title: Use of fluorescent substrates for characterization of Gaucher disease mutations.
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.
authors.affiliation: Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, 69978, Israel.
authors.forename: Idit
authors.initials: I
authors.lastname: Ron
authors.forename: Arie
authors.initials: A
authors.lastname: Dagan
authors.forename: Shimon
authors.initials: S
authors.lastname: Gatt
authors.forename: Metzada
authors.initials: M
authors.lastname: Pasmanik-Chor
authors.forename: Mia
authors.initials: M
authors.lastname: Horowitz
mesh_terms: D002460:Cell Line; D005456:Fluorescent Dyes; D005776:Gaucher Disease / Q000175:diagnosis / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D005962:Glucosylceramidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011237:Predictive Value of Tests; D020871:RNA Stability / Q000235:genetics; D012720:Severity of Illness Index; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D005962:Glucosylceramidase