Biosynthesis of α-galactosidase A in cultured chang liver cells

An investigation of the structure and biosynthesis of α-galactosidase A (α- d-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2- 3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with M r = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the M r = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of α-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man 8–9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the α-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19–39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.