 1 containing proteins which are retained in theendoplasmic reticulum if they do not undergo C-mannosylation[14] This explains why a type ofcytokine receptors,erythropoietin receptor remained in theendoplasmic reticulum if it lacked C-mannosylation sites.[17]
Glypiation is a special form of glycosylation that features the formation of aGPI anchor. In this kind of glycosylation a protein is attached to a lipid anchor, via a glycan chain. (See alsoprenylation.)
Glycosylation can also be effected using the tools ofsynthetic organic chemistry. Unlike the biochemical processes, synthetic glycochemistry relies heavily on protecting groups[18] (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, α/β orcis/trans. Generally, the α- orcis-glycoside is more challenging to synthesis.[19] New methods have been developed based on solvent participation or the formation of bicyclic sulfonium ions as chiral-auxiliary groups.[20]
The non-enzymatic glycosylation is also known asglycation or non-enzymatic glycation. It is a spontaneous reaction and a type ofpost-translational modification of proteins meaning it alters their structure and biological activity. It is thecovalent attachment between thecarbonil group of a reducing sugar (mainly glucose and fructose) and the amino acidside chain of the protein. In this process the intervention of an enzyme is not needed. It takes place across and close to the water channels and the protruding tubules.[21]
At first, the reaction forms temporary molecules which later undergo different reactions (Amadori rearrangements,Schiff base reactions,Maillard reactions,crosslinkings...) and form permanent residues known asAdvanced Glycation end-products (AGEs).[citation needed]
AGEs accumulate in long-lived extracellular proteins such ascollagen[22] which is the most glycated and structurally abundant protein, especially in humans. Also, some studies have shownlysine may trigger spontaneous non-enzymatic glycosylation.[23]
AGEs are responsible for many things. These molecules play an important role especially in nutrition, they are responsible for the brownish color and the aromas and flavors of some foods. It is demonstrated that cooking at high temperature results in various food products having high levels of AGEs.[24]
Having elevated levels of AGEs in the body has a direct impact on the development of many diseases. It has a direct implication indiabetes mellitus type 2 that can lead to many complications such as:cataracts,renal failure, heart damage...[25] And, if they are present at a decreased level, skin elasticity is reduced which is an important symptom of aging.[22]
They are also the precursors of manyhormones and regulate and modify their receptor mechanisms at theDNA level.[22]
There are differentenzymes to remove theglycans from theproteins or remove some part of thesugar chain.[citation needed]
Notch signalling is a cell signalling pathway whose role is, among many others, to control thecell differentiation process in equivalentprecursor cells.[26] This means it is crucial in embryonic development, to the point that it has been tested on mice that the removal of glycans in Notch proteins can result inembryonic death or malformations of vital organs like the heart.[27]
Some of the specific modulators that control this process areglycosyltransferases located in theendoplasmic reticulum and theGolgi apparatus.[28] The Notch proteins go through these organelles in their maturation process and can be subject to different types of glycosylation:N-linked glycosylation andO-linked glycosylation (more specifically: O-linked glucose and O-linked fucose).[26]
All of the Notch proteins are modified by an O-fucose, because they share a common trait: O-fucosylationconsensus sequences.[26] One of the modulators that intervene in this process is the Fringe, a glycosyltransferase that modifies the O-fucose to activate or deactivate parts of the signalling, acting as a positive or negative regulator, respectively.[28]
There are three types of glycosylation disorders sorted by the type of alterations that are made to the glycosylation process: congenital alterations, acquired alterations and non-enzymatic acquired alterations.
All these diseases are difficult to diagnose because they do not only affect one organ, they affect many of them and in different ways. As a consequence, they are also hard to treat. However, thanks to the many advances that have been made innext-generation sequencing, scientists can now understand better these disorders and have discovered new CDGs.[31]
It has been reported that mammalian glycosylation can improve the therapeutic efficacy ofbiotherapeutics. For example, therapeutic efficacy of recombinanthuman interferon gamma, expressed inHEK 293 platform, was improved against drug-resistantovarian cancer cell lines.[32]