Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase.
The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, 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 (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-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 approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc 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 approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lipari
F
F
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Herscovics
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004271:DNA, Fungal; D006026:Glycoside Hydrolases / Q000378:metabolism; D008361:Mannosidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004271:DNA, Fungal; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase