SUBSTRATE SPECIFICITIES OF RECOMBINANT MURINE GOLGI ALPHA-1,2-MANNOSIDASES IA AND IB AND COMPARISON WITH ENDOPLASMIC-RETICULUM AND GOLGI PROCESSING ALPHA-1,2-MANNOSIDASES

The catalytic domains of murine Golgi alpha 1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted p roteins in P.pastoris, Recombinant mannosidases IA and IB both require d 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 inve rting glycosidase, Recombinant mannosidases IA and IB were used to cle ave Man(9)GlcNAc and the isomers produced were identified by high perf ormance liquid chromatography and proton-nuclear magnetic resonance sp ectroscopy, 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 f ormed by both enzymes. These results combined with proton-nuclear magn etic resonance spectroscopy data demonstrate that it is the terminal a lpha 1,2-mannose residue missing in the Man8B isomer that is cleaved f rom Man9GlcNAc at a much slower rate. When rat liver endoplasmic retic ulum membrane extracts were incubated with Man(9)GlcNAc(2), Man(8)GlcN Ac(2) was the major product and Man8B was the major isomer, In contras t, 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 i somers of Man(8)GlcNAc(2) mere formed as intermediates, but a distinct ive isomer, Man8A, was predominant. Antiserum to recombinant mannosida se LA immunoprecipitated an enzyme from Golgi extracts with the same s pecificity as recombinant mannosidase IA, These immunodepleted membran es were enriched in a Man(9)GlcNAc(2) to Man(8)GlcNAc(2)-cleaving acti vity forming predominantly the Man8B isomer. These results suggest tha t mannosidases IA and IB in Golgi membranes prefer the Man8B isomer ge nerated by a complementary mannosidase that removes a single mannose f rom Man(9)GlcNAc(2).