Journal Article

Expression of Mutated Glucocerebrosidase Alleles in Human Cells

Metsada Pasmanik-Chor, Liora Madar-Shapiro, Orna Elroy Stein, Hans Aerts, Shimon Gatt and Mia Horowitz

in Human Molecular Genetics

Volume 6, issue 6, pages 887-895
Published in print June 1997 | ISSN: 0964-6906
Published online June 1997 | e-ISSN: 1460-2083 | DOI:
Expression of Mutated Glucocerebrosidase Alleles in Human Cells

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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.

Journal Article.  5248 words.  Illustrated.

Subjects: Genetics and Genomics

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