Glycosylation and processing of carbohydrate side chains of ecto-5'-nucleotidase in cultured human chorionic cells

Glycosylation and carbohydrate processing of ecto-5'-nucleotidase were studied in cultured human chorionic cells using metabolic labelling and immunoprecipitation with monoclonal antibodies. Tunicamycin blocks glycosylation altogether leading to a reduction in molecular mass of 9,500 Da. The same result is obtained by digesting the mature 72,000-Da protein with endoglycosidase F. Using various inhibitors of the carbohydrate-trimming reactions like deoxynojirimycin, deoxymannojirimycin and swainsonine smaller molecular mass reductions are observed and the oligosaccharide side chains are kept in a configuration sensitive to endoglycosidase H digestion. Digestion of mature 5'-nucleotidase with endoglycosidase H leads to a much smaller (2,000 Da) reduction in molecular mass. It is calculated that, in addition to the phosphatidylinositol-glycan anchor structure, ecto-5'-nucleotidase of human chorionic cells should carry 4 oligosaccharide side chains per subunit, 3 of which should be of the complex and one of the high mannose type. Interference with carbohydrate processing by various inhibitors does not seem to influence the distribution of ecto-5'-nucleotidase between the cell surface and intracellular membranes nor does it block the transfer of the enzyme to the phosphatidylinositol glycan anchor.