J. Biol. Chem., Vol. 263, Issue 33, 17278-17283, 11, 1988

Selective inhibition of glycoprotein-processing enzymes. Differential inhibition of glucosidases I and II in cell culture

GP Kaushal, YT Pan, JE Tropea, M Mitchell, P Liu and AD Elbein
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.

In this study, we compared the effects of 2,6-dideoxy-2,6-imino-7-O- (beta-D-glucopyranosyl)-D-glycero-L-gulohep titol (MDL) to those of the glucosidase I inhibitor, castanospermine, on the purified processing enzymes glucosidases I and II. WE also compared the effects of these two inhibitors on glycoprotein processing in cell culture using influenza virus-infected Madin-Darby canine kidney cells as a model system. With the purified processing enzymes, castanospermine was a better inhibitor of glucosidase I than of glucosidase II, whereas MDL is more effective against glucosidase II than glucosidase I. In cell culture at the appropriate dose, MDL also preferentially affected glucosidase II. Thus, at 250 micrograms/ml MDL, the major [3H]glucose- labeled (or [3H]mannose-labeled) glycopeptide from the viral hemagglutinin was susceptible to endoglucosaminidase H, and the oligosaccharide liberated by this treatment was characterized as a Glc2Man7-9GlcNAc on the basis of size, resistance to digestion by glucosidase I (but sensitivity to glucosidase II), methylation analysis, and Smith degradation studies. These data indicate that at appropriate concentrations of MDL (250 micrograms/ml), one can selectively inhibit glucosidase II in Madin-Darby canine kidney cells. However, at higher concentrations of inhibitor (500 micrograms/ml), both enzymes are apparently affected. Since MDL did not greatly inhibit the synthesis of lipid-linked saccharides or the synthesis of protein or RNA, it should be a useful tool for studies on the biosynthesis and role of N-linked oligosaccharides in glycoprotein function.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?