1. Free oligosaccharides as biological markers of endoplasmic reticulum-associated degradation and the role of endomannosidase.
I admit that the title of my project it is a bit cumbersome, and so for the purpose of this presentation ‘free oligosaccharides’ will abbreviated to ‘FOS’ and endoplasmic reticulum associated degradation as ‘ERAD’. The context of this project is within the field of protein folding and so the next few slides will be a v brief tour of protein folding in the ER…
Emily Dennis - Trinity College, Oxford.

2. N-linked glycosylation
1. Dolichol-pyrophosph-ate carries G3M9N2 (lipid-linked oligosaccharide).
CYTOSOL
ER-membrane 3.
ER-LUMEN
2. Glycosylation recognition sequence = Asn-X-Ser/Thr on nascent polypeptide, detected by OST.
Protein folding is aided by N-linked glycosylation. This increases protein solubility and so decreases the likelihood of aggregation. Oligosaccharide in the form of 3Glc, 9Man and 2GlcNAc(represented as triangles, circles and squares respectively) is transferred from a dolichol carrier to a specific a. a motif (asparagine-X-Ser/Thr) on the nascent polypeptide.
4. Polypeptide released from ribosome and N-linked oligosaccharide is subject to processing which facilitates protein folding.

3. The Calnexin/Calreticulin cycle
4. Glucosyl-transferase senses folding state and reglucosylates misfolded proteins.
3. Glucosidase II cleaves 3rd Glc residue. Protein dissociates from CNX/CRT.
2. G1M9N2 is recognition for CNX/CRT.
1. Glucosidase trimming.
Once the polypeptide has been released from the ribosome, the oligosaccharide unit undergoes further processing by glucosidases. This facilitates interactions with lectins, conventionally CNX/CRT. These aid protein folding by bringing them into association with components such as ERp57…which catalyzes disulphide bind formation until the protein is correctly folded and can enter the secretory pathway. However if the protein is terminally misfolded it is targeted in a time-dependent manner for degradation in the cytosol in the process of ERAD.
5. ER- mannosidase trims oligosaccharide in a time-dependent manner if persistently misfolded.
6. EDEM binds the de-mannosylated misfolded substrate and protein is transported to the cytosol.

4. Endoplasmic reticulum-associated degradation (ERAD): generation of FOS
1. Alongside EDEM, Yos9p plays role in binding misfolded proteins via a MRH domain: stabilizes the protein which is then translocated to cytosol.
2. FOS produced when oligosaccharide chain cleaved by PNGase enzyme.
4. FOS converted from GlcNAc2 to GlcNAc1 species by ENGase action.
3. Protein degradation via Ub-dependent 26S proteosome pathway.
Once in the cytosol, the oligosaccharide unit is cleaved by a PNGase enzyme and released as FOS. The FOS is subject to further trimming and finally removed by the lysosome; on the other hand, the protein is degraded via the Ub-dependent 26S proteosome pathway.
Interestingly, if entry into the CNX/CRT cycle is blocked, mature glycoproteins are still been secreted which suggests that there is an alternative quality control pathway…
6. However: Glc-FOS cannot be discarded via lysosome. Fate still unknown.
5. Trimmed FOS removed in lysosome.

5. Alternative quality control pathwayX X X
NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2
Folded:  secretory pathway
? Misfolded:  shuttled back into ER?
This alternative pathway involves a Golgi enzyme called endomannosidase. This pathway can be induced by blocking entry to the CNX/CRT cycle by the imino sugar inhibitor NB-DNJ, which prevents the removal of glucose residues from the bound oligosaccharide. The enzyme cleaves the a-1,2-mannosidic bond shown by the green arrow, releasing the tetrasaccharide G3M. A 2nd endomannosidase product M7N2 is also detected. The N2 species being indicative of ER localization. Enzymes capable of cleaving FOS from protein have only been detected in the cytosol and the ER, not the Golgi and so somehow the protein must return to the ER from the Golgi network. This has led to the hypothesis that endomannosidase could also play a role in chaperoning misfolded proteins from the Golgi, back to the ER. In the light of al this the aims of my project were as follows…

6. Show that FOS are markers for protein misfolding (ERAD)
Project Aims
Show that FOS are markers for protein misfolding (ERAD)
Determine the origin of FOS (protein-linked vs. dolichol lipid-linked)
1st: I proposed that the inhibition of glucosidases by NB-DNJ would decrease entry into CNX/CRT. Misfolding would increase and therefore so would ERAD. I aimed to show that, as a product of ERAD, FOS were suitable markers for such degradation. This is not a new idea, however would provide supporting evidence to existing ideas.
2nd:The first claim is assuming that FOS are derived from misfolded glycoprotein but there is little evidence to suggest that they couldn’t also be derived from glycolipids (such as the original dolichol carrier). So I sought to extract FOS from both proteins and lipids under protein synthesis inhibition with puromycin.
3rd: I aimed to investigate the role of endomannosidase (as both enzyme and hypothesized chaperone) in protein quality control, using the technique of RNAi which is a technique not greatly explored in this area.
Investigate the role of endomannosidase using RNAi

7. 1&2. Methods: FOS extraction
Ion-exchange (mixed bed) chromatography and 2-AA labelling
24hr incubation
+/- 1mM NB-DNJ
Affinity chromatography (ConA)
eluate
wash
Isolation of dolichol-LLO in chloroform:methanol
Large FOS (e.g. Man5GlcNAc1, Glc3Man7GlcNAc2)
Small FOS (e.g. Glc1-3Man)
Purified by ion-exchange chromatography
HPLC
FOS were extracted from cells after growth in the presence or absence of 1mM NBDNJ, then harvested. The cell homogenate was subjected to ion-exchange chromatography, fluorescently labelled and further purified by affinity chromatography using ConA. The small frees, such as the predicted G3M endomannosidase product, do not bind ConA and so the wash from the column was also collected and analysed. The final fractions were run on HPLC and the profiles used to interpret the FOS species and their relative amounts. Similarly for the detection of lipid linked oligosaccharides, the same principles were applied except the extraction was achieved in organic solvents and the oligosaccharide cleaved by acid hydrolysis as not already existing as free oligosaccharide.
Oligosaccharides released by acid cleavage. Further purified by ion exchange and 2-AA labelled

8. Results: Effect of NB-DNJ treatment on FOS production in HL60smV 50
100
150
200
Minutes 20 22 24 26 28 30 32 34 36 38 40 42
M4N
M5N
G1M5N
Control mV 50
100
150
200
Minutes 20 22 24 26 28 30 32 34 36 38 40 42
G3M5N
NB-DNJ treated
These results show the HPLC chromatograms from FOS extraction in HL60s: both control cells (in black) and NBDNJ treated cells (in red). The presence of the glucosylated frees, indicates that successful glucosidase inhibition has occurred. The total amount of FOS can be determined by calculating the total peak areas. When the 2 were compared it was found that FOS produced under NBDNJ treated was nearly x3 greater than in the controls. As a product of ERAD, this increase shows that the amount of substrate passing through ERAD is increasing.
But were these FOS originating from glycoprotein?
FOS produced under NB-DNJ treatment ~ x3 greater than controls
Increased FOS = increased ERAD activity

9. Results: Effect of inhibiting protein synthesis on FOS production in HL60s
Puromycin  large, progressive decrease in FOS with increasing [puromycin] (0-4 mg ml-1).
Controls: %  27%  11%
1mM NB-DNJ: 100%  7%  4%  3%
~maximum inhibition achieved with 4mg ml-1 puromycin concentration
Adding a protein synthesis inhibitor sought to test this out. Maximum inhibition was found to be achieved at concentrations of 4ug/ml. The major FOS species, being M5N in the case of controls and G3M5N under NBDNJ treatment, were monitored. Problems with protein assay sensitivity has meant that I haven’t been able to get absolute figures for the amount of FOS and so the results presented only truly reflect relative amounts. However, despite, this a trend is still clearly visible. The FOS levels dropped dramatically with puromycin present. The next question is ‘was this the same in the case of the LLO??

10. Results: Effect of inhibiting protein synthesis on LLO-FOS production
1mM NB-DNJ treated cells, FOS levels remain same +/-puromycin
control cells: ~50% increase in LLO-FOS (+puromycin)
? pool of dolichol increases to compensate for lack of protein for oligosaccharide transfer.
? enzymes involved in dolichol synthesis appear not to be affected by protein synthesis inhibition.
A very different result was obtained from the dolichol-LLO experiment. The FOS investigated in this case was G3M9N2. Under 1mM NBDNJ the FOS levels with/without puromycin remain the same within experimental error. In the control cells, however, there was an almost 50% increase in FOS levels! It is possible that the pool of dolichol increases to compensate for the lack of protein. However, this would also suggest that the enzymes involved in dolichol synthesis are not affected by the addition of puromycin. Further experiments investigating protein levels, turnover rates and their activity is therefore essential before a satisfactory conclusion can be made about this observed increase. Nevertheless…puromycin certainly didn’t cause a decrease in LLO and so it is more than likely that the FOS observed in my experiments are cleaved from misfolded glycoprotein undergoing ERAD.

11. Results: Investigating endomannosidase function
Controls: MDBK (left); RAW (right) 50 mV 10 20 30
Minutes 7 8 9 11 12 13 14 15 16 17 18 19 40 1 22 25 27 32 35 37 42 45
NB-DNJ treated: MDBK (left); RAW (right) mV 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45 50 2
Minutes mV 10 20 30 40 50
Minutes 7 8 9 11 12 13 14 15 16 17 18 19
G3M
Moving on to the investigation into endomannosidase function…the same method of FOS extraction was applied to 2 adherent cells lines: MDBK (non-catalytically active enzymes) and RAW (functional endomannosidase). Adherent cells were essential for my RNAi protocol and so this is why I no longer used HL60 as my endomannosidase-functional cell line
The test for endomannosidase catalytic function is the detection of the small FOS: G3M. In both sets of controls and the NBDNJ treated MDBK cells, this was not detected. However a separate peak at ~17.6 min appeared with the RAW cells. This has been previously identified (by previous work in this building) as G3M. I had hoped to confirm its identity with glucosidase digests, however the data has not been included because 100% digestion wasn’t achieved with my enzyme. Therefore further digest experiments are needed before complete confidence can be put in the identity of this species. However, in agreement with previously published work, it is more than likely to represent G3M and thus confirms that fact that endomannosidase in RAW is catalytically functional.
MDBK: endomannosidase NOT catalytically active
RAW: catalytically function enzyme  G3M detected

12. Results: Investigating endomannosidase function cont’d
Controls: MDBK (left); RAW (right) mV 50
100
150
200
250 20 22 24 26 28 30 32 34 36 38 40 42
Minutes
-50
300 1 10 12 14 16 18 45
M5N
G1M5N
M4N
NB-DNJ treated: MDBK (left); RAW (right) 50
100
150
200
250
Minutes 20 22 24 26 28 30 32 34 36 38 40 42
G3M7N2 10 12 14 16 18 45
-50 2
G3M5N mV
When analysing the results from the large FOS extraction, the pattern was very similar to that achieved with HL60s. However at ~40mins a peak correlating to G3M7N2 was detected firstly in the MDBK cells but then with RAWs.. This confirmed the observation that has led to the hypothesis that endomannosidase could also have a chaperone function. However, also suggests endomannosidase isn’t as catalytically efficient in RAWs as in HL60s. When, comparing the peak areas, the relative amount of G3M7N2 in RAW is much smaller than with MDBKs and because the small FOS G3M was detected I still conclude that endomannosidase is functional in RAWs. I then attempted to knockdown enzyme activity by RNAi.
G3M7N2: FOS in ER Does endomannosidase play role in shuttling FOS Golgi  ER?
G3M7N2: endomannosidase inefficiency
G3M7N7RAW < G3M7N2MDBK

13. 2 endomannosidase-specific siRNAs
3. Methods: RNAi
2 endomannosidase-specific siRNAs
Negative control: no siRNA introduced
Positive, non-specific control: siRNA targeted against ubiquitously expressed protein kinase MAPK1
Non-silencing control: siRNA with no known homology, labelled with Alexa Fluor 488
RNAi is a gene silencing technique and relies on siRNAs to cause mRNA degradation of a target gene transcript. The target gene in this case was endomannosidase and I used 2 specific siRNAs against it. My negative control was my RAW cells with no siRNA added. In addition I had a positive control which seeks to achieve a high level of knockdown against a ubiquitously expressed target gene, in this case MAPK1 (on the recommendation of the manufacturers). This should however have no effect on my target gene, but confirm the success of the technique. Finally I had a non-silencing control which was a siRNA with no known homology labelled with a flurophore which confirm successful transfection of the cells.
Despite all this, the experiment has a few limitations. In particular, I have been unable to successfully determine the degree of knockdown quantitatively. I aimed to quantify mRNA levels by RT, RT-PCR. However accurate results eluded me due to issues with contamination. If more time had allowed I hope I may have succeeded in this (then again I may have not!!) Also there is high potential, non-specific effects.
The results of my transfections are as follows:
Limitations: Unsuccessful quantitative RT-PCR attempts for knockdown confirmation
Potential for non-specific, off-target effects

14. Results: Non-silencing control  transfection efficiency of siRNAs
1. Non-transfected overlay
2. Transfected overlay
The fluorescence microscopy pictures show that successful transfection occurred. On the left are the negative control cell and on the right are the cells transfected with my non-silencing control. The areas of bright fluorescence indicate transfection. Again, this is limited by the lack of exact quantification of what proportion of my cells did receive the siRNA.
As G3M is a specific product of endomannosidase, I first looked at the effect of siRNAs on this…
No bright spots detected in negative control cells
Bright spots of fluorescence (A495nm) = successful transfection of siRNA into RAWs
Limitations: Very difficult to get accurate quantitation of transfection efficiency.

15. Results: Effect of siRNA on G3M levels
Prediction: G3M in controls < 1mM NB-DNJ treated.
 G3M decrease (siRNAs 1&2)
 No effect with MAPK
G3M levels in controls < NB-DNJ treated cells
Inconsistent results with siRNAs 1&2 probe2 = more effective.
G3M decrease with MAPK siRNA
I predicted that G3M would be at a higher level under NBDNJ treatment and G3M should decrease if endomannosidase was successfully knocked down. If an effective control…no effect should be detected with the MAPK.
G3M levels in NBDNJ treated RAWs were ~x10 greater than in controls, however the effects with the siRNAs were far from straight forward. siRNA2 produced a decrease under both conditions and so was deemed a more effective probe than siRNA1. However, MAPK siRNA also produced decreases; most significantly, a 71% decrease in G3M levels under NBDNJ treatment. I then went on to look at M5N levels…

16. Results: Effect of siRNA on M5N levels
M5N is common product and unrelated to endomannosidase.
M5N is unrelated to endomannosidase and therefore shouldn’t be massively affected by the introduction of siRNAs. Levels were expected, as observed, to be lower in NBDNJ treated cells because the major species are likely to be glucosylated because of the inhibition. Here, variable decreases were observed with all the siRNAs, MAPK included! Overall: No consistent effect. The 3rd FOS investigated was G3M7N2, relating to the proposed chaperone function of endomannosidase…
Decreases observed with all probes, e.g. 4% siRNA1 and 57% MAPK.
Similar variable decrease in M5N levels under NB-DNJ treatment e.g. 49% siRNA1 but 27% MAPK.
Overall:
No consistent effect.

17. Results: Effect of siRNA on G3M7N2 levels
Prediction:
 Decrease in G3M7N (siRNAs 1&2)
 No change with MAPK
G3M7N2 = endomannosidase product detected in MDBKs.
siRNA1  19% decrease
siRNA2  63% decrease
However…
MAPK  50% decrease
Here, it was predicted that G3M7N2 levels would decrease if endomannosidase was successfully knocked down. But MAPK shouldn’t induce any effects. Decreases were observed with the specific probes: 19%, siRNA1 and 63% siRNA2. yet again…decreases were induced with the MAPK siRNA as well.
This must lead to the question: does the MAPK control siRNA itself directly affect endomannosidase? Or is the technique of RNAi generally causing widespread effects? The level of knockdown, as already mentioned, has not been determined due to unsuccessful RT-PCR attempts. This problem must be overcome; maybe using Northern blotting instead of RT-PCR. Also much evidence has been published indicating off-target effects. These include apoptosis, translation inhibition, protein binding and IFN-mediated signalling cascade induction (mainly because dsRNA is a common viral intermediate). Therefore, further experiments such as gene expression profiling to detect changes in mRNA on a global scale could provide a better insight into what’s going on. In addition, W blot analysis could provide more information on the effect on proteins. Still, these effects may be cell line or reagent specific and so running the same experiment in different cell lines and using different transfection reagent may shed some light on these issues. Nonetheless, however weakly, the siRNA did result in a decrease in G3M and G3M7N2 and so they don’t necessarily go against the ideas of endomannosidase function.
MAPK results  Is the MAPK siRNA directly effecting endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

18. FOS are suitable biological markers for ERAD.
Conclusions
FOS are suitable biological markers for ERAD.
FOS are derived from glycoproteins rather than dolichol lipids.
RNAi technique not optimized to make strong conclusions about the function of endomannosidase Further investigation essential.
Overall, what are the conclusions.
1st: The increase of FOS under NBDNJ treatment have shown that they are suitable marker for ERAD and protein misfolding.
2nd: the puromycin expts have shown conclusively that FOS originate from glycoprotein and not glycolipid
3rd: RNAi technique is not optimized to make strong conclusion about the function of endomannosidase and so further investigation is essential!!

19. References Diagrams (in order of appearance) taken and adapted from:
Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S; Neville, D.C.A; Butters, T.D)
Annual Review of Biochemistry, Vol. 73 pg : Roles of N-linked glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)
Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)
The EMBO Journal, Vol 16 pg : The solution NMR structure of glucosylated N-glycans involved in early stages of glycoprotein biosynthesis and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S; Platt. F.M; Dwek, R.A; Wormald, M.R)
These are the refs relating to the diagrams used.

20. Acknowledgements I would like to thank: Dr Terry Butters
Dominic Alonzi
Dr David Neville, Gabriele Reinkensmeier, Stephanie Boomkamp
Dr Steve Woodhouse, Dr Narayan Ramamurthy
Finally, I would like to thank
Terry, for providing this project and his support throughout
Dom for his supervision
Dave, Gabi and Steph for their lab expertise
Steve, Narayan and others on the 2nd floor for their assistance and use of their equipment during my freq, unsuccessful attempts at PCR!!
Thanks very much for listening.