Differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture

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To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of alpha 1-protease inhibitor, ceruloplasmin, and alpha 2-macroglobulin, but had no effect on the export of fibronectin, alpha-fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized alpha 1-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized alpha 1-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on alpha 1-protease inhibitor, ceruloplasmin and alpha 2-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.