Use of fluorescent substrates for characterization of Gaucher disease mutations.
Blood Cells Mol Dis
; 35(1): 57-65, 2005.
Article
em En
| MEDLINE
| ID: mdl-15916907
ABSTRACT
Gaucher disease results from impaired activity of the lysosomal enzyme beta-glucocerebrosidase. More than 200 mutations within the glucocerebrosidase gene have been associated with this disease. In this study we tested the effect of several mutations (K157Q, D140H, E326K, D140H+E326K, V394L and R463C) on RNA stability, protein stability and activity toward four different fluorescent substrates (LR-12-GC, Bodipy-12-GC, LR-0-PAP-glucose and 4-MUG), using the vaccinia-derived expression system. The results indicated that the K157Q mutation leads to RNA instability, causing low protein levels and a concomitant reduction in beta-glucocerebrosidase activity. All other tested mutations led to production of glucocerebrosidase RNA and protein with stabilities comparable to those of the normal counterpart. The D140H variant exhibited a high activity toward the tested substrates while the variant enzymes containing either the E326K or D140H and E326k mutations together expressed low beta-glucocerebrosidase activity. The V394L variant exhibited low activity toward the tested substrates, while a higher activity was presented by the R463C containing glucocerebrosidase variant. Our results strongly indicated that the LR-12-GC substrate distinguishes between severities of different mutant glucocerebrosidase variants overexpressed in a heterologous system.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Índice de Gravidade de Doença
/
Doença de Gaucher
/
Glucosilceramidase
/
Mutação
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Blood Cells Mol Dis
Assunto da revista:
HEMATOLOGIA
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Israel