Processing of Lysosomal β-Galactosidase

Abstract

Lysosomal ? -D-galactosidase ( ? -gal), the enzyme defi- cient in the autosomal recessive disorders GM1 gangli- osidosis and Morquio B, is synthesized as an 85-kDa precursor that is C-terminally processed into a 64–66- kDa mature form. The released ?20-kDa proteolytic fragment was thought to be degraded. We now present evidence that it remains associated to the 64-kDa chain after partial proteolysis of the precursor. This polypep- tide was found to copurify with ? -gal and protective protein/cathepsin A from mouse liver and Madin-Darby bovine kidney cells and was immunoprecipitated from human fibroblasts but not from fibroblasts of a GM1 gan- gliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosia- lidosis fibroblasts resulted in a significant increase of mature and active ? -gal and its C-terminal fragment. Expression in COS-1 cells of mutant cDNAs encoding either the N-terminal or the C-terminal domain of ? -gal resulted in the synthesis of correctly sized polypeptides without catalytic activity. Only when co-expressed, the two subunits associate and become catalytically active. Our results suggest that the C terminus of ? -gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal ? -galactosidase is a two-subunit molecule. These data may give new signifi- cance to mutations in GM1 gangliosidosis patients found in the C-terminal part of the molecule.