Lorna Pierce. To generate free oligosaccharides characteristic of protein misfolding due to...
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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 IICNX/CRT
GT
EDEMSec61
G3M5N1
-glc II
Golgi
ER lumen
ER man I
ER man I
PNGase
Proteasome
Plasma membrane
ER man I
-N2M9G3 -N2M9G2 -N2M9G1
-N2M9
-N2M8
-N2M8G1-3
-N2M8
ERAD
Glycosylation maturation pathway
EnGase & Cytosolic mannosidase
• To generate free oligosaccharides characteristic of protein misfolding due to treatment with NB-DNJ
-N2M9G3 -N2M9G2
-N2M9G1
CNX/CRT
GT
-N2M8
EDEMSec61
-N2M8G1-3
ER lumen
ER man I
ER man I
PNGase
Proteasome
ER man I
-N2M8
EnGase & Cytosolic mannosidase
Endomannosidase Pathway
NB-DNJ Inhibition in the Protein Folding Pathway
G3M5N1
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
Minutes20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00
G3M5NG3M7N2
G3M5N
G3M7N2
M7N2
The Free Oligosaccharides Generated by the -glucosidase blockage Caused
by NB-DNJ1mM 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
20.00 24.00 28.00 32.00 36.00 40.00 44.00
FOS produced on treatment with NB-DNJ in Endomannosidase +/- cells
Control
Functional Endomannosidase (HL60)
Non-utilised Endomannosidase (MDBK)
G3M5N G3M7N2
• 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
Minutes
12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00
G3M5N
G3M7N2
G3M7N2
G3M5N
1mM NB-DNJ
1mM NB-DNJ and 10M Thapsigargin
0
0.2
0.4
0.6
0.8
1.0
1.2
0 0.01 0.1 1 10 20
Mean G3M5N
Mean G3M7N2
(pm
ol)
per
ug
of
pro
tein
Thapsigargin (µM)
Triglucosylated FOS produced upon treatment with Thapsigargin
Thapsigargin + NBDNJ
-2 -1 0 125
50
75
100
48h +
24h +
6h +
4h +
2h +
1h +
log [Thapsigargin µM]
% o
f co
ntr
ol
leu
cin
e i
nco
rpo
rati
on
Effect of Thapsigargin on Protein Synthesis
MDBK Cells incubated with NB-DNJ
Minutes
12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00
G3M5NG3M7N2
G3M7N2
G3M5N
1mM NB-DNJ
1mM NB-DNJ and 10µg/ml Brefeldin A
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.001 0.01 0.1 1 10
Mean G3M5N
Mean G3M7N2
(pm
ol)
per
ug
of
pro
tein
Brefeldin µg/ml
Triglucosylated FOS produced upon treatment with Brefeldin A
Brefeldin + NBDNJ
-3 -2 -1 0
025
50
75
100
125
48h +
24h +
6h +
4h +
2h +
1h +
log [Brefeldin µg/ml]
% o
f co
ntr
ol
leu
cin
e i
nco
rpo
rati
on
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
Effect of 1 mM NBDNJ
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
4000.0
0 6 12 18 24 30 36 42 48
time (h)
leu
cin
e i
nco
rpo
rati
on
(cp
m/µ
g
pro
tein
)
control
+ NB-DNJ
