Glycan

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The terms glycans and

of monosaccharide residues, and can be linear or branched.

Glycans and proteins

Glycans can be found attached to proteins as in glycoproteins and proteoglycans. In general, they are found on the exterior surface of cells. O- and N-linked glycans are very common in

.

N-Linked glycans

Introduction

N-Linked glycans are attached in the

, and other monosaccharides.

Assembly

In eukaryotes, N-linked glycans are derived from a core 14-sugar unit assembled in the cytoplasm and endoplasmic reticulum. First, two N-acetylglucosamine residues are attached to dolichol monophosphate, a lipid, on the external side of the endoplasmic reticulum membrane. Five mannose residues are then added to this structure. At this point, the partially finished core glycan is flipped across the endoplasmic reticulum membrane, so that it is now located within the reticular lumen. Assembly then continues within the endoplasmic reticulum, with the addition of four more mannose residues. Finally, three glucose residues are added to this structure. Following full assembly, the glycan is transferred en bloc by the glycosyltransferase oligosaccharyltransferase to a nascent peptide chain, within the reticular lumen. This core structure of N-linked glycans, thus, consists of 14 residues (3 glucose, 9 mannose, and 2 N-acetylglucosamine).

Image: https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.figgrp.469

Dark squares are N-acetylglucosamine; light circles are mannose; dark triangles are glucose.

Processing, modification, and diversity

Once transferred to the nascent peptide chain, N-linked glycans, in general, undergo extensive processing reactions, whereby the three glucose residues are removed, as well as several mannose residues, depending on the N-linked glycan in question. The removal of the glucose residues is dependent on proper protein folding. These processing reactions occur in the Golgi apparatus. Modification reactions may involve the addition of a phosphate or acetyl group onto the sugars, or the addition of new sugars, such as neuraminic acid. Processing and modification of N-linked glycans within the Golgi does not follow a linear pathway. As a result, many different variations of N-linked glycan structure are possible, depending on enzyme activity in the Golgi.

Functions and importance

N-linked glycans are extremely important in proper protein folding in eukaryotic cells. Chaperone proteins in the endoplasmic reticulum, such as calnexin and calreticulin, bind to the three glucose residues present on the core N-linked glycan. These chaperone proteins then serve to aid in the folding of the protein that the glycan is attached to. Following proper folding, the three glucose residues are removed, and the glycan moves on to further processing reactions. If the protein fails to fold properly, the three glucose residues are reattached, allowing the protein to re-associate with the chaperones. This cycle may repeat several times until a protein reaches its proper conformation. If a protein repeatedly fails to properly fold, it is excreted from the endoplasmic reticulum and degraded by cytoplasmic proteases.

N-linked glycans also contribute to protein folding by steric effects. For example, cysteine residues in the peptide may be temporarily blocked from forming disulfide bonds with other cysteine residues, due to the size of a nearby glycan. Therefore, the presence of a N-linked glycan allows the cell to control which cysteine residues will form disulfide bonds.

N-linked glycans also play an important role in cell-cell interactions. For example, tumour cells make N-linked glycans that are abnormal. These are recognized by the

as a sign that the cell in question is cancerous.

Within the immune system the N-linked glycans on an immune cell's surface will help dictate that migration pattern of the cell, e.g. immune cells that migrate to the skin have specific glycosylations that favor homing to that site.^[3] The glycosylation patterns on the various immunoglobulins including IgE, IgM, IgD, IgE, IgA, and IgG bestow them with unique effector functions by altering their affinities for Fc and other immune receptors.^[3] Glycans may also be involved in "self" and "non self" discrimination, which may be relevant to the pathophysiology of various autoimmune diseases;^[3] including rheumatoid arthritis ^[4] and type 1 diabetes.^[5]

The targeting of degradative

) and CD-MPR (cation-dependent mannose-6-phosphate receptor).

O-Linked glycans

Introduction

In eukaryotes, O-linked glycans are assembled one sugar at a time on a serine or threonine residue of a peptide chain in the Golgi apparatus. Unlike N-linked glycans, there is no known consensus sequence yet. However, the placement of a proline residue at either -1 or +3 relative to the serine or threonine is favourable for O-linked glycosylation.

Assembly

The first monosaccharide attached in the synthesis of O-linked glycans is N-acetyl-galactosamine. After this, several different pathways are possible. A Core 1 structure is generated by the addition of galactose. A Core 2 structure is generated by the addition of N-acetyl-glucosamine to the N-acetyl-galactosamine of the Core 1 structure. Core 3 structures are generated by the addition of a single N-acetyl-glucosamine to the original N-acetyl-galactosamine. Core 4 structures are generated by the addition of a second N-acetyl-glucosamine to the Core 3 structure. Other core structures are possible, though less common.

Images:

https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.figgrp.561 : Core 1 and Core 2 generation. White square = N-acetyl-galactosamine; black circle = galactose; Black square = N-acetyl-glucosamine. Note: There is a mistake in this diagram. The bottom square should always be white in each image, not black.

https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.figgrp.562 : Core 3 and Core 4 generation.

A common structural theme in O-linked glycans is the addition of polylactosamine units to the various core structures. These are formed by the repetitive addition of galactose and N-acetyl-glucosamine units. Polylactosamine chains on O-linked glycans are often capped by the addition of a sialic acid residue (similar to neuraminic acid). If a fucose residue is also added, to the next to penultimate residue, a Sialyl-Lewis X (SLex) structure is formed.

Functions and importance

blood antigen determination.

SLex is also important to proper immune response. P-

of these cells into the surrounding tissue during infection.

O-linked glycans, in particular mucin, have been found to be important in developing normal intestinal microflora. Certain strains of intestinal bacteria bind specifically to mucin, allowing them to colonize the intestine.

Examples of O-linked glycoproteins are:

• cell membranes
• Mucin, a protein in saliva involved in formation of dental plaque
• Notch
  , a transmembrane receptor involved in development and cell fate decisions
• Thrombospondin
• Factor VII
• Factor IX
• Urinary type plasminogen activator

Glycosaminoglycans

Another type of cellular glycan is the

).

Glycoscience

A 2012 report from the

The report presents a roadmap for transforming glycoscience from a field dominated by specialists to a widely studied and integrated discipline.

Glycans and lipids

See

glycolipids

GPI-Anchors

See

glycophosphatidylinositol

Tools used for glycan research

The following are examples of the commonly used techniques in glycan analysis:[8]^[9]

High-resolution mass spectrometry (MS) and high-performance liquid chromatography (HPLC)

The most commonly applied methods are MS and HPLC, in which the glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis.^[10] In case of glycolipids, they can be analyzed directly without separation of the lipid component.

N-

A large variety of different labels were introduced in the recent years, where 2-aminobenzamide (AB), anthranilic acid (AA), 2-aminopyridin (PA), 2-aminoacridone (AMAC) and 3-(acetylamino)-6-aminoacridine (AA-Ac) are just a few of them.[12] Different labels have to be used for different ESI modes and MS systems used. ^[13]

O-

are usually analysed without any tags, due to the chemical release conditions preventing them to be labeled.

Fractionated glycans from

In recent years, high performance liquid chromatography online coupled to mass spectrometry became very popular. By choosing porous graphitic carbon as a stationary phase for liquid chromatography, even non derivatized glycans can be analyzed. Detection is here done by mass spectrometry, but in instead of

Multiple reaction monitoring (MRM)

Although MRM has been used extensively in metabolomics and proteomics, its high sensitivity and linear response over a wide dynamic range make it especially suited for glycan biomarker research and discovery. MRM is performed on a triple quadrupole (QqQ) instrument, which is set to detect a predetermined precursor ion in the first quadrupole, a fragmented in the collision quadrupole, and a predetermined fragment ion in the third quadrupole. It is a non-scanning technique, wherein each transition is detected individually and the detection of multiple transitions occurs concurrently in duty cycles. This technique is being used to characterize the immune glycome.[3]^[18]

Table 1:Advantages and disadvantages of mass spectrometry in glycan analysis

Advantages	Disadvantages
Applicable for small sample amounts (lower fmol range) Useful for complex glycan mixtures (generation of a further analysis dimension). Attachment sides can be analysed by tandem MS experiments (side-specific glycan analysis). Glycan sequencing by tandem MS experiments.	Destructive method. Need of a proper experimental design.

Arrays

Lectin and antibody arrays provide high-throughput screening of many samples containing glycans. This method uses either naturally occurring

, where both are immobilized on a certain chip and incubated with a fluorescent glycoprotein sample.

Glycan arrays, like that offered by the Consortium for Functional Glycomics and Z Biotech LLC, contain carbohydrate compounds that can be screened with lectins or antibodies to define carbohydrate specificity and identify ligands.

Metabolic and covalent labeling of glycans

Metabolic labeling of glycans can be used as a way to detect glycan structures. A well-known strategy involves the use of

. This method has been used for in vitro and in vivo imaging of glycans.

Tools for glycoproteins

, etc.).

See also

• Glycoside
• Glycoside hydrolase
• Glycosylation
• Glycosyltransferase

Resources

• National Center for Functional Glycomics (NCFG) The focus of the NCFG is the development in the glycosciences, with an emphasis on exploring the molecular mechanisms of glycan recognition by proteins important in human biology and disease. They have a number of resources for glycan analysis as well as training in glycomics and protocols for glycan analysis
• GlyTouCan, Glycan structure repository
• Glycosciences.DE, German glycan database
• Carbohydrate Structure Database, Russian glycan database
• UniCarbKB, Australian glycan database
• GlycoSuiteDB, glycan database by Swiss Institute of Bioinformatics
• GlyGen, NIH funded glycoinformatics resource
• The
  Nature Publishing Group
  .
• Transforming Glycoscience: A Roadmap for the Future Archived 2014-10-20 at the
  U.S. National Research Council
  's reports and workshops on glycoscience.

References

• Varki, Ajit; Cummings, Richard; Esko, Jeffrey; Freeze, Hudson; Hart, Gerald; Marth, Jamey, eds. (1999). Essentials of Glycobiology. Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press.
  ISBN 978-0-87969-559-0
  . NBK20709.

External links

• Emanual Maverakis; et al. (2015). "Glycans in the immune system and The Altered Glycan Theory of Autoimmunity". Journal of Autoimmunity. 57: 1–13.
  PMID 25578468
  .