Expression and processing of recombinant human galactosylceramidase.
Stable transformants of CHO cells that overexpress human galactosylceramidase (GALC) were established. The GALC within the cell consisted of 50- and 30-kDa proteins. The active GALC secreted into the culture medium in large amounts consisted of the 80-kDa precursor enzyme. We confirmed that the precursor enzyme was taken up by fibroblasts via the mannose-6-phosphate receptor and processed into the 50- and 30-kDa fragments. Fragmentation was inhibited by the lysosomotropic agents chloroquine and NH4Cl, suggesting that it occurs within the lysosome. GALC mutations identified in globoid cell leukodystrophy suppressed fragmentation. Neither the 50- or 30-kDa fragment expressed had GALC activity, indicative that the entire structure is necessary for enzyme activity and that fragments expressed separately cannot associate to form the active enzyme.
Department of Neurology, Neurological Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
S
S
Nagano
T
T
Yamada
N
N
Shinnoh
H
H
Furuya
T
T
Taniwaki
J
J
Kira
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004705:Endocytosis; D005347:Fibroblasts / Q000201:enzymology; D005698:Galactosylceramidase / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D005698:Galactosylceramidase