Lecture 12 - N-glycosylation (ER) Flashcards
Why is glycosylation important?
Most proteins in secretory pathway have covalently added sugar.
Perturbations in the addition of correct sugars to a glycoprotein has dramatic consequences on biological function.
N-linked glycosylation is important in ER protein folding.
Addition of sugars are important in their trafficking and their function
What is the difference between alpha and beta anomers?
alpha anomers have the hydroxy group of the 1st carbon under the plane of the ring
What is the glycosyl transferase enzyme specific for?
Nucleotide sugar
Linkage formed
Where is it unlikely to find sugars facing in the plasma membrane?
Sugars are never found on the cytoplasmic face of the plasma membrane. They are typically found on the external surface or within the membrane.
What are some structures that proteins can bind to in membranes?
Some proteins are embedded into membranes by a covalently bound lipid anchor
What is the function of the transaminase enzyme?
Transaminase enzyme transfers protein from hydrophobic C-terminal peptide anchor of the plasma membrane to a glycolipid anchor.
- Not to be confused with the signal peptide which is found at the N-terminus. (proteins produced from C terminus to N terminus)
- This is the method by which GPI anchored proteins are attached to the cell membrane.
Does glycosylation happen post or co-translationally?
Glycosylation can happen post or co tranlsationally. N-linked glycosylation occurs cotranlsationally.
What happens to a presynthesized oligosaccharide during N-linked glycosylation?
preformed oligosaccharide is transferred from a dolichol lipid to asparagine residue of protein.
What is the consensus sequence for N-linked glycosylation?
-Asn.Xaa.Ser/Thr-
What are the first sugar molecules of the oligosaccharide added during N-linked glycosylation.
first monosaccharides of the oligosaccharide chain are the GlcNAc sugars.
What are the steps of N-linked glycosylation that occur in the ER?
1) Formation of a large precursor oligosaccharide linked to dolichol. This occurs via addition of 1 sugar at a time with different enzymes adding each sugar. Addition of the sugars occurs on the cytosolic side of the ER and then the oligosaccharide is flipped into the lumen by a flippase enzyme.
2) Oligosaccharide is transferred from dolichol to the asparagine residue of the protein. This is catalysed by a membrane bound oligosaccharide transferase complex.
3) Initial processing of N-linked oligosaccharides occurs in the ER.
What are the initial processing steps that occur in the ER?
1) Glucosidase 1 cleaves terminal glucose
2) glucosidase 2 cleaves terminal glucose twice
3) ER mannosidase cleaves the middle mannose.
How does glycosylation assist in protein folding?
1) Unfolded protein gets its terminal glucose subunits trimmed leaving only one glucose molecule.
2) Protein folds and then interacts with calnexin (a chaperone which ensures that only correctly folded proteins continue along the secretory pathway).
3) glucosidase then cleaves off the terminal glucose which can exit from the ER if properly folded.
4) Proteins that are incompletely folded go to a glucosyl transferase and are folded again.
5) UDP glucose is added for interaction with calnexin. And cycle occurs again from step 2.
What happens to proteins that never fold correctly in the ER?
They are first carried out of the ER via chaperones through an ER translocator.
They are then labelled with ubiquitin and broken down by the proteosome.
What can be said about the conserved nature of the N-glycosylation pathway?
Structure of dolichol-oligosaccharide precursor is the same in animals, plants, and yeast. The pathway is the same in the ER. However, the difference is seen when processing in the golgi occurs.
