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