Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.
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September 2007
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Research Article|
August 13 2007
Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin
Satoshi Ishii;
Satoshi Ishii
*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A.
†Department of Agricultural and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan
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Hui-Hwa Chang;
Hui-Hwa Chang
*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A.
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Kunito Kawasaki;
Kunito Kawasaki
†Department of Agricultural and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan
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Kayo Yasuda;
Kayo Yasuda
*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A.
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Hui-Li Wu;
Hui-Li Wu
*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A.
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Scott C. Garman;
Scott C. Garman
‡Department of Biochemistry and Molecular Biology, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, U.S.A.
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Jian-Qiang Fan
Jian-Qiang Fan
1
*Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, U.S.A.
1To whom correspondence should be addressed (email jian-qiang.fan@mssm.edu).
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Publisher: Portland Press Ltd
Received:
April 05 2007
Revision Received:
May 24 2007
Accepted:
June 08 2007
Accepted Manuscript online:
June 08 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 406 (2): 285–295.
Article history
Received:
April 05 2007
Revision Received:
May 24 2007
Accepted:
June 08 2007
Accepted Manuscript online:
June 08 2007
Citation
Satoshi Ishii, Hui-Hwa Chang, Kunito Kawasaki, Kayo Yasuda, Hui-Li Wu, Scott C. Garman, Jian-Qiang Fan; Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin. Biochem J 1 September 2007; 406 (2): 285–295. doi: https://doi.org/10.1042/BJ20070479
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