Demonstration that a kifunensine-resistant alpha-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells.
A novel alpha-mannosidase has been identified in rat liver endoplasmic reticulum (ER) which at neutral pH processes the Man9GlcNAc oligosaccharide of glycoproteins by specifically cleaving the terminal mannose residue of the alpha 1,6-linked chain to yield Man8GlcNAc, isomer C. This enzyme accounted for about half of the total ER alpha-mannosidase activity and was fully active at the concentration (0.25 microM) of kifunensine (KIF) completely inhibitory to the action of the ER enzyme which by removing the terminal sugar of the middle chain converts Man9GlcNAc to Man8GlcNAc isomer B; both ER enzymes, however, were inhibited in a similar manner by 1-deoxymannojirimycin (IC50 = 0.2 mM) and their action could not be distinguished with this agent. The KIF-resistant mannosidase which functioned optimally in the presence of 0.1-0.5% Triton X-100 did not show the high susceptibility to EDTA demonstrated by the KIF-sensitive enzyme and unlike the latter had the capacity to hydrolyze p-nitrophenyl-alpha-D-mannoside (Km = 0.45 mM); it had no specific cation requirements, but its activity was greatly reduced in the presence of Zn2+. In isolated ER membranes as well as in intact carbonyl cyanide m-chlorophenylhydrazone-treated cells, the processing pattern was substantially different in the presence of KIF than in its absence; while in the latter instance Man9GlcNAc was readily converted to Man6GlcNAc, the KIF-resistant enzyme was limited in its capacity to go beyond Man8GlcNAc. The KIF-resistant alpha-mannosidase was found in substantial amounts in all cell lines examined (HL-60, BW5147.3, MOLT-4, K-562, HepG2, Chinese hamster ovary, F-9, Madin-Darby canine kidney, FRTL-5). The finding that mannose removal from N-linked oligosaccharides can be initiated in two distinctive manners substantially broadens our concept of the processing events which can occur before a glycoprotein reaches the Golgi complex or to which ER resident molecules can be exposed.
Weng
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
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D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured; D043323:alpha-Mannosidase
D000470:Alkaloids; D009844:Oligosaccharides; C065629:kifunensine; D017485:1-Deoxynojirimycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D008361:Mannosidases; D043323:alpha-Mannosidase