The biosynthesis of
N-linked glycans occurs via three major steps: • 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. 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.
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 (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. 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 a protein, consists of two GlcNAc, nine mannose, and three
glucose molecules.
Transfer of glycan to protein 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 a glycan is transferred to a nascent polypeptide: • 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). • Asparagine must be located appropriately in the three-dimensional structure of the protein (Sugars are
polar molecules and 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. 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 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 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. • 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
secretory pathway. Thus, the organization of this machinery within a cell plays an important role in determining which glycans are made.
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:
In archaea and prokaryotes Similar
N-glycan biosynthesis pathway have been found in prokaryotes and Archaea. 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. == Function ==