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52-487: This type of linkage is important for both the structure and function of many eukaryotic proteins. The N -linked glycosylation process occurs in eukaryotes and widely in archaea , but very rarely in bacteria . The nature of N -linked glycans attached to a glycoprotein is determined by the protein and the cell in which it is expressed. It also varies across species . Different species synthesize different types of N -linked glycan. There are two types of bonds involved in

104-489: A pyrophosphate molecule. The biosynthesis of N -linked glycans occurs via three major steps: Synthesis, en bloc transfer and initial trimming of precursor oligosaccharide occurs in the endoplasmic reticulum (ER). Subsequent processing and modification of the oligosaccharide chain are carried out in the Golgi apparatus . The synthesis of glycoproteins is thus spatially separated in different cellular compartments. Therefore,

156-490: A consequence, they are also hard to treat. However, thanks to the many advances that have been made in next-generation sequencing , scientists can now understand better these disorders and have discovered new CDGs. 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,

208-465: A decreased level, skin elasticity is reduced which is an important symptom of aging. They are also the precursors of many hormones and regulate and modify their receptor mechanisms at the DNA level. There are different enzymes to remove the glycans from the proteins or remove some part of the sugar chain. Notch signalling is a cell signalling pathway whose role is, among many others, to control

260-452: A diverse range of N -glycan structures. The process of N -linked glycosylation starts with the formation of dolichol -linked GlcNAc sugar. Dolichol is a lipid molecule composed of repeating isoprene units. This molecule is found attached to the membrane of the ER. Sugar molecules are attached to the dolichol through a pyrophosphate linkage (one phosphate was originally linked to dolichol, and

312-414: A glycan is transferred to a nascent polypeptide: Oligosaccharyltransferase is the enzyme responsible for the recognition of the consensus sequence and the transfer of the precursor glycan to a polypeptide acceptor which is being translated in the endoplasmic reticulum lumen. N -linked glycosylation is, therefore, a co-translational event. N -glycan processing is carried out in endoplasmic reticulum and

364-400: A glycoprotein: bonds between the saccharides residues in the glycan and the linkage between the glycan chain and the protein molecule. The sugar moieties are linked to one another in the glycan chain via glycosidic bonds . These bonds are typically formed between carbons 1 and 4 of the sugar molecules. The formation of glycosidic bond is energetically unfavourable, therefore the reaction

416-413: A non-enzymatic reaction. 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. Glycosylation is also present in the cytoplasm and nucleus as the O -GlcNAc modification. Aglycosylation

468-404: A protein, consists of two GlcNAc, nine mannose, and three glucose molecules. Once the precursor oligosaccharide is formed, the completed glycan is then transferred to the nascent polypeptide in the lumen of the ER membrane. This reaction is driven by the energy released from the cleavage of the pyrophosphate bond between the dolichol-glycan molecule. There are three conditions to fulfill before

520-450: 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 can also modulate the thermodynamic and kinetic stability of the proteins. Glycosylation increases diversity in

572-524: A variety of diseases, most of which involve the nervous system . Many therapeutic proteins in the market are antibodies , which are N -linked glycoproteins. For example, Etanercept , Infliximab and Rituximab are N -glycosylated therapeutic proteins. The importance of N -linked glycosylation is becoming increasingly evident in the field of pharmaceuticals . Although bacterial or yeast protein production systems have significant potential advantages such as high yield and low cost, problems arise when

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624-463: Is a feature of engineered antibodies to bypass glycosylation. Five classes of glycans are produced: 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

676-504: 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

728-399: Is a spontaneous reaction and a type of post-translational modification of proteins meaning it alters their structure and biological activity. It is the covalent attachment between the carbonil group of a reducing sugar (mainly glucose and fructose) and the amino acid side chain of the protein. In this process the intervention of an enzyme is not needed. It takes place across and close to

780-416: Is added to the first tryptophan residue in the sequence W–X–X–W (W indicates tryptophan; X is any amino acid). A C-C bond is formed between the first carbon of the alpha-mannose and the second carbon of the tryptophan. 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

832-458: Is another group of proteins that undergo C -mannosylation, type I cytokine receptors . C -mannosylation is unusual because the sugar is linked to a carbon rather than a reactive atom such as nitrogen or oxygen . In 2011, the first crystal structure of a protein containing this type of glycosylation was determined—that of human complement component 8. Currently it is established that 18% of human proteins , secreted and transmembrane undergo

884-399: Is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase complex . The active site of OST is located about 4 nm from the lumenal face of the ER membrane. It usually acts during translation as the nascent protein is entering the ER, but this cotranslational glycosylation

936-404: Is attached to an asparagine (Asn) residue in the sequence Asn-X- Ser or Asn-X- Thr where X is any amino acid except proline . This sequence is called a glycosylation sequon . The reaction catalyzed by OST is the central step in the N -linked glycosylation pathway . OST is a component of the translocon in the endoplasmic reticulum (ER) membrane . A lipid-linked core-oligosaccharide

988-426: Is coupled to the hydrolysis of two ATP molecules. On the other hand, the attachment of a glycan residue to a protein requires the recognition of a consensus sequence . N -linked glycans are almost always attached to the nitrogen atom of an asparagine (Asn) side chain that is present as a part of Asn–X– Ser / Thr consensus sequence, where X is any amino acid except proline (Pro). In animal cells,

1040-440: Is nevertheless called a posttranslational modification . A few examples have been found of OST activity after translation is complete. Current opinion is that post-translational activity may occur if the protein is poorly folded or folds slowly. Yeast OST is composed of eight different membrane-spanning proteins in three subcomplexes (one of them is OST4 ). These octomers do not form higher order oligomers , and three of

1092-422: Is the reaction in which a carbohydrate (or ' glycan '), i.e. a glycosyl donor , is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor ) in order to form a glycoconjugate . In biology (but not always in chemistry), glycosylation usually refers to an enzyme-catalysed reaction, whereas glycation (also 'non-enzymatic glycation' and 'non-enzymatic glycosylation') may refer to

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1144-402: Is the stereoselectivity that each glycosidic linkage has two stereo-outcomes, α/β or cis / trans . Generally, the α- 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. The non-enzymatic glycosylation is also known as glycation or non-enzymatic glycation. It

1196-454: The Golgi apparatus . The Notch proteins go through these organelles in their maturation process and can be subject to different types of glycosylation: N-linked glycosylation and O-linked glycosylation (more specifically: O-linked glucose and O-linked fucose). All of the Notch proteins are modified by an O-fucose, because they share a common trait: O-fucosylation consensus sequences . One of

1248-460: 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

1300-422: The cell differentiation process in equivalent precursor cells . 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 in embryonic death or malformations of vital organs like the heart. Some of the specific modulators that control this process are glycosyltransferases located in the endoplasmic reticulum and

1352-471: The cis -Golgi, a series of mannosidases remove some or all of the four mannose residues in α-1,2 linkages. Whereas in the medial portion of the Golgi, glycosyltransferases add sugar residues to the core glycan structure, giving rise to the three main types of glycans: high mannose, hybrid and complex glycans. The order of addition of sugars to the growing glycan chains is determined by the substrate specificities of

1404-610: The galactose-alpha-1,3-galactose epitope, which can induce serious allergenic reactions, including anaphylactic shock , in people who have Alpha-gal allergy . These drawbacks have been addressed by several approaches such as eliminating the pathways that produce these glycan structures through genetic knockouts. Furthermore, other expression systems have been genetically engineered to produce therapeutic glycoproteins with human-like N -linked glycans. These include yeasts such as Pichia pastoris , insect cell lines, green plants, and even bacteria. Glycosylation Glycosylation

1456-559: 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

1508-456: The proteome , because almost every aspect of glycosylation can be modified, including: There are various mechanisms for glycosylation, although most share several common features: 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

1560-471: The Golgi body. Initial trimming of the precursor molecule occurs in the ER and the subsequent processing occurs in the Golgi. Upon transferring the completed glycan onto the nascent polypeptide, two glucose residues are removed from the structure. Enzymes known as glycosidases remove some sugar residues. These enzymes can break glycosidic linkages by using a water molecule. These enzymes are exoglycosidases as they only work on monosaccharide residues located at

1612-454: 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. Having elevated levels of AGEs in the body has a direct impact on the development of many diseases. It has a direct implication in diabetes mellitus type 2 that can lead to many complications such as: cataracts , renal failure , heart damage... And, if they are present at

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1664-412: The cell, e.g. immune cells that migrate to the skin have specific glycosylations that favor homing to that site. The glycosylation patterns on the various immunoglobulins including IgE, IgM, IgD, IgA, and IgG bestow them with unique effector functions by altering their affinities for Fc and other immune receptors. Glycans may also be involved in "self" and "non self" discrimination, which may be relevant to

1716-506: 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

1768-455: The eight proteins are glycosylated themselves. OST in mammals is known to have a similar composition. OST is thought to require many subunits because it must: The catalytically active subunit of the OST is called STT3. Two paralogs exist in eukaryotes, termed STT3A and STT3B . STT3A is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of

1820-408: The endoplasmic reticulum whereas STT3B can also mediate posttranslational glycosylation. The structure of PglB , the prokaryotic homolog of STT3 has been solved. The high sequence similarity between the prokaryotic and the eukaryotic STT3 suggests that their structures are similar. CDG syndromes are genetic disorders of the glycosylation pathway. They are labelled "Type I" if the defective gene

1872-504: The enzymes and their access to the substrate as they move through secretory pathway . Thus, the organization of this machinery within a cell plays an important role in determining which glycans are made. Golgi enzymes play a key role in determining the synthesis of the various types of glycans. The order of action of the enzymes is reflected in their position in the Golgi stack: Similar N -glycan biosynthesis pathway have been found in prokaryotes and Archaea. However, compared to eukaryotes,

1924-435: The final glycan structure in eubacteria and archaea does not seem to differ much from the initial precursor made in the endoplasmic reticulum. In eukaryotes, the original precursor oligosaccharide is extensively modified en route to the cell surface. N -linked glycans have intrinsic and extrinsic functions. Within the immune system, the N -linked glycans on an immune cell's surface will help dictate that migration pattern of

1976-415: The glycan attached to the asparagine is almost inevitably N -acetylglucosamine (GlcNAc) in the β-configuration. This β-linkage is similar to glycosidic bond between the sugar moieties in the glycan structure as described above. Instead of being attached to a sugar hydroxyl group, the anomeric carbon atom is attached to an amide nitrogen. The energy required for this linkage comes from the hydrolysis of

2028-619: 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 dystroglycan . 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

2080-529: 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. O -linked glycosylation is a form of glycosylation that occurs in eukaryotes in the Golgi apparatus , but also occurs in archaea and bacteria . Xylose , fucose , mannose , and GlcNAc phosphoserine glycans have been reported in

2132-411: The machinery required to add complex, human-type glycans. However, glycans produced in these systems can differ from glycans produced in humans, as they can be capped with both N -glycolylneuraminic acid (Neu5Gc) and N -acetylneuraminic acid (Neu5Ac), whereas human cells only produce glycoproteins containing N -acetylneuraminic acid. Furthermore, animal cells can also produce glycoproteins containing

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2184-674: 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. 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

2236-401: The non-reducing end of the glycan. This initial trimming step is thought to act as a quality control step in the ER to monitor protein folding . Once the protein is folded correctly, two glucose residues are removed by glucosidase I and II. The removal of the final third glucose residue signals that the glycoprotein is ready for transit from the ER to the cis -Golgi. ER mannosidase catalyses

2288-471: The pathophysiology of various autoimmune diseases. In some cases, interaction between the N-glycan and the protein stabilizes the protein through complex electronic effects. Changes in N -linked glycosylation has been associated with different diseases including rheumatoid arthritis , type 1 diabetes , Crohn's disease , and cancers. Mutations in eighteen genes involved in N -linked glycosylation result in

2340-422: The process of C-mannosylation. Numerous studies have shown that this process plays an important role in the secretion of Trombospondin type 1 containing proteins which are retained in the endoplasmic reticulum if they do not undergo C-mannosylation This explains why a type of cytokine receptors , erythropoietin receptor remained in the endoplasmic reticulum if it lacked C-mannosylation sites. Glypiation

2392-582: The protein of interest is a glycoprotein. Most prokaryotic expression systems such as E. coli cannot carry out post-translational modifications . On the other hand, eukaryotic expression hosts such as yeast and animal cells, have different glycosylation patterns. The proteins produced in these expression hosts are often not identical to human protein and thus, cause immunogenic reactions in patients. For example, S.cerevisiae (yeast) often produce high-mannose glycans which are immunogenic. Non-human mammalian expression systems such as CHO or NS0 cells have

2444-493: The removal of this final glucose. However, if the protein is not folded properly, the glucose residues are not removed and thus the glycoprotein can't leave the endoplasmic reticulum. A chaperone protein ( calnexin / calreticulin ) binds to the unfolded or partially folded protein to assist protein folding. The next step involves further addition and removal of sugar residues in the cis-Golgi. These modifications are catalyzed by glycosyltransferases and glycosidases respectively. In

2496-485: The second amino 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. Thrombospondins are one of the proteins most commonly modified in this way. However, there

2548-458: The second phosphate came from the nucleotide sugar). The oligosaccharide chain is then extended through the addition of various sugar molecules in a stepwise manner to form a precursor oligosaccharide. The assembly of this precursor oligosaccharide occurs in two phases: Phase I and II. Phase I takes place on the cytoplasmic side of the ER and Phase II takes place on the luminal side of the ER. The precursor molecule, ready to be transferred to

2600-447: The type of N -glycan synthesized, depends on its accessibility to the different enzymes present within these cellular compartments. However, in spite of the diversity, all N -glycans are synthesized through a common pathway with a common core glycan structure. The core glycan structure is essentially made up of two N -acetyl glucosamine and three mannose residues. This core glycan is then elaborated and modified further, resulting in

2652-692: The water channels and the protruding tubules. 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 as Advanced Glycation end-products (AGEs). AGEs accumulate in long-lived extracellular proteins such as collagen which is the most glycated and structurally abundant protein, especially in humans. Also, some studies have shown lysine may trigger spontaneous non-enzymatic glycosylation. AGEs are responsible for many things. These molecules play an important role especially in nutrition, they are responsible for

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2704-427: Was improved against drug-resistant ovarian cancer cell lines. Oligosaccharyltransferase Oligosaccharyltransferase or OST ( EC 2.4.1.119 ) is a membrane protein complex that transfers a 14- sugar oligosaccharide from dolichol to nascent protein . It is a type of glycosyltransferase . The sugar Glc 3 Man 9 GlcNAc 2 (where Glc= Glucose , Man= Mannose , and GlcNAc= N -acetylglucosamine )

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