Abstract
Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.