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 cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as 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 in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction 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 of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway.
Ogier-Denis
E
E
Institut National de la Santé et de la Recherche Médicale (INSERM) U504 Glycobiologie et Signalisation Cellulaire, 16 Avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France.
Bauvy
C
C
Cluzeaud
F
F
Vandewalle
A
A
Codogno
P
P
D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D016466:CHO Cells / Q000187:drug effects; D002236:Carbohydrate Conformation; D002277:Carcinoma / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005959:Glucosidases / Q000037:antagonists & inhibitors; D006023:Glycoproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D054836:Indolizidines
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D005947:Glucose; D005959:Glucosidases; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D007212:Indolizines; D008358:Mannose; D018832:Molecular Chaperones; D009844:Oligosaccharides; C037806:castanospermine; D054836:Indolizidines