Slide 1

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 2

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 3

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 4

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 5

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 6

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 7

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 8

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 9

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 10

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 11

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 12

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 13

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 14

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 15

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 16

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 17

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 18

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 19

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy


Slide 20

Free oligosaccharides as
biological markers of
endoplasmic reticulumassociated degradation
and the role of
endomannosidase.
Emily Dennis - Trinity College, Oxford.

N-linked glycosylation
1. Dolicholpyrophosphate carries
G3M9N2 (lipid-

CYTOSOL

linked
oligosaccharide).

ER-membrane
3.

ER-LUMEN

2. Glycosylation
recognition
sequence = AsnX-Ser/Thr on
nascent
polypeptide,
detected by OST.
4. Polypeptide released
from ribosome and Nlinked oligosaccharide is
subject to processing
which facilitates protein
folding.

The Calnexin/Calreticulin cycle
4. Glucosyltransferase
senses folding
state and
reglucosylates
misfolded
proteins.

3. Glucosidase
II cleaves 3rd
Glc residue.
Protein
dissociates from
CNX/CRT.

2. G1M9N2 is
recognition
for CNX/CRT.

1. Glucosidase
trimming.

6. EDEM binds the de-mannosylated
misfolded substrate and protein is
transported to the cytosol.

5. ERmannosidase
trims
oligosacchari
de in a timedependent
manner if
persistently
misfolded.

Endoplasmic reticulum-associated
degradation (ERAD): generation of FOS
1. Alongside
EDEM, Yos9p
plays role in
binding
misfolded
proteins via a
MRH domain:
stabilizes the
protein which is
then
translocated to
cytosol.

3. Protein degradation
via Ub-dependent 26S
proteosome pathway.

6. However: Glc-FOS
cannot be discarded via
lysosome. Fate still
unknown.

2. FOS produced
when
oligosaccharide
chain cleaved by
PNGase enzyme.

4. FOS converted
from GlcNAc2 to
GlcNAc1 species
by ENGase action.

5. Trimmed FOS
removed in
lysosome.

Alternative quality control pathway



NB-DNJ = a-glucosidases I & II inhibitor  no CNX/CRT

cycle in ER
Endomannosidase:
 Glc3Man
 Man7GlcNAc2

 Folded:  secretory pathway
? Misfolded:  shuttled back into ER?

Project Aims
1. Show that FOS are markers for
protein misfolding (ERAD)
2. Determine the origin of FOS
(protein-linked vs. dolichol lipidlinked)

3. Investigate the role of
endomannosidase using RNAi

1&2. Methods: FOS extraction
24hr incubation
+/- 1mM NB-DNJ

Ion-exchange
(mixed bed)
chromatography
and 2-AA labelling

Affinity chromatography
(ConA)
eluate

wash

Isolation of dolicholLLO in
chloroform:methanol

Purified by ionexchange
chromatography

Oligosaccharides
released by acid
cleavage. Further
purified by ion
exchange and 2-AA
labelled

Small FOS (e.g.
Glc1-3Man)

Large FOS (e.g.
Man5GlcNAc1,
Glc3Man7GlcNAc2)

HPLC

Results: Effect of NB-DNJ treatment
on FOS production in HL60s
200
150

mV

Control

M5N
G1M5N

100

M4N

50
0

20

22

24

26

28

30

200

32
Minutes

34

36

38

G3M5N

40

42

NB-DNJ treated

mV

150

100

50
0

20

22

24

26

28

30

32
Minutes

34

36

38

40

 FOS produced under NB-DNJ treatment ~ x3 greater than controls
 Increased FOS = increased ERAD activity

42

Results: Effect of inhibiting protein
synthesis on FOS production in HL60s
Effects of varying puromycin concentrations on G3M5N
levels in 1mM NB-DNJ trated HL60s

120

120

100

100
% Peak Area

% peak area

Effect of varying puromycin concentration on M5N
levels in HL60s

80
60
40

80
60
40

20

20

0

0
0

2

4

0

Puromycin concentration (ug/ml)

2

4

Puromycin concentration (ug/ml)

 Puromycin  large, progressive decrease in FOS with increasing
[puromycin] (0-4 mg ml-1).
Controls:

100%  27%  11%

1mM NB-DNJ: 100%  7%  4%  3%
 ~maximum inhibition achieved with 4mg ml-1 puromycin concentration

5

Results: Effect of inhibiting protein
synthesis on LLO-FOS production
Graph to show the effect of puromycin on lipidlinked oligosaccharide (G3M9N2) levels

 1mM NB-DNJ treated
cells, FOS levels remain
same +/-puromycin
 control cells: ~50%
increase in LLO-FOS
(+puromycin)

5.00E+06
4.50E+06

peak area (uV/sec)

4.00E+06
3.50E+06

? pool of dolichol increases

3.00E+06
2.50E+06

to compensate for lack of
protein for oligosaccharide
transfer.

2.00E+06
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Control

4 ug/ml
puromycin

NB-DNJ

NB-DNJ+4ug/ml

? enzymes involved in

dolichol synthesis appear
not to be affected by
protein synthesis inhibition.

Results: Investigating
endomannosidase function
Controls: MDBK (left); RAW (right)
50
40

mV

mV

30
20

30

20

10

10 1

0
7

8

9

10

11

12

13

14

15

16

17

18

19

20

0 10 12 15 17 20 22 25 27 30 32 35 37 40 42 45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
50

40

40

30

30

mV

mV

50

20
10

20
10 2

0
7

G3M

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

 MDBK: endomannosidase NOT catalytically
active

0
10

12

15

17

20

22

25

27

30

32

35

37

40

Minutes

 RAW: catalytically function enzyme 
G3M detected

42

45

Results: Investigating endomannosidase
function cont’d
Controls: MDBK (left); RAW (right)
300

mV

250
200
150
100

200

M5N

100

50
0

20

22

24

26

28

30

M5N

32

34

36

38

40

42

G1M5N

M4N

1
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

45

Minutes

Minutes

NB-DNJ treated: MDBK (left); RAW (right)
G3M7N2
250

G3M7N2

200

mV

250

150

G3M5N

100

100
50
020

22

24

26

28

30

32
34
Minutes

36

38

 G3M7N2: FOS in ER.
Does endomannosidase play role in
shuttling FOS Golgi  ER?

40

42

2
-50
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Minutes

 G3M7N2: endomannosidase inefficiency
 G3M7N7RAW < G3M7N2MDBK

45

3. Methods: RNAi
 2 endomannosidase-specific siRNAs

 Negative control: no siRNA introduced
 Positive, non-specific control: siRNA targeted
against ubiquitously expressed protein kinase MAPK1
 Non-silencing control: siRNA with no known
homology, labelled with Alexa Fluor 488

Limitations: Unsuccessful quantitative RT-PCR attempts for
knockdown confirmation
Potential for non-specific, off-target effects

Results: Non-silencing control  transfection
efficiency of siRNAs
1. Non-transfected overlay

No bright spots
detected in negative
control cells

2. Transfected overlay

Bright spots of fluorescence (A495nm)
= successful transfection of siRNA into
RAWs

 Limitations: Very difficult to get accurate quantitation of transfection efficiency.

Results: Effect of siRNA on G3M levels
Graph to show the effect of siRNAs on G3M levels in
control cells
20

Peak Area (mV/min)

18

Prediction: G3M in controls < 1mM
NB-DNJ treated.

16

 G3M decrease (siRNAs 1&2)

14
12
10

 No effect with MAPK

8
6
4
2
0
control

siRNA1

siRNA2

MAPK

Graph to show the effect of siRNAs on G3M in 1mM NBDNJ treated cells
140

G3M levels in controls < NB-DNJ
treated cells

Peak Area (mV/min)

120
100

 Inconsistent results with siRNAs 1&2
probe2 = more effective.

80
60
40

 G3M decrease with MAPK siRNA

20
0
NBDNJ

si1+NBDNJ

si2+NBDNJ

MAPK+NBDNJ

Results: Effect of siRNA on M5N levels
Graph to show the effect of siRNAs on M5N levels in
HL60s

Peak Area (uV/sec)

1400000

M5N is common product
and unrelated to
endomannosidase.

1200000
1000000
800000
600000
400000
200000

Decreases observed with all
probes, e.g. 4% siRNA1 and
57% MAPK.

Overall:
No consistent
effect.

N
J

K+
N
BD

N
J
M
AP

2+

N
BD

N
J
si

si

1+

N
BD

N
BD

N

J

K
M
AP

2
N
A
si
R

N
A
si
R

Co
nt
r

ol

1

0

Similar variable decrease in
M5N levels under NB-DNJ
treatment e.g. 49% siRNA1
but 27% MAPK.

Results: Effect of siRNA on G3M7N2 levels
Graph to show the effect of siRNAs on G3M7N2 levels in
1mM NB-DNJ treated HL60s

Prediction:
 Decrease in G3M7N2
(siRNAs 1&2)

 siRNA1  19% decrease
 siRNA2  63% decrease
However…
 MAPK  50% decrease



G3M7N2 =
endomannosidase
product detected in
MDBKs.

200000

Peak Area (uV/sec)

 No change with MAPK

250000

150000
100000
50000
0
NBDNJ

si1+NB

MAPK results  Is the MAPK siRNA directly effecting
endomannosidase knockdown?
 RNAi technique affecting the data?
 Further investigation required!

si2+NB

MAPK+NB

Conclusions
1. FOS are suitable biological markers for
ERAD.
2. FOS are derived from glycoproteins
rather than dolichol lipids.
3. RNAi technique not optimized to make strong
conclusions about the function of
endomannosidase.
Further investigation essential.

References
Diagrams (in order of appearance) taken and adapted from:


Roles of N-linked oligosaccharides in protein folding and ERAD (Alonzi, D.S;
Neville, D.C.A; Butters, T.D)



Annual Review of Biochemistry, Vol. 73 pg 1019-1049: Roles of N-linked
glycans in the endoplasmic reticulum (Helenius, A; Aebi, M)



Glycobiology, Vol. 15 pg 43R-52R: Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses (Butters, T.D; Dwek, R.A; Platt, F.M)



The EMBO Journal, Vol 16 pg 4302-4310: The solution NMR structure of
glucosylated N-glycans involved in early stages of glycoprotein biosynthesis
and folding (Petrescu, A.J; Butters, T.D; Reinkensmeier, G; Petrescu, S;
Platt. F.M; Dwek, R.A; Wormald, M.R)

Acknowledgements
I would like to thank:
 Dr Terry Butters
 Dominic Alonzi
 Dr David Neville, Gabriele Reinkensmeier, Stephanie
Boomkamp
 Dr Steve Woodhouse, Dr Narayan Ramamurthy