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.