Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme

Abstract

An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa.