Free oligosaccharides Free oligosaccharides as biological markers of as biological markers of endoplasmic reticulum-endoplasmic reticulum-associated degradation associated degradation

and the role of and the role of endomannosidase.endomannosidase.

Emily Dennis - Trinity College, Oxford.

N-linked glycosylationN-linked glycosylation

1. Dolichol-pyrophosph-ate carries G3M9N2 (lipid-linked oligosaccharide).

2. Glycosylation recognition sequence = Asn-X-Ser/Thr on nascent polypeptide, detected by OST.

3.

4. Polypeptide released from ribosome and N-linked oligosaccharide is subject to processing which facilitates protein folding.

CYTOSOL

ER-LUMEN

ER-membrane

The Calnexin/Calreticulin cycleThe Calnexin/Calreticulin cycle

1. 1. Glucosidase Glucosidase trimming.trimming.

2. G1M9N2 2. G1M9N2 is is recognition recognition for for CNX/CRTCNX/CRT..

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

4. Glucosyl-4. Glucosyl-transferase transferase senses senses folding folding state and state and reglucosylatreglucosylates es misfolded misfolded proteins.proteins.

5. ER- 5. ER- mannosidamannosidase trims se trims oligosaccholigosaccharide in a aride in a time-time-dependent dependent manner if manner if persistentlpersistently y misfolded.misfolded.

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

Endoplasmic reticulum-associated Endoplasmic reticulum-associated degradation (ERAD): generation of FOSdegradation (ERAD): generation of FOS

1. Alongside 1. Alongside EDEM, Yos9p EDEM, Yos9p plays role in plays role in binding binding misfolded misfolded proteins via a proteins via a MRH domain: MRH domain: stabilizes the stabilizes the protein which protein which is then is then translocated translocated to cytosol.to cytosol.

2. FOS 2. FOS produced produced when when oligosaccharidoligosaccharide chain e chain cleaved by cleaved by PNGase PNGase enzyme.enzyme.

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

4. FOS 4. FOS converted from converted from GlcNAcGlcNAc22 to to GlcNAcGlcNAc11 species species by ENGase by ENGase action.action.

5. Trimmed FOS 5. Trimmed FOS removed in removed in lysosome.lysosome.

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

Alternative quality control pathwayAlternative quality control pathway

NNB-DNJ = B-DNJ = -glucosidases I & II inhibitor -glucosidases I & II inhibitor no no CNX/CRT cycle in ERCNX/CRT cycle in ER

EndomannosidaseEndomannosidase::

GlcGlc33ManMan

ManMan77GlcNAcGlcNAc2 2

Folded: Folded: secretory pathway secretory pathway

? Misfolded: ? Misfolded: shuttled back into ER? shuttled back into ER?

Project AimsProject Aims

1.1. Show that FOS are markers for Show that FOS are markers for protein misfolding (ERAD)protein misfolding (ERAD)

2.2. Determine the origin of FOS Determine the origin of FOS (protein-linked vs. dolichol lipid-(protein-linked vs. dolichol lipid-linked)linked)

3.3. Investigate the role of Investigate the role of endomannosidase using RNAiendomannosidase using RNAi

1&2. Methods: FOS extraction1&2. Methods: FOS extraction24hr incubation24hr incubation

+/- 1mM NB-DNJ+/- 1mM NB-DNJ

Ion-exchange Ion-exchange (mixed bed) (mixed bed)

chromatography chromatography and 2-AA and 2-AA labellinglabelling

Affinity Affinity chromatography chromatography

(ConA)(ConA)

Small FOSSmall FOS (e.g. Glc(e.g. Glc1-1-

33Man)Man)

Large FOSLarge FOS (e.g. (e.g. ManMan55GlcNAcGlcNAc11, ,

GlcGlc33ManMan77GlcNAGlcNAcc22))

HPLCHPLC

washwash eluateeluate

Isolation of Isolation of dolichol-LLO in dolichol-LLO in

chloroform:methanchloroform:methanolol

Purified by ion-Purified by ion-exchange exchange

chromatographychromatography

Oligosaccharides Oligosaccharides released by acid released by acid cleavage. Further cleavage. Further

purified by ion purified by ion exchange and 2-AA exchange and 2-AA

labelledlabelled

Results: Effect of NB-DNJ Results: Effect of NB-DNJ treatment on FOS production in treatment on FOS production in HL60sHL60s

mV

50

100

150

200

0

Minutes20 22 24 26 28 30 32 34 36 38 40 42

M4M4NN

M5M5NN

G1M5G1M5NN

ControlControl

mV

50

100

150

200

0

Minutes20 22 24 26 28 30 32 34 36 38 40 42

G3M5NG3M5N NB-DNJ NB-DNJ treatedtreated

FOS produced under NB-DNJ treatment ~ x3 greater than controlsFOS produced under NB-DNJ treatment ~ x3 greater than controls

Increased FOS = increased ERAD activityIncreased FOS = increased ERAD activity

Results: Effect of inhibiting protein Results: Effect of inhibiting protein synthesis on FOS production in synthesis on FOS production in HL60sHL60s

Effects of varying puromycin concentrations on G3M5N levels in 1mM NB-DNJ trated HL60s

0

20

40

60

80

100

120

0 2 4 5

Puromycin concentration (ug/ ml)

% P

eak

Are

a

Effect of varying puromycin concentration on M5N levels in HL60s

0

20

40

60

80

100

120

0 2 4

Puromycin concentration (ug/ ml)

% p

eak a

rea

Puromycin Puromycin large, progressive decrease in FOS with increasing large, progressive decrease in FOS with increasing [puromycin] (0-4 [puromycin] (0-4 g mlg ml-1-1).).

Controls: 100% Controls: 100% 27% 27% 11% 11%

1mM NB-DNJ: 100% 1mM NB-DNJ: 100% 7% 7% 4% 4% 3% 3%

~maximum inhibition achieved with ~maximum inhibition achieved with g mlg ml-1-1 puromycin puromycin concentrationconcentration

Results: Effect of inhibiting protein Results: Effect of inhibiting protein synthesis on LLO-FOS productionsynthesis on LLO-FOS production

Graph to show the effect of puromycin on lipid-linked oligosaccharide (G3M9N2) levels

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

4.00E+06

4.50E+06

5.00E+06

Control 4 ug/ mlpuromycin

NB-DNJ NB-DNJ+4ug/ ml

peak a

rea (

uV/se

c)

1mM NB-DNJ treated 1mM NB-DNJ treated cells, FOS levels remain cells, FOS levels remain same +/-puromycinsame +/-puromycin

control cells: ~50% control cells: ~50% increase in LLO-FOS increase in LLO-FOS (+puromycin) (+puromycin)

? ? pool of dolichol pool of dolichol increases to increases to compensate for lack of compensate for lack of protein for protein for oligosaccharide oligosaccharide transfer. transfer.

? ? enzymes involved in enzymes involved in dolichol synthesis dolichol synthesis appear not to be appear not to be affected by protein affected by protein synthesis inhibition.synthesis inhibition.

Results: Investigating Results: Investigating endomannosidase functionendomannosidase function

50

mV

0

10

20

30

Minutes

7 8 9 10 11 12 13 14 15 16 17 18 19 20 0

10

20

30

40

1

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

Minutes

mV

mV

0

10

20

30

40

50

Minutes

7 8 9 10 11 12 13 14 15 16 17 18 19 20

Controls: MDBK (left); RAW (right)Controls: MDBK (left); RAW (right)

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

mV

10 12 15 17 20 22 25 27 30 32 35 37 40 42 450

10

20

30

40

50

2

Minutes

G3MG3M

MDBK: endomannosidase NOT MDBK: endomannosidase NOT catalytically activecatalytically active

RAW: catalytically function enzyme RAW: catalytically function enzyme G3M detected G3M detected

Results: Investigating endomannosidase Results: Investigating endomannosidase function cont’dfunction cont’d

mV

Controls: MDBK (left); RAW Controls: MDBK (left); RAW (right)(right)

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

mV

0

50

100150200

250

20 22 24 26 28 30 32 34 36 38 40 42

Minutes

-50

100

200

300

1

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 45

Minutes

M5NM5N

M5M5NN

G1M5G1M5NNM4M4

NN

50

100

150

200

250

0

Minutes20 22 24 26 28 30 32 34 36 38 40 42

G3M7NG3M7N22

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 45-50

100

250

2

Minutes

G3M5G3M5NN

G3M7N2G3M7N2

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

G3M7N2: endomannosidase G3M7N2: endomannosidase inefficiencyinefficiency

G3M7N7G3M7N7RAW RAW < G3M7N2< G3M7N2MDBKMDBK

3. Methods: RNAi3. Methods: RNAi

2 endomannosidase-specific siRNAs2 endomannosidase-specific siRNAs

Negative controlNegative control: no siRNA introduced: no siRNA introduced

Positive, non-specific controlPositive, non-specific control: siRNA : siRNA targeted against ubiquitously expressed protein targeted against ubiquitously expressed protein

kinase MAPK1kinase MAPK1

Non-silencing controlNon-silencing control: siRNA with no known : siRNA with no known homology, labelled with Alexa Fluor 488homology, labelled with Alexa Fluor 488

Limitations:Limitations: Unsuccessful quantitative RT-PCR attempts for Unsuccessful quantitative RT-PCR attempts for knockdown confirmation knockdown confirmation

Potential for non-specific, off-target effectsPotential for non-specific, off-target effects

Results: Non-silencing control Results: Non-silencing control transfection efficiency of siRNAstransfection efficiency of siRNAs

1. Non-transfected overlay1. Non-transfected overlay 2. Transfected overlay2. Transfected overlay

Bright spots of fluorescence Bright spots of fluorescence (A(A495nm495nm) = successful transfection ) = successful transfection of siRNA into RAWsof siRNA into RAWs

No bright spots No bright spots detected in negative detected in negative control cellscontrol cells

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

Results: Effect of siRNA on G3M Results: Effect of siRNA on G3M levelslevels

Graph to show the effect of siRNAs on G3M levels in control cells

0

2

4

6

8

10

12

14

16

18

20

control siRNA1 siRNA2 MAPK

Peak A

rea (

mV/m

in)

Graph to show the effect of siRNAs on G3M in 1mM NB-DNJ treated cells

0

20

40

60

80

100

120

140

NBDNJ si1+NBDNJ si2+NBDNJ MAPK+NBDNJ

Peak A

rea (

mV/m

in)

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

G3M decrease (siRNAs G3M decrease (siRNAs 1&2)1&2)

No effect with MAPKNo effect with MAPK

G3M levels in controls < NB-DNJ G3M levels in controls < NB-DNJ treated cellstreated cells

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

G3M decrease with MAPK siRNA G3M decrease with MAPK siRNA

Results: Effect of siRNA on M5N Results: Effect of siRNA on M5N levelslevels

Graph to show the effect of siRNAs on M5N levels in HL60s

0

200000

400000

600000

800000

1000000

1200000

1400000

Peak A

rea (

uV/sec)

M5N is common M5N is common product and product and unrelated to unrelated to endomannosidase.endomannosidase.

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

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

Overall: Overall:

No consistent No consistent effect.effect.

Results: Effect of siRNA on G3M7N2 Results: Effect of siRNA on G3M7N2 levelslevels

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

0

50000

100000

150000

200000

250000

NBDNJ si1+NB si2+NB MAPK+NBPeak A

rea (

uV/se

c)

G3M7N2 = G3M7N2 = endomannosidase endomannosidase product detected in product detected in MDBKs.MDBKs.

PredictionPrediction: :

Decrease in G3M7N2 Decrease in G3M7N2 (siRNAs 1&2)(siRNAs 1&2)

No change with MAPKNo change with MAPK

siRNA1 siRNA1 19% decrease 19% decrease siRNA2 siRNA2 63% decrease 63% decrease

However…However… MAPK MAPK 50% decrease 50% decrease

MAPK resultsMAPK results Is the MAPK siRNA directly effecting Is the MAPK siRNA directly effecting endomannosidase knockdown? endomannosidase knockdown?

RNAi technique affecting the data?RNAi technique affecting the data?

Further investigation required!Further investigation required!

ConclusionsConclusions

3.3. RNAi technique not optimized to make RNAi technique not optimized to make strong conclusions about the function of strong conclusions about the function of endomannosidase. endomannosidase. Further investigation essential.Further investigation essential.

1.1. FOS are suitable biological FOS are suitable biological markers for ERAD.markers for ERAD.

2.2. FOS are derived from FOS are derived from glycoproteins rather than dolichol glycoproteins rather than dolichol lipids.lipids.

ReferencesReferences

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

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

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

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

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

AcknowledgementsAcknowledgements

I would like to thank:I would like to thank:

Dr Terry ButtersDr Terry Butters

Dominic AlonziDominic Alonzi

Dr David Neville, Gabriele Reinkensmeier, Stephanie Dr David Neville, Gabriele Reinkensmeier, Stephanie

BoomkampBoomkamp

Dr Steve Woodhouse, Dr Narayan RamamurthyDr Steve Woodhouse, Dr Narayan Ramamurthy