Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)9(GlcNAc)2 oligosaccharides.
A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-1-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of high-mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1-deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 microM, 75 microM, and 140 microM, respectively. Man9 mannosidase cleaves three of the four alpha 1,2-linked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrolyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl alpha-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum.
D017485:1-Deoxynojirimycin; D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002845:Chromatography; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
D002413:Cations, Divalent; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D008361:Mannosidases; D005944:Glucosamine; D008358:Mannose; D000117:Acetylglucosamine