The catalytic domains of murine Golgi alpha 1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris, Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Man alpha 1,2Man alpha-O-CH3 as substrate. Mannosidase LA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase, Recombinant mannosidases IA and IB were used to cleave Man(9)GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy, Man(9)GlcNAc was rapidly cleaved by both enzymes to Man(6)GlcNAc, followed by a much slower conversion to Man(5)GlcNAc. The same isomers of Man(7)GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man(8)GlcNAc mere formed, When Man(8)GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man(5)GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha 1,2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man(9)GlcNAc(2), Man(8)GlcNAc(2) was the major product and Man8B was the major isomer, In contrast, rat liver Golgi membranes rapidly cleaved Man(9)GlcNAc(2) to Man(6)GlcNAc(2) and more slowly to Man(5)GlcNAc(2). In this case all three isomers of Man(8)GlcNAc(2) mere formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase LA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA, These immunodepleted membranes were enriched in a Man(9)GlcNAc(2) to Man(8)GlcNAc(2)-cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man(9)GlcNAc(2).
