TY - JOUR
T1 - Expression and processing of recombinant human galactosylceramidase
AU - Nagano, Sukehisa
AU - Yamada, Takeshi
AU - Shinnoh, Nobue
AU - Furuya, Hirokazu
AU - Taniwaki, Takayuki
AU - Kira, Jun Ichi
N1 - Funding Information:
This work was supported in part by the memorial fund for the late Professor Takuro Kobayashi, as well as by grants from the Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare of Japan.
PY - 1998/8/10
Y1 - 1998/8/10
N2 - 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. Copyright (C) 1998 Elsevier Science B.V.
AB - 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. Copyright (C) 1998 Elsevier Science B.V.
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U2 - 10.1016/S0009-8981(98)00095-3
DO - 10.1016/S0009-8981(98)00095-3
M3 - Article
C2 - 9760019
AN - SCOPUS:0031841041
VL - 276
SP - 53
EP - 61
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
SN - 0009-8981
IS - 1
ER -