Evaluation of the early processing routes of N-linked oligosaccharides of glycoproteins through the characterization of Man(8)GlcNAc(2) isomers: Evidence that endomannosidase functions in vivo in the absence of a glucosidase blockade

Since it has become apparent that the early processing of the N-linked oligosaccharides of glycoproteins can proceed by several routes, we undertook to determine whether the isomeric nature of Man(8)GlcNAc(2), which is the first intermediate with the potential for structural diversity, can provide information relating to the pathways utilized in various intact cultured cells as well as in the total membrane fraction derived from these cells (BW5147.3, HepG2, HL-60, F-9, and MDCK), With the use of kifunensine (KIF) to block processing by Golgi mannosidase I, it could be shown that a substantial amount of Man(8)GlcNAc(2) components in which the terminal mannose is missing in the alpha 1,3-linked and alpha 1,6-linked chain (isomers A and C, respectively) are produced, although in the absence of the inhibitor only the B-isomer, in which the mannose of the middle chain has been excised, was apparent, Our findings in vivo and in vitro suggest that the distinctive Man(8)GlcNAc(2) product of endomannosidase (isomer A) and of ER mannosidase II (isomer C) are not evident in the absence of KIF, since they are rapidly degraded by Golgi mannosidase I, which is located in an intracellular compartment distal to the other two enzymes and itself exclusively generates the Man(8)GlcNAc(2) isomer B. Investigations carried out in HepG2 cells indicated that glycoproteins with N-linked oligosaccharides whose processing has been blocked by KIF at the Man(8)GlcNAc(2) isomer A and C stage can nevertheless be effectively secreted, The observation that isomer A of Man(8)GlcNAc(2) is a specific product of endomannosidase action made it possible to demonstrate the action of this enzyme in vivo without employing a glucosidase blockade and to show that a substantial amount of the deglucosylation of N-linked oligosaccharides is carried out by this enzyme.