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Stress and Responses to the Environment

In our lectures we have discussed that reglucosylation of the protein-linked oligosaccharide
mannose9-N-acetylglucosamide2 (M9N2, M = mannose, N = N-acetylglucosamine) by UDPglucose glycoprotein glucosyltransferase (UGGT) using UDP-glucose as glucose donor can
trigger a second round of interaction of a folding protein with the lectin chaperones calnexin
and calreticulin. We have also discussed that comparatively slow demannosylation of proteinlinked M9N2 to successively demannosylated species such M8N2, M7N2, M6N2, and M5N2, are
superimposed over the deglucosylation-reglucosylation cycle and ultimately targets slowly
folding glycoproteins to the ER-associated protein degradation machinery.
In this summative assessment you will analyse and interpret some of the experimental evidence
for this model of the calnexin/calreticulin cycle.
1. Figure 1 shows the profile of protein-linked oligosaccharides that were radioactively
labelled in vivo by incubation of rat liver cells (Figure 1A), common bean seedlings (Figure
1B), or yeast cells (Figure 1C) for ~20 min with [U14C]-D-glucose (U stands for
‘universal’, meaning that the radioactive isotope 14C of carbon has been incorporated
equally into all six positions of the carbon atom in the glucose molecule).
After lysis of cells, protein-linked oligosaccharides were released from proteins by
digestion with endoglycosidase H (Endo H). Release of oligosaccharides from proteins
with Endo H leaves the terminal N-acetylglucosamine on the protein. For example, for the
protein-linked oligosaccharide G3M9N2, Endo H will release the oligosaccharide G3M9N,
for the protein-linked oligosaccharide M9N2 Endo H will release M9N, and for proteinlinked M8N2 Endo H will release M8N.
The released oligosaccharides were then separated by a chromatographic technique, and
their carbohydrate (sugar) composition identified by comparing their migration during the
chromatography to standards of known carbohydrate composition and structure (Figure 1).
a) Based on the data shown in Figure 1, which cells show incorporation of [U14C]-
glucose into the protein-linked oligosaccharide glucose1-mannose9-Nacetylglucosamine2 (G1M9N2, G = glucose, M = mannose, N = Nacetylglucosamine) (10 % of the marks)?
b) For yeast cells (Figure 1C), how may you explain the formation of radioactively
labelled M9N and M8N when the cells are labelled with [U14C]-glucose (10 % of
the marks)?
2. Further investigation shows that all of the radioactivity in G1M8N and G1M7N, where
synthesised (Figure 1) resides in the glucose residue; no radioactivity is found in any of the
mannose residues or the N-acetylglucosamine residue. Two explanations for formation of
G1M8N2 and G1M7N2 are proposed:

To distinguish between these two possible explanations, rat liver microsomes [Microsomes
are vesicles isolated from both the endoplasmic reticulum and the Golgi complex. They
contain all the enzymes for protein glycosylation] were labelled with UDP-[U14C]-glucose
under conditions that fully inhibit transfer of oligosaccharides from the lipid-linked
precursor G3M9N2-P-P-dolichol (P = phosphate group) onto proteins (i.e. in the absence of
detergents). Protein-linked oligosaccharides were then released by digestion with Endo H
and chromatographically separated (Figure 2A). The experiment was repeated in the
presence of increasing concentrations of oligosaccharides of mannose, which act as
inhibitors of α1,2-mannosidases (Figures 2B, C).
Discuss how the data shown in Figure 2 support either mechanism i., mechanism ii., both
mechanisms, or none of the two mechanisms (25 % of the marks).
3. Figure 1 shows evidence that the protein-linked oligosaccharide G1M9N2 exists. In the
calnexin/calreticulin cycle this protein-linked oligosaccharide (G1M9N2) is deglucosylated
to terminate the interaction with calnexin/calreticulin.
Discuss how the data shown in Figure 3 either support the hypothesis that protein-linked
G1M9N2 can be deglucosylated to M9N2, do not support this hypothesis (i.e. support the
hypothesis that G1M9N2, once formed, is stable, and cannot be deglucosylated), or are
inconclusive with regard to distinguishing between these two hypotheses. How might the
red curve look if it would support the alternative hypothesis (i.e. the hypothesis that you
decided that has been ruled out by the data in Figure 3)? (25 % of the marks).
4. Degradation of the protein-linked oligosaccharide M8N2 to species with a lower mannose
content such as M7N2, M6N2, and M5N2, is thought to terminate the cycling of a
glycoprotein in the calnexin/calreticulin cycle and to target the affected glycoprotein to the
ER-associated protein degradation (ERAD) machinery. It is now very worthwhile to compare the information from question 3, as well as some information from the formative
assessment, with the stability of protein-linked M8N2.
To do this we determine the half-life of protein-linked M8N2 by labelling cells with
radioactive [2-
H]-mannose for 10 min, and then incubating them in a vast excess of nonradioactive mannose for another 10, 20, or 40 min. Protein-linked oligosaccharides are then
released with Endo H, separated by gel filtration, and the radioactivity of individual
fractions is measured and plotted against the fraction number (Figure 4).
Are the half-lives of protein-linked M8N2 and protein-linked G3M9N2 (determined in the
formative assessment) consistent with the model of the calnexin/calreticulin cycle or not?
Explain your answer (30% of the marks).