Question

5. Many proteins are glycosylated via 0- or N-linked sugars. Describe N-linked protein glycosylation and its processing. 3 marks

Answer 1

N-linked glycosylation starts in rough endoplasmic reticulum (RER) as a co-translational process while O-linked glycosylation begins and gets completed in Golgi apparatus (GA). N-linked glycosylation occurs on asparagine (N) residues in sequence N-X-S/T (S - Serine, T - Threonine; X can be any amino acid except Proline or Aspartic acid). N-linked glycosylation begins with transfer of a 14 sugar residue moiety when the translating polypeptide enters in RER lumen. The resides present in this moiety are as follows - 2 n-acetylglucosamine + 9 mannose + 3 glucose. All the sugar added are nucleotide sugars i.e... they are part of nucleotides before being donated to form the oligosaccharide.

The synthesis of the oligosaccharide begins at the cytoplasmic face of RER and completes in the lumen of RER. At the cytoplasmic surface 2 molecules of n-acetylglucosamine are added to the phosphate group of dolichol to which 5 mannose residues are added one by one. Both the processes are energy dependent, energy is extracted in the form breakage of phosphodiester bonds. A total 7 residues are added in the cytoplasmic face after which the moiety is flipped to lumen side. In lumen of RER, 4 more mannose resides are added before adding 3 glucose residues. The oligosaccharide is transferred by severing the bond between first n-acetylglucosamine and phosphate group of dolichol pyrophospate, this reaction is catalyzed by glycosyl transferases.

The N-linked glycosylation plays important role in determining the proper folding of glycoproteins. For proper folding, glucosidase removes two of the last glucose residues. The last glucose is required for the proteins attachment to Calnexin (or Calreticulin) which holds the protein to allow and determine proper folding. In case of proper folding, Glucosidase-ll removes the last glucose reside. In case of improper folding, UDP-glucose:glycoprotein glucosyltransferase adds back a single glucose molecule to direct the protein into second round of folding.

After proper folding is achieved the protein is transported to Golgi in COP-ll coated vesicles. In Golgi, the oligosaccharide branch can be remodeled based on the type of protein and its target. There are variety of glycosidases and glycosyltransferases present in Golgi which adds or removes residues off the protein.