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J Biol Chem, Vol. 275, Issue 14, 10035-10040, April 7, 2000

Processing of Lysosomal -Galactosidase
THE C-TERMINAL PRECURSOR FRAGMENT IS AN ESSENTIAL DOMAIN OF THE MATURE ENZYME^*

Aarnoud van der Spoel^§¶, Erik Bonten^¶, and Alessandra d'Azzo

From the  Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Lysosomal -D-galactosidase (-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 ~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 -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 -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 significance to mutations in G_M1 gangliosidosis patients found in the C-terminal part of the molecule.

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