The effect of tunicamycin on the glycosylation of lactating-rabbit mammary glycoproteins

Abstract

Tunicamycin inhibited the incorporation of D-[2-3H]mannose into dolichol-linked oligosaccharide and glycoprotein of lactating-rabbit mammary explants by approximately the same extent approximately 30% of control value), suggesting that lipid-linked intermediates are involved in the mannosylation of mammary glycoproteins. The incorporation radioactivity from N-acetyl-D-[1-14C] glucosamine into dolichol-linked oligosaccharide was inhibited by tunicamycin to 32% of the control value, whereas the incorporation of the radiolabel into glycoprotein was only inhibited to 72% of the control value. Considerable redistribution of label from N-acetylglucosamine to N-acetylgalactosamine was found to occur in the explants. In the presence of tunicamycin .apprx. 76% of the radioactivity incorporated into glycoprotein from N-acetyl-D-[1-14C]glucosamine was present as N-acetylgalactosamine, compared with .apprx. 61% in the absence of the inhibitor. Tunicamycin selectively inhibits the incorporation of N-acetylglucosamine into glycoprotein. Radioactivity from N-acetyl-D-[1-14C]glucosamine was incorporated into a glycoprotein that was identified as casein by the use of a casein-specific antiserum, and also into a group of glycopolypeptides with apparent MW ranging between 40,000-80,000. N-Acetylgalactosamine was the only radioactive sugar released on strong-acid hydrolysis of the immunoprecipitated casein, whereas N-acetylglucosamine was the major radioactive residue present in the non-casein glycoproteins. Glucosamine and galactosamine were the only radiolabelled sugars detected by paper chromatography of the strong-acid hydrolysate of the protein fraction. Tunicamycin inhibited the incorporation of radioactivity from N-acetyl-D-[1-14C]glucosamine into the glycopolypeptides with MW between 40,000 and 80,000 as described by polyacrylamide-gel electrophoresis, but did not affect the incorporation of label into casein. Tunicamycin may inhibit the incorporation of mannose and N-acetylglucosamine into a number of mammary glycoproteins by inhibiting the formation of lipid-linked intermediates, but it does not inhibit the incorporation of N-acetylgalactosamine into casein.