Human Molecular Genetics, Vol 6, 887-895, Copyright © 1997 by Oxford University Press
M Pasmanik-Chor, L Madar-Shapiro, EO Stein, H Aerts, S Gatt and M Horowitz
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 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.
ARTICLES
Expression of mutated glucocerebrosidase alleles in human cells
Department of Cell Research and Immunology, Tel-Aviv University, Ramat- Aviv, Israel.
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