1. Analysis of N-linked Glycoprotein misfolding Derek Wan Christ Church

2. The N-glycoproteins folding pathway Step 1 Assembly of the precursor complex, Glc 3 Man 9 GlcNAc 2

3. The assembly of the precursor complex Flippase OST

4. The N-glycoproteins folding pathway Step 2 Transfer of Precursor onto the Asn-X-Thr motif on the emerging protein

5. The N-glycoproteins folding pathway Step 3 Trimming of the first two glucose residues by ER glucosidase I and II

6. The N-glycoproteins folding pathway Step 4 The protein enters the Calnexin/Calreticulum cycle

7. The Calnexin/Calreticulum cycle

8. The N-glycoproteins folding pathway Step 5A Secretion of Mature proteins to Golgi Step 5B Degradation of terminally misfolded proteins through ER- Associated Degradation (ERAD)

9. ER-Associated Degradation (ERAD)

10. Fate of Free Oligosaccharides (FOS)

11. Effect of NB-DNJ on the N-glycoprotein folding pathway

12. Project Aim To investigate the effect of NB-DNJ on the following glycan pool: 1. Lipid-linked oligosaccharides (Dolichol) 2. Intracellular and Extracellular FOS 3. Intracellular and Extracellular Protein-linked oligosaccharides (PLO) To investigate the amount of protein misfolding related to N- linked glycosylation in one individual HL60 cell, in 24 hours

13. Results and Discussion

14. R&D – Lipid Linked Oligosaccharides Species Detected Control 1mM NB-DNJ

15. R&D – Lipid Linked Oligosaccharides Quantitative Amount of oligosaccharides (pmol/mg of proteins) Standard Deviation Control Cells (without NB-DNJ) 3.17 1.62 Treated Cells (with 1mM NB-DNJ) 3.20 0.39

16. R&D – Intracellular Free Oligosaccharides Species Detected Control 1mM NB-DNJ

17. R&D – Intracellular FOS Quantitative Amount of oligosaccharide (pmol/mg of protein) Standard Deviation Control Cells (without NB-DNJ)180.915.71 Treated Cells (with 1mM NB-DNJ)509.4430.03 Cellular Fractionation To identify the origin of these intracellular FOS Result: 50% from Cytosol 10% from Lysosome The rest (40%) from other cell compartments

18. R&D – Extracellular Free Oligosaccharides In 24 hours, no FOS were detected in the extracellular medium by our methods Suggesting FOS were retained in the cells for the first 24 hours Next Question… How long does the cell retain the FOS before discharging them into the extracellular medium?

19. Control Cells Medium only 24 hours 36 hours 48 hours 72 hours 96 hours

20. 1mM NB-DNJ Medium only 24 hours 36 hours 48 hours 72 hours 96 hours

21. Conclusion In both control and treated cells, the cell retains FOS species for at least 72 hours 96 hours intracellular v.s. extracellular FOS R&D – Extracellular Free Oligosaccharides Does this suggest that the excretion system favours G3M4N over G3M5N??

22. R&D – Protein-linked Oligosaccarides (Intracellular) Species Detected Control 1mM NB-DNJ

23. R&D – Protein-Linked Oligosaccharides (Intracellular) Quantitative Amount of oligosaccharides (pmol/mg of proteins) Standard Deviation Control Cells (without NB-DNJ)42.7011.65 Treated Cells (with 1mM NB-DNJ)61.5544.51 BUT….

24. R&D – Protein-Linked Oligosaccharides (Intracellular) Quantitative (corrected) Amount of oligosaccharides (pmol/mg of proteins) Standard Deviation Control Cells (without NB-DNJ)128.111.65 Treated Cells (with 1mM NB-DNJ)184.6544.51

25. Bottled Medium were de-glycoproteinated by passage through a Concanavalin A (Con-A)-Sepharose 4B column PLO extracted as per normal Results: No PLO can be detected after 24 hours incubation (with and without 1mM NB- DNJ) Samples were concentrated by passage through an QAE-sephadex column Still, no PLO can be detected R&D – Protein-Linked Oligosaccharides (Extracellular) Does this suggest that no glycoproteins were secreted into the extracellular medium in 24 hours??

26. Effect of NB-DNJ on the N-glycoprotein folding pathway Control Cells1mM NB-DNJ LLO3 major + 1 minor species 3.17 pmol/mg 1 major species 3.20 pmol/mg Intracellular FOS3 major + numerous glucosylated species 180.91 pmol/mg Major species in control cells decreased in amount Major species of Glc 1 Man 5 GlcNAc 1 + minor peaks 509.44 pmol/mg Extracellular FOS Retain in the cell for at least 72 hours Intracellular PLONumerous species identified 128.1 pmol/mg Species in control cells still present, but decrease in amount 3 major glucosylated species identified 184.65 pmol/mg Extracellular PLO Cannot be detected with the methods used

27. Quantitative Measure of N-Glycoprotein misfolding Objective: To investigate the amount of protein misfolding related to N-linked glycosylation in one individual HL60 cell, in 24 hours Total Amount of proteins = sum of all detected oligosaccharides in different oligosaccharide pool Misfolded protein = Cytosolic FOS + Lysosomal FOS (60% of total FOS) Therefore…

28. Quantitative Measure of N-Glycoprotein misfolding Dolichol pool Total FOS (amount of cytosolic and lysosomal FOS) Glycoproteins*Total Amount Amount of oligosaccharides (pmol/mg) in Control cells 3.17180.91(108.54)128.1312.18 Amount of oligosaccharides (pmol/mg) in Treated cells 3.20509.44(305.66)184.65697.29 By applying these data to the equation… Control Cells 34.7% Treated Cells 43.8%

29. Conclusion Effects of NB-DNJ to: LLO – Affect glycan structure but not the amount Intracellular FOS – Affect both qualitatively and quantitatively Intracellular PLO – Affect glycan structure but not the amount Extracellular FOS & PLO – cannot be detected in 24 hours 34.7% of N-glycoproteins are degraded through ERAD by a single HL60 cells over 24 hours In the presence of 1mM NB-DNJ, the amount of N-glycoproteins degraded through ERAD increased to 43.8%