Figure 12–47 N-linked protein glycosylation in the rough ER. (a) almost as soon as a polypeptide chain enters the er lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide (shown in color) is attached only to asparagines in the sequences asnX-ser and asn-X-Thr (where X is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at too many sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. for many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus. (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme. one copy of this enzyme is associated with each protein translocator in the er membrane. (The translocator is not shown.) oligosaccharyl transferase contains 13 transmembrane α helices and a large er lumenal domain that contains its substrate-binding sites. The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol–oligosaccharide. The structure shown is of a prokaryotic homolog that closely resembles the catalytic subunit of the eukaryotic oligosaccharyl transferase. (pDB code: 3rCe.) oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. A special lipid molecule called dolichol anchors the precursor oligosaccharide in the ER membrane. The precursor oligosaccharide is transferred to the target asparagine in a single enzymatic step immediately after that amino acid has reached the ER lumen during protein translocation. The precursor oligosaccharide is linked to the dolichol lipid by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction (Figure 12–47B). One copy of oligosaccharyl transferase is associated with each protein translocator, allowing it to scan and glycosylate the incoming polypeptide chains efficiently.
The precursor oligosaccharide is built up sugar by sugar on the membrane-bound dolichol lipid and is then transferred to a protein. The sugars are first activated in the cytosol by the formation of nucleotide (UDP or GDP)-sugar intermediates, which then donate their sugar (directly or indirectly) to the lipid in an orderly sequence. Part way through this process, the lipid-linked oligosaccharide is flipped, with the help of a transporter, from the cytosolic to the lumenal side of the ER membrane (Figure 12–48).
All of the diversity of the N-linked oligosaccharide structures on mature glycoproteins results from the later modification of the original precursor oligosaccharide. While still in the ER, three glucoses (see Figure 12–47) and one mannose are quickly removed from the oligosaccharides of most glycoproteins. We shall return to the importance of glucose trimming shortly. This oligosaccharide “trimming,” or “processing,” continues in the Golgi apparatus, as we discuss in Chapter 13.