N-linked glycosylation
N-linked glycosylation, is the attachment of an oligosaccharide, a carbohydrate consisting of several sugar molecules, sometimes also referred to as glycan, to a nitrogen atom (the amide nitrogen of an asparagine (Asn) residue of a protein), in a process called N-glycosylation, studied in biochemistry.[1] The resulting protein is called an N-linked glycan, or simply an N-glycan.
This type of linkage is important for both the structure. Different species synthesize different types of N-linked glycan.
Energetics of bond formation
There are two types of bonds involved in a glycoprotein: bonds between the
The sugar
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).[4]
In animal cells, the glycan attached to the asparagine is almost inevitably
Biosynthesis
The biosynthesis of N-linked glycans occurs via 3 major steps:[4]
- Synthesis of dolichol-linked precursor oligosaccharide
- En bloc transfer of precursor oligosaccharide to protein
- Processing of the oligosaccharide
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, 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.[4] 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 a diverse range of N-glycan structures.[4]
Synthesis of precursor oligosaccharide
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[4] (one phosphate was originally linked to dolichol, and 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.
The precursor molecule, ready to be transferred to a protein, consists of 2 GlcNAc, 9 mannose, and 3 glucose molecules.
Phase I
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Steps |
Location
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Cytoplasmic side of ER
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At this point, the lipid-linked glycan is
translocated across the membrane making it accessible to enzymes in the endoplasmic reticulum lumen. This translocation process is still poorly understood, but it is suggested to be performed by an enzyme known as flippase . | |
Phase II | |
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Luminal side of ER
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Transfer of glycan to protein
Once the precursor oligosaccharide is formed, the completed glycan is then transferred to the nascent
- Asparagine must be located in a specific consensus sequence in the primary structure (Asn–X–Ser or Asn–X–Thr or in rare instances Asn–X–Cys).[5]
- Asparagine must be located appropriately in the three-dimensional structure of the protein (Sugars are polar moleculesand thus need to be attached to asparagine located on the surface of the protein and not buried within the protein)
- Asparagine must be found in the luminal side of the endoplasmic reticulum for N-linked glycosylation to be initiated. Target residues are either found in secretory proteins or in the regions of transmembrane protein that face the lumen.
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
Processing of glycan
N-glycan processing is carried out in endoplasmic reticulum and 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 the non-reducing end of the glycan.[4] 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
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 the cis-Golgi, a series of mannosidases remove some or all of the four mannose residues in α-1,2 linkages.[4] 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.
- High-mannose is, in essence, just two N-acetylglucosamines with many mannose residues, often almost as many as are seen in the precursor oligosaccharides before it is attached to the protein.
- Complex oligosaccharides are so named because they can contain almost any number of the other types of saccharides, including more than the original two N-acetylglucosamines.
- Hybrid oligosaccharides contain a mannose residues on one side of the branch, while on the other side a N-acetylglucosamine initiates a complex branch.
The order of addition of sugars to the growing glycan chains is determined by the substrate specificities of the enzymes and their access to the substrate as they move through
Enzymes in the Golgi
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:
Enzymes | Location within Golgi |
---|---|
Mannosidase I | cis-Golgi |
GlcNAc transferases | medial Golgi |
Galactosyltransferase and Sialyltransferase | trans-Golgi |
In archaea and prokaryotes
Similar N-glycan biosynthesis pathway have been found in prokaryotes and Archaea.[6] However, compared to eukaryotes, 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.[4]
Function
N-linked glycans have intrinsic and extrinsic functions.[4][7]
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.[8] 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.[8] Glycans may also be involved in "self" and "non self" discrimination, which may be relevant to the pathophysiology of various autoimmune diseases.[8]
Intrinsic | |
Extrinsic |
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In some cases, interaction between the N-glycan and the protein stabilizes the protein through complex electronic effects.[11]
Clinical significance
Changes in N-linked glycosylation has been associated with different diseases including rheumatoid arthritis,[12] type 1 diabetes,[13] Crohn's disease,[14] and cancers.[15][16]
Mutations in eighteen genes involved in N-linked glycosylation result in a variety of diseases, most of which involve the nervous system.[3][16]
Importance in therapeutic proteins
Many
The importance of N-linked glycosylation is becoming increasingly evident in the field of
Non-human mammalian expression systems such as
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 and even bacteria.
See also
References
- ^ "Glycosylation". UniProt: Protein sequence and functional information.
- PMID 10600722.
- ^ PMID 16584073.
- ^ ISBN 978-0-19-928278-4.
- PMID 9578569.
- PMID 21490701.
- ^ GlyGen. "GlyGen glycan structure dictionary". GlyGen. Retrieved 1 Apr 2021.
- ^ PMID 25578468.
- PMID 15959882.
- PMC 10830103.
- PMID 33723379.
- PMID 17392038.
- PMID 29146600.
- PMID 25895110.
- S2CID 254501034.
- ^ S2CID 11131196.
- S2CID 206548002.
- S2CID 38981893.
- PMID 25000187.
External links
- GlycoEP: In silico Platform for Prediction of N-, O- and C-Glycosites in Eukaryotic Protein Sequences
- Maverakis E, Kim K, Shimoda M, Gershwin ME, Patel F, Wilken R, Raychaudhuri S, Ruhaak LR, Lebrilla CB (February 2015). "Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review". Journal of Autoimmunity. 57: 1–13. PMID 25578468.