![PRELIMINAR STUDY ABOUT THE
CHAPERONE EFFECT ON MUTATED
GLUCOCEREBROSIDASES AS A
TREATMENT FOR GAUCHER DISEASE
G. Sánchez-Ollé, M. Egido-Gabás*, J. Duque**, J. Casas*, A. Chabás**, L. Vilageliu & D. Grinberg
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona.
*RUBAM (Research Unit on BioActive Molecules); Departament de Química Orgànica Biològica, Institut d'Investigacions
Químiques i Ambientals de Barcelona (IIQAB‑CSIC), Barcelona.
**Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Barcelona.
Barcelona
INTRODUCTION
Gaucher disease (GD) is a glycosphingolipid disorder, B) Results on the GBA activiy of iminosugar-treated
caused by deficiency of lysosomal acid β-glucosidase (GBA), enzymes:
resulting in progressive accumulation of glucosylceramide in
macrophages of liver, spleen, bone marrow and, sometimes,
central nervous system. It has been reported that some
iminosugar compounds are able to work as chemical
chaperones, at subinhibitor concentrations, and increase the
N188S;E326K
K
326
activity of wild-type and mutated GBAs.
;E S
188
N
P
44
L4
L444P
OBJETIVES
H
409
D
NN-DNJ
NB-DNJ
T
02
I4
The aim of this work is to express and characterize mutant N188S
S
70
GBA alleles and assay the chaperone effect of the
3
N
iminosugars N-(n-nonyl)deoxynojirimycin (NN-DNJ) and N-
S
188
N
WT
(n-butyl)deoxynojirimycin (NB-DNJ), on the expressed
T
W
proteins.
1,5
3,5
2,5
0,5
4
3
2
1
0
10
9
8
7
6
5
4
3
2
1
0
RELATIVE INCREASE RELATIVE INCREASE
2,52,5 µM
µM 5 µM µM
5 5 5 µM
µM 10 µM µM
10
MATERIAL AND METHODS
COS-7 cells have been transfected with the pcDNA3
expression vector carrying the wild-type or the mutated
cDNAs.
CONCLUSIONS
Stable transfected cells, selected with geneticin, were treated
-The residual enzymatic activities, given as percentage of
for 6 days with NN-DNJ (2.5 and 5 µM) or NB-DNJ (5 and 10
the wild-type enzyme activity are the following: N188S:
µM).
53.84%, G202R: 24.84%, H255Q: 77.09%, E326K: 30.51%,
N370S: 8.11%, G377S: 11.06%, I402T: 19.37%, D409H:
RESULTS 16.46%, L444P: 2.42%, [N188S;E326K]: 12.78% and
[H255Q;D409H]: 16.05%.
A) Eleven different mutated alleles have been studied.
Results on the GBA activity of mutated enzymes are
indicated in the figure below. - The preliminary results obtained after treating the cells for 6
days with NN-DNJ show an increase of enzymatic activity of
TRANSIENT EXPRESSION the wild-type protein (at 2,5 and 5 µM) and themutated
120 N188S (at 2,5 µM) and N188S;E326K (at 5 µM) GBAs.
100 Besides, a decrease in protein activity is observed for N188S
% A OF ACTIVITY COMPARED TO
80 (at 5 µM) and N188S;E326K (at 2,5 µM) mutated proteins.
WILD TYPE ALLELE
60
40
20 - After treating the cells for 6 days with NB-DNJ, not only an
0 increase of the wild-type protein activity (at 10 µM) is
observed, but also an increase in the mutated proteins
T
9H
W
T
P
8S
0S
K
9H
6K
5Q
7S
2R
02
44
26
40
18
37
40
32
37
20
25
I4
L4
E3
;D
N
N
D
;E
G
G
H
5Q
8S
N188S (at 5 and 10 µM), N370S (at 10 µM) and
25
18
H
N
N188S;E326K (at 10 µM) activity.
SPHINGOLIPID CLUB 5th ANNUAL MEETING
Calella, November 2006](https://image.slidesharecdn.com/slc06poster-120810040239-phpapp02/85/Sphyngolipid-club-2006-1-320.jpg)
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- 1. PRELIMINAR STUDY ABOUT THE CHAPERONE EFFECT ON MUTATED GLUCOCEREBROSIDASES AS A TREATMENT FOR GAUCHER DISEASE G. Sánchez-Ollé, M. Egido-Gabás*, J. Duque**, J. Casas*, A. Chabás**, L. Vilageliu & D. Grinberg Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona. *RUBAM (Research Unit on BioActive Molecules); Departament de Química Orgànica Biològica, Institut d'Investigacions Químiques i Ambientals de Barcelona (IIQAB‑CSIC), Barcelona. **Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Barcelona. Barcelona INTRODUCTION Gaucher disease (GD) is a glycosphingolipid disorder, B) Results on the GBA activiy of iminosugar-treated caused by deficiency of lysosomal acid β-glucosidase (GBA), enzymes: resulting in progressive accumulation of glucosylceramide in macrophages of liver, spleen, bone marrow and, sometimes, central nervous system. It has been reported that some iminosugar compounds are able to work as chemical chaperones, at subinhibitor concentrations, and increase the N188S;E326K K 326 activity of wild-type and mutated GBAs. ;E S 188 N P 44 L4 L444P OBJETIVES H 409 D NN-DNJ NB-DNJ T 02 I4 The aim of this work is to express and characterize mutant N188S S 70 GBA alleles and assay the chaperone effect of the 3 N iminosugars N-(n-nonyl)deoxynojirimycin (NN-DNJ) and N- S 188 N WT (n-butyl)deoxynojirimycin (NB-DNJ), on the expressed T W proteins. 1,5 3,5 2,5 0,5 4 3 2 1 0 10 9 8 7 6 5 4 3 2 1 0 RELATIVE INCREASE RELATIVE INCREASE 2,52,5 µM µM 5 µM µM 5 5 5 µM µM 10 µM µM 10 MATERIAL AND METHODS COS-7 cells have been transfected with the pcDNA3 expression vector carrying the wild-type or the mutated cDNAs. CONCLUSIONS Stable transfected cells, selected with geneticin, were treated -The residual enzymatic activities, given as percentage of for 6 days with NN-DNJ (2.5 and 5 µM) or NB-DNJ (5 and 10 the wild-type enzyme activity are the following: N188S: µM). 53.84%, G202R: 24.84%, H255Q: 77.09%, E326K: 30.51%, N370S: 8.11%, G377S: 11.06%, I402T: 19.37%, D409H: RESULTS 16.46%, L444P: 2.42%, [N188S;E326K]: 12.78% and [H255Q;D409H]: 16.05%. A) Eleven different mutated alleles have been studied. Results on the GBA activity of mutated enzymes are indicated in the figure below. - The preliminary results obtained after treating the cells for 6 days with NN-DNJ show an increase of enzymatic activity of TRANSIENT EXPRESSION the wild-type protein (at 2,5 and 5 µM) and themutated 120 N188S (at 2,5 µM) and N188S;E326K (at 5 µM) GBAs. 100 Besides, a decrease in protein activity is observed for N188S % A OF ACTIVITY COMPARED TO 80 (at 5 µM) and N188S;E326K (at 2,5 µM) mutated proteins. WILD TYPE ALLELE 60 40 20 - After treating the cells for 6 days with NB-DNJ, not only an 0 increase of the wild-type protein activity (at 10 µM) is observed, but also an increase in the mutated proteins T 9H W T P 8S 0S K 9H 6K 5Q 7S 2R 02 44 26 40 18 37 40 32 37 20 25 I4 L4 E3 ;D N N D ;E G G H 5Q 8S N188S (at 5 and 10 µM), N370S (at 10 µM) and 25 18 H N N188S;E326K (at 10 µM) activity. SPHINGOLIPID CLUB 5th ANNUAL MEETING Calella, November 2006