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