Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins

The macroautophagic-lysosomal pathway is a bulk degradative process for cyt osolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell populati on is characterized by a high rate of autophagic degradation of N-linked gl ycoproteins substituted with ER-type glycans. In the present work we demons trate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimyc in] have a stabilizing effect on newly synthesized glucosylated N-linked gl ycoproteins and impaired their lysosomal delivery as shown by subcellular f ractionation on Percoll gradients. The inhibition of macroautophagy was res tricted to N-linked glycoproteins because macroautophagic parameters such a s the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and i n Lec23 cells (a CHO mutant deficient in glucosidase I activity). The inter action of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans o f glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glu cosylated N-linked glycoproteins from being sequestered by the autophagic p athway.