Expression of mutated glucocerebrosidase alleles in human cells

Human Molecular Genetics
M Pasmanik-ChorM Horowitz

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, recNcil, 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 recNcil-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 enz...Continue Reading

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Citations

Jul 16, 2009·Neuroradiology·Ahmed Abdel Khalek Abdel RazekAshraf Abdel Rahman
May 27, 2005·Blood Cells, Molecules & Diseases·Idit RonMia Horowitz
Jan 23, 1999·The Biochemical Journal·L Madar-ShapiroM Horowitz
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