Overexpression of human glucocerebrosidase containing different-sized leaders.
Gaucher disease results from impaired activity of the lysosomal enzyme glucocerebrosidase. Aiming at overexpressing the human glucocerebrosidase and testing the efficiency of the two in-frame ATGs of its gene in directing synthesis of an active enzyme, it was coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). cDNAs containing either one or both ATGs of the glucocerebrosidase mRNA were linked to the T7 polymerase promoter. Recombinant viruses were produced and used for infecting human cells in tissue culture. The results demonstrated that both ATGs directed translation of active glucocerebrosidase, resulting in a 10-fold increase in enzymic activity. Most of the protein remained sensitive to endoglycosidase H. The active enzyme represented a small fraction of the expressed glucocerebrosidase. The recombinant enzyme had the same Km and optimal pH towards the artificial substrate 4-methylumbelliferyl glucopyranoside as the authentic endogenous human enzyme. Measurements of intracellular enzymic activity directed by the cDNAs with either one or both ATGs in cells loaded with a fluorescent glucosylceramide demonstrated a 30% increase in activity directed by the cDNAs containing the first ATG over that containing the second ATG. This indicates that the protein synthesized from the first ATG, with a 38 amino acid leader, is translocated through the endoplasmic reticulum more readily than its counterpart directed by the second ATG, with a 19 amino acid leader. The elevation in glucocerebrosidase activity and the reproducibility of the data leads us to propose the use of the vaccinia virus-derived expression system as a tool for studying glucocerebrosidase mutants in Gaucher disease.
Pasmanik-Chor
M
M
Department of Cell Research and Immunology, Tel-Aviv University, Ramat-Aviv, Israel.
Elroy-Stein
O
O
Aerts
H
H
Agmon
V
V
Gatt
S
S
Horowitz
M
M
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D005776:Gaucher Disease / Q000201:enzymology*; D005962:Glucosylceramidase / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000096:biosynthesis* / Q000235:genetics; D012313:RNA / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D014616:Vaccinia virus / Q000235:genetics
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012313:RNA; D005962:Glucosylceramidase