Expression of Mutated Glucocerebrosidase Alleles in Human Cells
Open Access
- 1 June 1997
- journal article
- research article
- Published by Oxford University Press (OUP) in Human Molecular Genetics
- Vol. 6 (6) , 887-895
- https://doi.org/10.1093/hmg/6.6.887
Abstract
Gaucher disease is a heterogeneous disease characterized by impaired activity of the lysosomal enzyme glucocerebrosidase. This heterogeneity is attributed to a large number of mutations in the corresponding gene. In order to test the biochemical properties of some mutations prevalent among Israeli populations, the normal human glucocerebrosidase cDNA and cDNAs carrying mutations N370S, L444P, D409H, recTL, recNciI, P415R and 84GG were coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). Recombinant viruses were produced and used to infect human tissue culture cells. RNA and protein stability, recognition by anti-glucocerebrosidase monoclonal antibodies and intracellular enzymatic activity were measured. The results demonstrated that the D409H allele directed synthesis of cytoplasmic RNA with decreased stability compared with its normal counterpart or other mutated forms. The D409H and L444P mutated proteins had lower stability than that of their normal counterpart, while the recNciI-mutated protein was more stable. Only glucocerebrosidase forms harboring leucine at position 444 were recognized by the anti-glucocerebrosidase monoclonal antibodies used (8E4 and 2C7). Measurements of enzymatic activity of the recombinant proteins in cells loaded with a fluorescent glucosylceramide demonstrated that the N370S mutated enzyme had activity similar to that of the normal enzyme. The other mutated enzymes exhibited varying degrees of activities, generally corresponding to the phenotypes with which they are associated. The results presented demonstrate the use of the vaccinia virus-derived expression system and of loading living cells with fluorescent substrate as efficient tools for studying mutants in Gaucher disease and in other lysosomal diseases.Keywords
This publication has 16 references indexed in Scilit:
- RecTL: A complex allele of the glucocerebrosidase gene associated with a mild clinical course of Gaucher diseaseAmerican Journal of Medical Genetics, 1994
- Fluorescence-based diagnosis of lipid storage diseases by analysis of the culture medium of skin fibroblastsClinica Chimica Acta; International Journal of Clinical Chemistry, 1993
- Identification of the second common Jewish Gaucher disease mutation makes possible population-based screening for the heterozygous state.Proceedings of the National Academy of Sciences, 1991
- Prevalent and rare mutations among Gaucher patientsGene, 1990
- Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.Proceedings of the National Academy of Sciences, 1989
- Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.Proceedings of the National Academy of Sciences, 1988
- EfficientIn VitroandIn VivoExpression of Human Glucocerebrosidase cDNADNA, 1987
- A Mutation in the Human Glucocerebrosidase Gene in Neuronopathic Gaucher's DiseaseNew England Journal of Medicine, 1987
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970