The N-linked oligosaccharides are by far the most common oligosaccharides, being found on 90% of all glycoproteins. Less frequently, oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine, or hydroxy-lysine amino acid. A first sugar of these O-linked oligosaccharides is added in the ER and the oligosaccharide is then further extended in the Golgi apparatus (see Figure 13–32).
lipid bilayer of ER membrane
Figure 12–48 Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane. The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol (a polyisoprenoid; see panel 2–5, pp. 98–99). Dolichol is long and very hydrophobic: its 22 five-carbon units can span the thickness of a lipid bilayer more than three times, so that the attached oligosaccharide is firmly anchored in the membrane. The first sugar is linked to dolichol by a pyrophosphate bridge. This high-energy bond activates the oligosaccharide for its eventual transfer from the lipid to an asparagine side chain of a growing polypeptide on the lumenal side of the rough er. as indicated, the synthesis of the oligosaccharide starts on the cytosolic side of the er membrane and continues on the lumenal face after the (man)5(Glcnac)2 lipid intermediate is flipped across the bilayer by a transporter (which is not shown). all the subsequent glycosyl transfer reactions on the lumenal side of the er involve transfers from dolichol-p-glucose and dolichol-pmannose; these activated, lipid-linked monosaccharides are synthesized from dolichol phosphate and UDp-glucose or GDp-mannose (as appropriate) on the cytosolic side of the er and are then flipped across the er membrane. Glcnac = N-acetylglucosamine; man = mannose; Glc = glucose.
oligosaccharides are Used as Tags to mark the state of protein folding
It has long been debated why glycosylation is such a common modification of proteins that enter the ER. One particularly puzzling observation has been that some proteins require N-linked glycosylation for proper folding in the ER, yet the precise location of the oligosaccharides attached to the protein’s surface does not seem to matter. A clue to the role of glycosylation in protein folding came from studies of two ER chaperone proteins, which are called calnexin and calreticulin because they require Ca2+ for their activities. These chaperones are carbohydrate-binding proteins, or lectins, which bind to oligosaccharides on incompletely folded proteins and retain them in the ER. Like other chaperones, they prevent incompletely folded proteins from irreversibly aggregating. Both calnexin and calreticulin also promote the association of incompletely folded proteins with another ER chaperone, which binds to cysteines that have not yet formed disulfide bonds.
Calnexin and calreticulin recognize N-linked oligosaccharides that contain a single terminal glucose, and they therefore bind proteins only after two of the three glucoses on the precursor oligosaccharide have been removed during glucose trimming by ER glucosidases. When the third glucose has been removed, the glycoprotein dissociates from its chaperone and can leave the ER.