Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)₉(GlcNAc)₂ oligosaccharides

Abstract

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 highmannose 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 pM, 75 pM, and 140 pM, respectively.Man9 mannosidase cleaves three of the four a1 ,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 a-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 (Gl~),(Man),(GlcNAc)~ intermediate, we assume that the enzyme is located in the endoplasmic reticulum.The structural diversity of N-linked oligosaccharides is the result of extensive modifications of a common (Gl~)~(Man),-(GlcNAc)2 precursor which is preassembled on a lipid carrier and, after transfer en bloc to the polypeptide chain, processed to high-mannose, hybrid and/or complex-type oligosaccharides. Processing begins with the stepwise removal of the three glucose residues by presumably two enzymes, glucosidase I and glucosidase 11. The resulting high-mannose intermediate is either preserved as such or acted upon by several u-mannosidases before other sugars, such as N-acetylglucosamine, galactose, sialic acid and fucose, are added [I].Up to now, four different mannosidase activities assumed to be involved in the mannose trimming reactions (endoplasmic reticulum mannosidase, Golgi mannosidase IA, IB and 11), have been identified and partially characterized on the basis of differences in their detergent extractability, response toward various inhibitors and some enzymatic properties [2-61. Here, we report on the isolation and purification to homogeneity of a processing mannosidase (Man9 mannosidase) from calf liver microsomes. Though similar in some respects, the calf liver enzyme differs from the abovementioned mannosidases especially in its substrate specificity. Thus, Man9 mannosidase hydrolyses three of the four a1,2-linked mannose residues from the natural (Man)9(GlcNAc)2 oligosaccharide, but does not cleave the remaining (Man)6-(GlcNAc)2 structure and is not active against aryl a-mannoCorrespondence to E. Bause, Institut fur Biochemie, Universitat Koln, Ziilpicher Stra...

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