As to glycomics (see proteomics).  As to characterization and detection see outline    As to Glycoproteins generally see outline

Glycosylation is a form of post- or co-translational modification occurring in all eukaryotic proteins. Glycosylation stands for an enzymatic reaction that allows the chemical linkage synthesis of monosaccharides or polysaccharides (glycans) onto proteins or lipids. More than 50% of all known proteins are estimated to be glycosylated.

Glycosylation involves the attachment of sugar chains to Asn, Ser and Thr residues on the peptide. Oligosaccharides attached to an asparagine are said to be N-linked while those attached to serine or theonine are O-linked. N-linked glycans have a core region of two N-acetylglucosamine linking the rest of the chain, usually a number of mannose residues and can contain other sugars. Mannose side chain decorations have been observed to be over 200 residues. By contrast, O-glycan side chains are usually between 1 and 5 sugar residues which are exclusively mannose. Schnorr (WO2004/090549)

Structural Complexity and Heterogeneity 

The structure of these highly branched oligosaccahrides is often very complex because their constitutuent monosaccharides can be linked in many differnt way. Consequently, the potential information encoded into an oligosaccharide via its monosaccharid sequence and 3D structure is considerable. Each glycoform may be involved in different and unique cellular functions (Rademacher, Springer Semin Immunopathol 10, (1988), 231-249. Protein glycosylation can be very diverse and dynamic. A survey suggests that there are at least 41 different types of sugar-amino acid linkages, with N-glycosylation (at the side chain of Asn), O-GalNAcglycosylation (at the Ser/Thr residues), and O-GlcNAcglycosylation (at the Ser/Thr residues) as the major forms. An important feature of protein glycosylation is the structural complexity of glycans. The number of glycan variants can grow very rapidly when the glycan core is further branched and decorated with various termianl sugars (e.g., sialic acids, and noncarbohydrate functional groups such as sulfate, phosphate, and acetate. Another common feature of glycosylation is structural heterogeneity. In contrast to nucleic acids and proteins that are biosynthetically assembled on templates and under direct transcriptional control, the biosynthesis of glycans on glycoproteins have no known template, and glycosylation patterns are dictated by many factors (amino acid sequences, local peptide conformations at the glycosylation sites, and the accessbility and localization of activated substrates, enzymes, and cofactors). As a result, glycoproteins are usually produced as mixtures of glycosylation variants (i.e., glycoforms that share the same polypeptide backbone but differ in the sites of glycosylation and/or in the structures of the pendant glycans (Wang et al. “Emerging Technologies for Making Glycan-Defined Glycoproteins” ACS Chem. Biol. 7, 2012, 110-122).

Variability in the initial attachment of N or O linked oligosacccharides leads to glycoforms with different numbers of oligosaccharide structures. For example, cultures mammalina cells secrete two types of t-PA, possessing either two or three N linked oligosaccharides due to variable site occupancy at Asn184. These two forms of t-PA have different in vitro activity. Another source of microheterogeneity results form competing glycosyltransferases in the golgi as with competition of GlcNAc transferase III (GnT III) and GlcNAc transferase IV (GnT IV) for a particular oligosaccharide substrate. If GnT III acts first, one resulting oligosaccharide structure is no longer an acceptable substrate for GnT IV and the oligosaccharide is committed to a processing pathway leading to a bisected, bi-antennary complex type structure. However, if Gnt IV acts first, the resulting structure is committed to a processing pathway potentially leading to a tri-antennary complex type structure. (Goochee, “Bioprocess factors affecting glycoprotein oligosaccharide structure” Develop biol standard, 76, 95-104, 1992).

A glycoprotein consists of a collection of glycosylated variants or glycoforms that arise as a consequence of the biosynthetic pathways which develop the O linked glycan chains and modify the Man9GlcNAc2 N linked oligosaccharide precursor. An oligosaccharide chain is modified through a series of interactions with glycosidase or glycosyl transferase enzymes, and at any time many proteins may be in the processing pathway, simultaneously competitng for the active sites of the enzymes. In general, conditions are such that some glycoprotein substrates are not processed by every enzyme, allowing microheterogeneity, or glycoforms to develop in a single protien porpulation. Tus, the sugars released form a pure glycoprotein often consist of heterogeneous population containing both neutral and charged oligosaccharides. For example, the single N-glycosylation site in human platelet CD59 is associated with more than 100 glycans. (Guile, Analytical Biochemistry 210-220, 1996). 

Where Glycosylation Sites are Located

Glycosylation of polypeptide is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyaino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. (Nadarajah, US14/355818 (US2014/0301977)) 

Most serum-derived and cell-surface proteins are N-glycosylated. However, antibodies (see below) have a glycosylation site on the Fc domain at the conserved N-glycosylation site of Asn 297. Consensus motifs, that is, the amino acid sequence recognized by various glycosyl transferases, have been described. For example, the consensus motif for an N-linked glcyosylation motif is frequently NXT or NXS, where X can be any amino acid except proline.

Mechanism of glycosylation in eukaryotic cells

The synthesis of N-linked and O-linked ogligosaccharides involves a series of enzyme catalysed events localized in several intraccelular compartments. 

The factors which specify protein linked glycan structure are the primary amino acid sequence, other occupied glycosylation sites, the cell/tissue type and the environmental/physiological state of the cell/tissue. It is the complex interplay of cell type and environmental (physiological) factors acting on an encrypted polypeptide instruction set that determines the identity and set of oligosaccharides attached to a protein. Different cell types have distinct complements of the glycosyltransferases and glycosidases which act upon glycoprotein biosynthetic intermediates. Thus, the same polypeptide produced in various cell lines can have glycan chains which differ in their detailed primary structure. (Cumming, Glycobiology, 1(2), 115-130, 991).

Peptides expressed in eukaryotic cells are typically N-glycoyslated on asparagine residues at sites in the peptide primary structure containing the sequence asparagine-X-serine/threonine where X can be any amino acid except proline and aspartic acid. The carbohydroate portion of such peptides is known as an N-linked glycan. The early events of N-glycosylation occur in the ER are identical in mammals, plants, insects and other higher eukaryotes. First, an oligoscaccharide chain comprising 14 sugar resiudes is contructed on a lipid carrier molecule. As the nascent peptide is translated and translocated into the ER, the entire oligosaccharide chain is transferred to the amide group of the asparagine residue in a reaction catalyzed by a membrane bound glycosyltransferase enzyme. The N-liniked glycan is further processed both in the ER and in the Glogi apparatus. The further processing generally entails removal of some of the sugar residues and addition of other sugar residues in reactions catalyzed by glycosidase and glycosyltransferases specific for the sugar residues removed and added. Typically, the final structures of the N-linked glycans are dependent upon the organism in which the peptide is produced. For example, in general, peptides produced in bacteria are completely unglycosylated. 

Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases), and have different substrates (nuceltoide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed ((U. S. application 12/696,314 p. 43, lines 10-19). Protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration.

Protein sialylation is an ezymatic process, and is the terminal reaction of glycosylation that proudces matured sialylated oligosaccharides on glycoproteins. In rough endoplasmic reticulum (ER), high mannose core is added to newly synthesized protein. The protein is then transported to the GA. There are at least 18 different introacellular, Golgi membrane bound glycosyltransferases which catalyze the reaciton for growing oligosaccharide chains by using nucleotide sugar precursors as substrates. For instand, sialytranferase ST3GAL4 (ST3 beta-galactoside alpha-2,3 sialytransferase 4) uses CMP sialic aicd as a substrate and added alpha-2,3 linked sialic acid to beta1,4 Galactose. (Xu, Mol Biotechno (2010) 45: 248-256)

Functions of Glycosylation

Protein glycosylation has two main roles. First, protein-linked glycans modulate biochemical attributes of proteins such as bioactivity, folding and immunogenicity. For example, carbohydrates can modulate the immunogenic potential of a glycoprotein either by defining all or part of an epitope, or by masking potential antigenic sites. Second, they can serve as determinants in molecular recognition evens such as the targeting of particular enzymes to lysosomes or the uptake of asialoglycoproteins by a hepatic receptor. (Cumming, Glycobiology, 1(2), 115-130, 991). 

Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy (Gharder, Biotechnology and Genetic Engineering Reviews – Vol. 28, 147-176 (2012).

Many glycans exist only for protective purposes against external physical stresses like freezing and biochemical attacks (e.g., proteases). Some glycans like the ABO blood group antigens, however, represent major histocompatibilitycomplex antigens which have crucial roles in cell-cell recognition events.

Glycoprotein oligosaccharides generally reside on the protein surface, where they may interact with both the protein surface and the solvent. The effects on protein surface chemistry are usually considerable. Proteins poessing oligosaccharides are almost always much more soluble than their aglycosyl counterparts. Differences in solubility among glycoforms are also likely, as demonstrated by the reduced solubility of EPO in the absence of terminal sialylation. Since sialic acid si negatively charged at neutral pH, differences in sialic acid content amoung clygoforms will influence their behaviro in IEX. (Goochee, “Bioprocess factors affecting glycoprotein oligosaccharide structure” Develop biol standard, 76, 95-104, 1992).

Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control and assembly of peptide loaded MHC antigens and the T cell receptor complex. Oligosaccharides attached to glycoproteins in the junction between T cells and APCs help to orient binding faces, provide protease protection and restrict nonspecific lateral protein protein interactions. In the humoral immuen system, all of the immunoglobulins and most of the complement components are glycosylated. (Rudde “glycosylation and the immune system” Science, 291, 2001) 

In Diseases:

Changes in post-translation modificaiton such as glycosylation are the hallmark of many common diseases. The most studies example is the lack of galactosylation of IgG in ververal autoimmune dsieases such as RN, SE and Crohn’s disease. Antoher example is the aberrant O-linked calactosylation and N-linked sialylation of serum IgG1 imprimary IgA nephropathy and primary Sjogren’s syndrome. Monitoring protein specific clysoylaton remains an active goal for monitoring dsieases because glycosylation is highly sensitive to local and global biologcial changes. (Hong, “A mehtod for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins” J of proteome research, 2015)

Use by Bacterial and Viruses to evade host cell response: 

Cell-surface glycans are taken advantage of by various microbes to achieve infection of their host cells. In fact, most bacterial toxins can be classified as AB toxins, consisting of a toxic A chain and a carbohydrate binding protein (lectin) B chain. These include cholera, diphteria, tubercular and cero toxins. Influenza virus also contains hemagglutinin (HA) lectin to enter the host cells.

Enveloped viruses such HIV can evade immuen recognition by exploiting the host glycosylation machinery to protein potentail protein antigenic epitopes. Envoloped viruses also use the host secretory pathway to fold and assemble their often heavily glycosylated caot proteins. A possible antiviral strategy involves the use of glycosylation inhibitors to interfer with the fiolding of viral envelope proteins, such as for HBV and HIV . 

(Rudde “glycosylation and the immune system” Science, 291, 2001)