Effect of tunicamycin and endoglycosidase H and F on the estrogen regulated 52000-Mr protein secreted by breast cancer cells.
The glycosylation and immunoreactivity of an estrogen regulated glycoprotein secreted by breast cancer cells in culture and defined by its molecular mass (52 000-Mr protein) have been studied indirectly using an inhibitor of glycosylation and specific endoglycosidases. The protein and its deglycosylated forms were immunoprecipitated with specific monoclonal antibodies to the 52 000-Mr protein and analyzed by SDS polyacrylamide gel electrophoresis. The 52 000-Mr protein was intensely labelled by [3H] mannose or [35S] methionine. Tunicamycin treatment of the cells, endoglycosidase H or endoglycosidase F digestion of conditioned media, gave two identical deglycosylated forms of 50 000-Mr and 48 000-Mr which remained immunoreactive. The 48 000-Mr protein, in contrast to the 52 000 and 50 000-Mr proteins, was unable to bind concanavalin A. The 52 000-Mr protein was resolved into five spots of decreasing pI on two-dimensional gels following immunoprecipitation. Endoglycosidase H treatment decreased the molecular weight and reduced the intensity of spots of lower pI, suggesting that the N-glycosylated chains contain acidic molecules. We conclude that: The 52 000-Mr secreted protein contains at least two high mannose or hybrid N-glycosylated chains of approximately 2,000 molecular weight corresponding to 8% of the mass of the 52 000-Mr protein. The two types of monoclonal antibodies (site 1 and 2) raised against the 52 000-Mr glycoprotein are still able to recognize the 48 000-Mr N-deglycosylated form indicating that they do not interact with the N-glycosylated moiety of the molecule.
D000118:Acetylglucosaminidase / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005643:Fucose / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives*; D006026:Glycoside Hydrolases / Q000494:pharmacology*; D006596:Hexosaminidases / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology*
D009363:Neoplasm Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin; D005643:Fucose; D004958:Estradiol; D008715:Methionine; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D005944:Glucosamine; D008358:Mannose