imino sugars and perturbation of protein folding pathways in the er dom alonzi

Imino sugars and perturbation of protein folding pathways

in the ER

Dom Alonzi

Imino Sugars

N CH2OH

OH

OHHO

Therapeutic roles

Lysosomal storage disorders:Gaucher, Sandhoff

Antiviral: HCV, HBV

ER

Golgi

Further GolgiProcessing toComplex-type

+

Cytoplasm

Proteosome

-Glc I & IICalnexin/

Calreticulin

+

Sec 61

Ceramideglucosyltransferase

Plasmamembrane

< 1 min

Chitobiase

N-alkyl-DNJs

N-alkyl-DNJs

N-alkyl-DNJs

N-alkyl-DNJs

N-alkyl-DNJs

Ceramide Glucosylceramide

Endoman’ase

GSLs

The effect of imino sugars on N-linked oligosaccharide processing in the cell

Free oligosaccharide (FOS) generation - a normal cellular process

Unconjugated polymannose-type oligosaccharides are, in the main, produced as a by-product of the proteasomal degradation of misfolded glycoproteins.

A smaller proportion are produced during the dolichol pathway of N-linked glycosylation. These are segregated from their glycoprotein-linked counterparts and follow a non-vesicular trafficking pathway that leads to their degradation in the lysosome.

-glucosidase I & II

ATPATP

-glucosidase II

Calnexin/calreticulin

Glucosyl transferase

Post ER modification

-SH

HS-

-s-s- -s-s-

HS-ERp57

-s-s-

NB-DNJ

NB-DNJ

Disruption of glycoprotein folding in the ER

ER lumen

Cytosol PNGase

Chitobiase

Cytosolic Mannosidase

Proteasome

Misfolded glycoprotein

?

Sec61/Der-1

Free oligosaccharides produced by glycoprotein misfolding

FOS is produced following treatment of HL60 cells with 1mM NB-DNJ for 24hrs

M4N

M5N

G1M5N

G3M5N

G3M6-9N1/2

Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

Control

1mM NB-DNJ 24hrs

G1M5NM5N

Build up of Mono-glucosylated FOS is time dependent

0 5 10 15 200

10

20

30

40

50

1mM Butyl-DNJ

Time (hours)

[G1M

5N]

rela

tive

to

[M5N

]

Build up of Tri-glucosylated FOS is time dependent

0 5 10 15 200

25

50

75

100

1mM Butyl-DNJ

Time (hours)

[G3M

5N]

rela

tive

to

[M5N

]

G3M5N build up is Butyl-DNJ concentration dependent

0 250 500 750 10000

25

50

75

100Butyl-DNJ

Inhibitor concentration (µM)

[G3M

5N]

rela

tive

to

[M5N

]

N CH2OH

OH

OHHO

N CH2OH

OH

OHHO

Butyl-DNJ Vs Nonyl-DNJ

Effect of chain length on glucosidase inhibition in the ER. Is it the result of differential inhibition of the enzymes or an access/localisation issue?

+

-glucosidase I

Potential Inhibitor

2AA 2AA2AA

+

100% 75% 25%

t= 30mins

In Vitro Glucosidase inhibition assay

40.00Minutes

30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00

G3M5N

G3M5N + -glucosidase I

G3M5N + -glucosidase I + 5M NN-DNJ

In vitro IC50 determination

IC50 NN-DNJ= 0.54M and NB-DNJ = 0.83M

0 5 10 15 20 250

25

50

75

100

Butyl-DNJ

Nonyl-DNJ

Inhibitor concentration (µM)0.01 0.1 1 10 1000

25

50

75

100

Butyl-DNJ

Nonyl-DNJ

Log Inhibitor concentration (µM)

FOS production: Nonyl-DNJ vs Butyl-DNJ

0 10 20 30 40 50 60 70 80 90 1000

10

20

30

40

50

60

70

80

90

Nonyl-DNJ

Butyl-DNJ

Inhibitor concentration (µM)

[G3M

5N]

rela

tive

to

[M5N

]

NB-DNJ and NN-DNJ treatment of MDBK cells shows differential inhibition of glucosidases

Control

100µM NB-DNJ 72hrs

100µM NN-DNJ 72hrs

G3M5N G3M7N2

Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

Differential inhibition

Minutes

20.00 24.00 28.00 32.00 36.00 40.00 44.00

Type of FOS produced on treatment with NB-DNJ is Endomannosidase dependent

Control

Functional Endomannosidase

Non-utilised Endomannosidase

G3M5N G3M7N2

ER ERGIC

cis-Golgi

+

The Endomannosidase pathway

Bulk flow or ERGIC-53 mediated

Retrograde transport

Cytosolic Mannosidase

Endomannosidase

Golgi Mannosidase I

Further Golgi processing to Complex N-links

?

PNGase in the ER or Cytosol or both?

Fate of FOS following long term 1mM NB-DNJ treatment of HL60 cells

Control

1mM NB-DNJ 24hrs

1mM NB-DNJ 72hrs

1mM NB-DNJ 48hrs

G3M5NG3M4N

Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

G3M5N is converted to G3M4N in long term recovery experiments in HL60s

Control

1mM NB-DNJ 24hrs

1mM NB-DNJ 24hrs + 72hrs recovery

1mM NB-DNJ 24hrs + 96hrs recovery

1mM NB-DNJ 24hrs + 120hrs recovery

G3M5NG3M4N

Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

Where?

Enzyme?

Fate of Glucosylated Man5 FOS?

Most likely theory: Cytosolic Mannosidase Glucosylated FOS cannot gain access to the lysosome {Saint-Pol A et al JBC (1999)}

lysosome

Mouse: G1M4N in serum is dependent on NB-DNJ dose

Control

300mg/kg/day

1200mg/kg/day

G1M4N

Minutes20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

G1M4N build up in serum following NB-DNJ treatment

0.0 2.5 5.0 7.5 10.0 12.5 15.00

25

50

75

100

2400mg/kg/day

days treated with 2400mg/kg/day

[G1M

4N]

rela

tive

to

[M3N

]

(Serum concentration ~ 40M)

Minutes

22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.0020.00

Control

Treated

[Serum] = 40.8M

G1M4N is excreted in mouse urine following NB-DNJ treatment

G1-3M7N2

G1M4N

FOS detected in serum from NPC patient treated with 100mg/day NB-DNJ

Untreated (day 0)

Treated (16 months)

G3M4N

G1M4N

Minutes

20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

Lysosome

ERGolgi

PNGaseDer-1/Sec61

Proteasome

Endomannosidase

Chitobiase

Cytosolic mannosidase

-glucosidase I & II

?

Retrograde transport

Pathway for FOS clearance

ConclusionsCharacterised cellular FOS produced in the presence/absence of glucosidase inhibitors

Demonstrated differential inhibition of glucosidases I and II by imino sugars

FOS as biological markers - provide a simple cell based assay for glucosidase inhibitor efficacy and entry into the ER, and calnexin/calreticulin mediated protein folding

Formation of glucosylated Man4 species in cells, as a result of the cytosolic mannosidase, is the metabolic endpoint

Glucosylated FOS, in serum and urine of mouse and human following NB-DNJ treatment, reveals pathway of clearance

Detection of two PNGases in the cell: (i) ER lumen and (ii) cytosolic

Trans-renal clearance of glucosylated FOS prevents adverse effects on normal cellular processes

Endomannosidase influences the FOS pathway due to differential activities on protein folding states

Acknowledgements

Dr David Neville

Neidys Sanchez-Hernandez

Dr Howard Mellor

Mark Hussey

Adam Pilling

Dr Terry Butters

Professor Raymond Dwek

1st floor