Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharideprocessing which through its capacity to cleave the internal linkagebetween the glucose-substituted mannose and the remainder of thepolymannose carbohydrate unit can provide an alternate pathway forachieving deglucosylation and thereby make possible the continued formationof complex oligosaccharides during a glucosidase blockade. In view of theimportant role which has been attributed to glucose on nascentglycoproteins as a regulator of a number of biological events, we chose tofurther define the in vivo action of endomannosidase by focusing on thewell characterized VSV envelope glycoprotein (G protein) which can beformed by the large array of cell lines susceptible to infection by thispathogen. Through an assessment of the extent to which the G protein wasconverted to an endo-beta-N- acetylglucosaminidase (endo H)-resistant formduring a castanospermine imposed glucosidase blockade, we found thatutilization of the endomannosidase-mediated deglucosylation route wasclearly host cell specific, ranging from greater than 90% in HepG2 and PtK1cells to complete absence in CHO, MDCK, and MDBK cells, with intermediatevalues in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of thelatter group the electrophoretic pattern after endo H treatment suggestedthat only one of the two N-linked oligosaccharides of the G protein wasprocessed by endomannosidase. In the presence of the specificendomannosidase inhibitor, Glcalpha1-->3(1- deoxy)mannojirimycin, theconversion of the G protein into an endo H- resistant form was completelyarrested. While the lack of G protein processing by CHO cells wasconsistent with the absence of in vitro measured endomannosidase activityin this cell line, the failure of MDBK and MDCK cells to convert the Gprotein into an endo H-resistant form was surprising since these cell lineshave substantial levels of the enzyme. Similarly, we observed thatinfluenza virus hemagglutinin was not processed in castanospermine-treatedMDCK cells. Our findings suggest that studies which rely on glucosidaseinhibition to explore the function of glucose in controlling such criticalbiological phenomena as intracellular movement or quality control should becarried out in cell lines in which the glycoprotein under study is not asubstrate for endomannosidase action.