1. Imino sugars and perturbation of protein folding pathways in the ER Dom Alonzi

2. Imino Sugars N CH 2 OH OH OH HO Therapeutic roles Lysosomal storage disorders: Gaucher, Sandhoff Antiviral: HCV, HBV

3. ER Golgi Further Golgi Processing to Complex-type + Cytoplasm Proteosome  -Glc I & II Calnexin/ Calreticulin + Sec 61 Ceramide glucosyltransferase Plasma membrane < 1 min Chitobiase N-alkyl- DNJs N-alkyl- DNJs N-alkyl- DNJs N-alkyl-DNJs CeramideGlucosylceramide Endoman’ase GSLs The effect of imino sugars on N-linked oligosaccharide processing in the cell

4. Free oligosaccharide (FOS) generation - a normal cellular process Unconjugated polymannose-type oligosaccharides are, in the main, produced as a by-product of the proteasomal degradation of misfolded glycoproteins. A smaller proportion are produced during the dolichol pathway of N-linked glycosylation. These are segregated from their glycoprotein-linked counterparts and follow a non-vesicular trafficking pathway that leads to their degradation in the lysosome.

5.  -glucosidase I & II ATP  -glucosidase II Calnexin/calreticulin Glucosyl transferase Post ER modification -SH HS- -s-s- HS- ERp57 -s-s- NB-DNJ Disruption of glycoprotein folding in the ER

6. ER lumen Cytosol PNGase Chitobiase Cytosolic Mannosidase Proteasome Misfolded glycoprotein ? Sec61/Der-1 Free oligosaccharides produced by glycoprotein misfolding

7. FOS is produced following treatment of HL60 cells with 1mM NB-DNJ for 24hrs M4N M5N G1M5N G3M5N G3M6-9N1/2 Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00 Control 1mM NB-DNJ 24hrs G1M5N M5N

8. Build up of Mono-glucosylated FOS is time dependent [G1M5N] relative to [M5N]

9. Build up of Tri-glucosylated FOS is time dependent [G3M5N] relative to [M5N]

10. G3M5N build up is Butyl-DNJ concentration dependent [G3M5N] relative to [M5N]

11. N CH 2 OH OH OH HO N CH 2 OH OH OH HO Butyl-DNJ Vs Nonyl-DNJ Effect of chain length on glucosidase inhibition in the ER. Is it the result of differential inhibition of the enzymes or an access/localisation issue?

12. +  -glucosidase I Potential Inhibitor 2AA + 100%75%25% t= 30mins In Vitro Glucosidase inhibition assay

13. In vitro IC 50 determination IC 50 NN-DNJ= 0.54  M and NB-DNJ = 0.83  M

14. FOS production: Nonyl-DNJ vs Butyl-DNJ [G3M5N] relative to [M5N]

15. NB-DNJ and NN-DNJ treatment of MDBK cells shows differential inhibition of glucosidases Control 100µM NB-DNJ 72hrs 100µM NN-DNJ 72hrs G3M5N G3M7N2 Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00 Differential inhibition

16. Minutes 20.0024.0028.0032.0036.0040.00 44.00 Type of FOS produced on treatment with NB-DNJ is Endomannosidase dependent Control Functional Endomannosidase Non-utilised Endomannosidase G3M5NG3M7N2

17. ER ERGIC cis-Golgi + The Endomannosidase pathway Bulk flow or ERGIC-53 mediated Retrograde transport Cytosolic Mannosidase Endomannosidase Golgi Mannosidase I Further Golgi processing to Complex N-links ? PNGase in the ER or Cytosol or both?

18. Fate of FOS following long term 1mM NB-DNJ treatment of HL60 cells Control 1mM NB-DNJ 24hrs 1mM NB-DNJ 72hrs 1mM NB-DNJ 48hrs G3M5NG3M4N Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00

19. G3M5N is converted to G3M4N in long term recovery experiments in HL60s Control 1mM NB-DNJ 24hrs 1mM NB-DNJ 24hrs + 72hrs recovery 1mM NB-DNJ 24hrs + 96hrs recovery 1mM NB-DNJ 24hrs + 120hrs recovery G3M5NG3M4N Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00

20. Where? Enzyme? Fate of Glucosylated Man 5 FOS ? Most likely theory: Cytosolic Mannosidase Glucosylated FOS cannot gain access to the lysosome {Saint-Pol A et al JBC (1999)} lysosome

21. Mouse: G1M4N in serum is dependent on NB-DNJ dose Control 300mg/kg/day 1200mg/kg/day G1M4N Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00

22. G1M4N build up in serum following NB-DNJ treatment [G1M4N] relative to [M3N] (Serum concentration ~ 40  M)

23. Minutes 22.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00 20.00 Control Treated [Serum] = 40.8  M G1M4N is excreted in mouse urine following NB-DNJ treatment G1-3M7N2 G1M4N

24. FOS detected in serum from NPC patient treated with 100mg/day NB-DNJ Untreated (day 0) Treated (16 months) G3M4N G1M4N Minutes 20.0022.0024.0026.0028.0030.0032.0034.0036.0038.0040.0042.00

25. Lysosome ER Golgi PNGase Der-1/Sec61 Proteasome Endomannosidase Chitobiase Cytosolic mannosidase  -glucosidase I & II ? Retrograde transport Pathway for FOS clearance

26. Conclusions Characterised cellular FOS produced in the presence/absence of glucosidase inhibitors Demonstrated differential inhibition of glucosidases I and II by imino sugars FOS as biological markers - provide a simple cell based assay for glucosidase inhibitor efficacy and entry into the ER, and calnexin/calreticulin mediated protein folding Formation of glucosylated Man 4 species in cells, as a result of the cytosolic mannosidase, is the metabolic endpoint Glucosylated FOS, in serum and urine of mouse and human following NB- DNJ treatment, reveals pathway of clearance Detection of two PNGases in the cell: (i) ER lumen and (ii) cytosolic Trans-renal clearance of glucosylated FOS prevents adverse effects on normal cellular processes Endomannosidase influences the FOS pathway due to differential activities on protein folding states

27. Acknowledgements Dr David Neville Neidys Sanchez-Hernandez Dr Howard Mellor Mark Hussey Adam Pilling Dr Terry Butters Professor Raymond Dwek 1st floor