Characterization of endomannosidase inhibitors and evaluation of their effect on N-linked oligosaccharide processing during glycoprotein biosynthesis.
Endo-alpha-D-mannosidase is a Golgi-located processing enzyme that achieves deglucosylation of N-linked carbohydrate units through its unique property of cleaving the oligosaccharide chain internally with the release of glucose-substituted mannose (Glc1-3Man). By chemically modifying the characteristic disaccharide product, Glc alpha 1-->3Man, a number of potent inhibitors of the endomannosidase were obtained, foremost among which were Glc alpha 1-->3(1-deoxy)mannojirimycin (Glc alpha 1-->3DMJ) and Glc alpha 1-->3(1,2-dideoxy)mannose (IC50 = 1.7 and 3.8 microM, respectively), which, while blocking the in vitro action of the enzyme, had negligible effect on other endoplasmic reticulum- and Golgi-processing glycosidases. Although preparation of a large number of Glc alpha 1-->3DMJ derivatives did not yield a more effective endomannosidase inhibitor it provided valuable information relating to the structural requirements for the enzyme-substrate interaction. Glc alpha 1-->3DMJ was found to be active not only on rat liver endomannosidase but also on the enzyme from a number of other sources including mouse lymphoma (BW5147.3), HepG2, baby hamster kidney, and Madin-Darby canine kidney cell lines. When tested in vivo in lymphoma and Madin-Darby canine kidney cells during a castanospermine-imposed glucosidase blockade, Glc alpha 1-->3DMJ interrupted the endomannosidase processing pathway as evident from a concomitant inhibition of complex oligosaccharide formation and Glc3Man release; similarly the capacity of the glucosidase II-deficient mouse lymphoma cell line (PHAR2.7) to synthesize complex oligosaccharides was blocked by Glc alpha 1-->3DMJ. Endomannosidase could not be detected in Chinese hamster ovary cells by in vitro assay and consistent with this these cells produced only glucosylated polymannose N-linked oligosaccharides during glucosidase blockade. It would appear that by acting in conjunction with a glucosidase inhibitor, Glc alpha 1-->3DMJ and related endomannosidase-blocking agents could have the potential of influencing the exit of glycoproteins from the endoplasmic reticulum and interfering with viral replication.
Hiraizumi
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02215.
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D017485:1-Deoxynojirimycin / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002250:Carbon Radioisotopes; D006224:Cricetinae; D004187:Disaccharides / Q000138:chemical synthesis / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008223:Lymphoma; D008297:Male; D008361:Mannosidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D004187:Disaccharides; D006023:Glycoproteins; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D006026:Glycoside Hydrolases; D008361:Mannosidases