title: Processing of lysosomal beta-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme.
abstract: Lysosomal beta-D-galactosidase (beta-gal), the enzyme deficient in the autosomal recessive disorders G(M1) gangliosidosis and Morquio B, is synthesized as an 85-kDa precursor that is C-terminally processed into a 64-66-kDa mature form. The released approximately 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 polypeptide was found to copurify with beta-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 G(M1) gangliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosialidosis fibroblasts resulted in a significant increase of mature and active beta-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 beta-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 beta-gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal beta-galactosidase is a two-subunit molecule. These data may give new significance to mutations in G(M1) gangliosidosis patients found in the C-terminal part of the molecule.
authors.affiliation: Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
authors.forename: A
authors.initials: A
authors.lastname: van der Spoel
authors.forename: E
authors.initials: E
authors.lastname: Bonten
authors.forename: A
authors.initials: A
authors.lastname: d'Azzo
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D016537:Gangliosidosis, GM1 / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007668:Kidney; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D046911:Macromolecular Substances; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012867:Skin / Q000166:cytology / Q000201:enzymology / Q000473:pathology; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D001616:beta-Galactosidase