EXPRESSION AND PROCESSING OF RECOMBINANT HUMAN GALACTOSYLCERAMIDASE

Stable transformants of CHO cells that overexpress human galactosylcer amidase (GALC) were established. The GALC within the cell consisted of 50- and 30-kDa proteins. The active GALC secreted into the culture me dium in large amounts consisted of the 80-kDa precursor enzyme. We con firmed that the precursor enzyme was taken up by fibroblasts via the m annose-6-phosphate receptor and processed into the 50- and 30-kDa frag ments. Fragmentation was inhibited by the lysosomotropic agents chloro quine and NH4Cl, suggesting that it occurs within the lysosome. GALC m utations identified in globoid cell leukodystrophy suppressed fragment ation. 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. (C) 1998 Elsevier Science B.V. All rights reserved.