Transcript Slides

Background: a bit about…
Important roles in:
•Glycoprotein biosynthesis, quality control & catabolism
•Multiple forms of α-mannosidases in mammalian cells, differing in specificity, function and
cellular location
Classified into 2 groups conserved through eukaryotic evolution
Class I
Class II
- Narrow specificity for α-1, 2mannosidic linkages
- Broad specificity: hydrolyse α(1→ 2),
α(1→3) and α(1→6) mannosidic
linkages
-Further specificity within family for
isomeric species produced
Man a1-2
Mana1-2
Mana1-6
Mana1-3
Mana1-6
Manß1-4GlcNAcß1-4GlcNAcß1-Asn
Man a 1-2
Mana1-2
Mana1-3
CELL
ERAD
Cytosol
persistently
misfolded
M8B
/ M8C
Monosaccharide
constituents
Lysosome
misfolded
ER
Protein and Oligosaccharide Processing in the Cytosol of ERAD
+ G1-3M8N2 + M8N2
Chitobiase
Proteasome
M8N1
Chitobiase
Cytosolicαmannosidase
Fast
M5N1
G1-3M8N1
Cytosolicαmannosidase
lysosome
G1-3M5N1
Cyt α-man ?
G1-3M4N1
Plasma
membrane
?
G1-3M4N1
Cyt α-man?
Slow
M4N1
Known α-mannosidase inhibitors
OH
H
N
HO
N
HO
HO
OH
OH
HO
Deoxymannojorimycin (DMJ)
HO
O
HO
H
N
O
N
H
Kifunensin (Kif)
Weak general inhibitor of
mammalian α-mannosidases
OH
HO
OH
HO
Swainsonine
(SW)
OH
1, 4 deoxy-1,4-imino Dmannitol (DIM)
Inhibits lysosomal
mannosidase and Golgi
mannosidase
ER mannosidase I and
Man9-mannosidase
Higher conc.: Will start to inhibit
other α-mannosidases
HO
H
H
Potent and specific
inhibitor of Golgi
mannosidase I
Low conc.: Golgi mannosidase I
and Man9-mannosidase
H
N
Novel 7-membered imino-sugars: inhibitory effect on cellular α-mannosidases
(Kukushkin & Butters; unpublished data)
N
HO
N
OH
HO
148
OH
N
HO
OH
HO
OH
149
N
OH
HO
266
OH
OH
HO
OH
265
Project Aims
The aims of my project were:
To define α-mannosidase targets of novel inhibitors in cells and compare these to known αmannosidase inhibitors
This will be accomplished by:
1.Evaluation of cytosolic and lysosomal α-mannosidase inhibition by measuring free
oligosaccharides (FOS) in HL60 and MDBK cell lines.
2.Increasing our knowledge of mannosidases within their cellular pathways in cell lines above.
3.Using glycoprotein precipitation, isolation and PNGase glycan release methods to observe effects
of inhibitors on glycan species of total cellular glycoprotein.
4.Identification of lysosomal FOS species following cellular fractionation. Two cell lines will be used
that have differing biosynthetic and catabolic pathways: HL60, a human promyelocytic leukaemia
cell line and MDBK, a bovine kidney cell line.
5.Estimation of ER and Golgi α-mannosidase inhibition by using Fluorescence-activated cell sorting
(FACS) to analysing glycoprotein derived oligosaccharides on the cell surfacee following treatment
with α-mannosidase inhibitors.
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
HL60 cells treated with known α-mannosidase inhibitors
Fluorescence (arbitrary units)
Untreated
HL60
M5N
M7N
M4N
DMJ
treated
(1mM)
DIM
treated
(100μM)
M9N
G1M5
M9N/N2
M5N
G1M5
M4N
M7N/N2 M7N/N2 M8N/N2
M8N
G1M9N?
M9N/N2
M7N/N2
M8N/N2
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
MDBK cells treated with known α-mannosidase inhibitors
3000
M5N
Fluorescence (arbitrary units)
Untreated
MDBK cells
M4N
G1M5N
M3N2
M4N2
M9N/N2
6000
DIM
treated
(100μM)
M3N2
M5N
M4N2
M5N2
M6N2 M7N/N2
M8N2
M9N/N2
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
MDBK cells treated with Kif at increasing concentrations (1μM- 100μM)
-
Untreated
M5N
G1M5
M4N2
M9N
Fluorescence (arbitrary units)
-
+ 1μM Kif
M5N
M4N2
G1M5
M6N2
M8N2
M7N2
M9N
+ 10μM Kif
M8N2
M7N2
M9N/N2
M8N2
+ 100μM Kif
M7N2
M9N/N2
22
23
24
25
26
27
28
29
30
31
32
Minutes
33
34
35
36
37
38
39
40
41
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
HL60 cells treated with 148 and 149
M5N
G1M5
Fluorescence (arbitrary units)
M4N
SATIN
M8N
M9N
- Fluorescence
M8N
M9N
M6N2
G1M5 M6N
G1M9
M7N/
N2
M5N
M4N
M5N
M4N
22
24
26
28
M8N
G1M5
M6N2 M7N/
M6N
N2
30
32
Minutes
34
M9N
G1M9
36
38
40
42
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
MDBK cells treated with 148 and 149
M5N
1200
Fluorescence (arbitrary units)
G1M5
M4N2
M3N2
-
M3N2
1800
M4N2
M5N
M8N2
M6N2
M7N2
M9N/N2
M5N2
6000
M4N2
M5N
M7N2
M6N2
M8N2
M9N/N2
M5N2
18
20
22
24
26
28
30
Minutes
32
34
36
38
40
42
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
HL60 cells treated with 265 and 266
1600
Untreated
M5N
Fluorescence (arbitrary units)
M9N
M3N
4000
M4N
M6N
+ 100μM 265
M3N
M5N
M4N
M6N
2000
+ 100μM 266
M5N
M4N
M3N
12
M9N
14
16
18
20
22
24
26
Minutes
M9N
28
30
32
34
36
38
40
42
HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from
MDBK cells treated with 265 and 266
1300
Fluorescence (arbitrary units)
M3N2
SATIN - Fluorescence
M3N2
6000
M3N2
6000
0
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Minutes
30.00
32.00
34.00
36.00
38.00
40.00
42.00
Flow cytometry data representing %
change in ConA binding in Kif and 148
treated HL60 cells over 24hrs and 72 hrs
Effect on total cellular glycoprotein upon
inhibitor treatment
Untreated HL60
600
1000
% increase in
ConA
binding
after
24hrs
% increase in
ConA
binding
after
72hrs
+ 100μM Kif
68.1%
136%
+ 100μM 148
13.9%
34.2%
+ DMJ (1mM)
SATIN - Fluorescence
fluorescence
1200
+ 148 (100μM)
+ 265 (100μM)
1000
1000 + 266 (100μM)
SATIN - Fluorescence
2500 + Kif (100μM)
22
24
26
M9N2
28
30
32
34
36
Elution time (minutes)
38
40
42
44
Cellular fractionation of untreated HL60 and MDBK cells
HL60 Cells Lysosomal Activity
MDBK Cells Lysosomal Activity
1.4
1.2
0.5
Fraction 9
lysosomal activity/ ug protein
Lysosomal Activity/ ug protein
0.6
0.4
0.3
0.2
0.1
Fraction 8
1
0.8
0.6
0.4
0.2
0
0
0
2
4
6
8
10
12
14
0
-0.1
2
4
6
8
10
12
14
-0.2
Cell Fraction Number
Fluorescence
(arbitrary units)
Fraction number
16
18
20
22
24
26
28
30
32
34
36
38
40
42
12
14
Elution time (minutes)
16
18
20
22
24
26
28
30
32
34
36
38
40
42
In Summary
Known α-mannosidase inhibitors:
-
DMJ: low potency, low specificity α-mannosidase inhibitor. Does not inhibit cytosolic mannosidase at high
concentration (1mM) but most likely inhibiting ER/Golgi and partially inhibiting lysosomal α-mannosidases.
-
Kif: Known strong inhibitor of ER Mannosidase I and Golgi mannosidases. No inhibition of ER Man II due to
accumulation of M8C.
-
Very high potency, inhibitory effects observed from 1μM conc. No inhibition of cytosolic/lysosomal αmannosidases. Selective for ER/Golgi mannosidases and the biosynthetic pathway for glycoprotein maturation.
-
DIM: Selective for lysosomal α-mannosidase and lysosomal α1,6 mannosidase. Partial inhibition of catabolism of
high mannose structures, strong inhibition of breakdown of core N-linked glycan isomer M3aN2.
Novel α-mannosidase inhibitors
-
148 & 149: Very similar inhibitory patterns in the cell. Inhibits cytosolic α-mannosidase, and lysosomal
mannosidase in a similar way to DIM. Subtle inhibition of conversion of oligomannose to complex glycan
maturation to the cell surface.
-
265: No inhibition of cytosolic α-mannosidase. Targets lysosomal α-mannosidase and lysosomal α1, 6
mannosidase with particularly potent inhibition for the catabolism of core M3aN2.
-
266: No inhibition of cytosolic α-mannosidase. Inhibits lysosomal α-mannosidase with particularly potent inhibition
for the catabolism of core M3aN2. Partial inhibition of catabolism of high oligomannose glycans by lysosomal αmannosidase . Low levels of inhibition for α1,6 mannosidase.
Future work
•
Cellular fractionation with added inhibitor followed by FOS isolation and characterisation to
confirm and clarify target compartments of novel inhibitors within the cell and to specify enzymes
inhibited.
•
Carry out Flow Cytometry experiment with 266 and 265 to analyse effect on glycoprotein
maturation at the cell surface
•
To confirm the cytosolic inhibition of 148 and 149. Proposed experiment: Addition of 148/149
following treatment of cells with glucosidase inhibitor such as NB-DMJ to create more triglucosylated species. If 148/149 inhibit in the cytosol, will generate more glucosylated highmannose structures from FOS analysis.
•
May want to develop a more selective or & more potent inhibitor of the cytosolic α-mannosidase:
drug screening with a range of 148,149-like structures.
Acknowledgements
Dr Terry Butters
Dr David Neville
Dr Dominic Alonzi
Stephanie Boomkamp
Professor Raymond Dwek