PRODUCTION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT YEAST PROCESSING ALPHA-1,2-MANNOSIDASE

The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man(9)GlcNAc to Man(8)GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S.cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc(1)Man(9)GlcNAc or Man(9)GlcNAc), the medium of cells transformed with this plasmid showed an increase in a-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of similar to 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution H-1 NMR analysis of the Man(8)GlcNAc formed from Man(9)GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by similar to 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations. The K-m for the Man(9)GlcNAc substrate was 0.3 mM. These results demonstrate that the recombinant alpha 1,2-mannosidase has the same properties as the endogenous processing enzyme.