Lorna Pierce. To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ To observe the effect of endomannosidase.

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Presentation transcript:

Lorna Pierce

To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ To observe the effect of endomannosidase activity on free oligosaccharides generated by protein misfolding To define the pathway for misfolded proteins between the ER, ERGIC and Golgi including Retrograde Transport

 -glc I  -glc II CNX/CRT GT EDEM Sec61 G3M5N1G3M5N1  -glc II Golgi ER lumen ER man I PNGase Proteasome Plasma membrane ER man I -N2M9G3-N2M9G3 -N 2 M 9 G 2 -N2M9G1-N2M9G1 -N2M9-N2M9 -N 2 M 8 -N 2 M 8 G 1-3 -N2M8-N2M8 ERAD Glycosylation maturation pathway EnGase & Cytosolic mannosidase

To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ

-N2M9G3-N2M9G3 -N 2 M 9 G 2 -N2M9G1-N2M9G1 CNX/CRT GT -N 2 M 8 EDEM Sec61 -N 2 M 8 G 1-3 ER lumen ER man I PNGase Proteasome ER man I -N2M8-N2M8 EnGase & Cytosolic mannosidase Endomannosidase Pathway NB-DNJ Inhibition in the Protein Folding Pathway G3M5N1G3M5N1

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 ? + NB-DNJ

Minutes G3M5N G3M7N2 G3M5N G3M7N2 M7N2 The Free Oligosaccharides Generated by the  - glucosidase blockage Caused by NB-DNJ 1mM NB-DNJ 24 hours 1mM NB-DNJ 24 hours, followed by 24 hours with no inhibitor

To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ To observe the effect of endomannosidase activity on free oligosaccharides generated by protein misfolding

Minutes FOS produced on treatment with NB-DNJ in Endomannosidase +/- cells Control Functional Endomannosidase (HL60) Non-utilised Endomannosidase (MDBK) G3M5NG3M7N2

In endomannosidase inactive cells the G3M8N2 constituent of a glycoprotein is cleaved to G3M7N2 by Golgi Mannosidase I or ER mannosidase II On re-entry to the ER, G3M7N2-containing proteins are acted upon by an ER located PNGase and G3M7N2 is released from its protein as a FOS The G3M7N2 FOS is not targeted for EDEM and hence becomes trapped within the ER G3M7N2 is a characteristic FOS of protein misfolding in endomannosidase inactive cells

To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ To observe the effect of endomannosidase activity on free oligosaccharides generated by protein misfolding To define the pathway for misfolded proteins between the ER, ERGIC and Golgi including Retrograde Transport

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 ? + NB-DNJ Thapsigargin / Brefeldin A

MDBK Cells Incubated with NB-DNJ

Mean G3M5N Mean G3M7N2 (pmol) per ug of protein Thapsigargin (µM) Triglucosylated FOS produced upon treatment with Thapsigargin

Effect of Thapsigargin on Protein Synthesis

MDBK Cells incubated with NB-DNJ Minutes G3M5N G3M7N2 G3M5N 1mM NB-DNJ 1mM NB-DNJ and 10µg/ml Brefeldin A

Mean G3M5N Mean G3M7N2 (pmol) per ug of protein Brefeldin µg/ml Triglucosylated FOS produced upon treatment with Brefeldin A

Effect of Brefeldin A on Protein Synthesis

Reduced levels of G3M7N2 following treatment with Thapsigargin and Brefeldin A indicate retrograde transport inhibition The reduction in G3M7N2 seen seems independent of effects on flux through the protein synthesis pathway caused by treatment with Thapsigargin and Brefeldin A Evidence for a cyclic pathway for glycoproteins between ER and Golgi compartments that is endomannosidase dependent

Dr T. Butters Dom Alonzi Dr D. Neville With Thanks to Prof Dwek

control + NB-DNJ