N-Glycan processing by a lepidopteran insect alpha 1,2-mannosidase

Protein glycosylation pathways are relatively poorly characterized in insec t cells. As part of an overall effort to address this problem, we previousl y isolated a cDNA from Sf9 cells that encodes an insect alpha 1,2-mannosida se (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimyci n. In the present study, we have characterized the substrate specificity of SfManI, A recombinant baculovirus was used to express a GST-tagged secrete d form of SfManI which was purified from the medium using an immobilized gl utathione column, The purified SflManI was then incubated with oligosacchar ide substrates and the resulting products were analyzed by HPLC. These anal yses showed that SfManI rapidly converts Man(9)GlcNAc(2) to Man(6)GlcNAc(2) isomer C, then more slowly converts Man(6)GlcNAc(2) isomer C to Man(5)GlcN Ac(2). The slow step in the processing of Man(9)GlcNAc(2) to Man(5)GlcNAc(2 ) by SfManI is removal of the alpha 1,2-linked mannose on the middle arm of Man(5)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha 1,2 -mannosidases IA and IB, However, additional HPLC and H-1-NMR analyses demo nstrated that SflManI converts Man(9)GlcNAc(2) to Man(5)GlcNAc(2) primarily through Man(9)GlcNAc(2) isomer C, the archetypal Man(9)GlcNAc(2) missing t he lower arm a1,2-linked mannose residues. In this respect, SflManI differs from mammalian al,2-mannosidases IA and IB, and is the first alpha 1,2-man nosidase directly shown to produce Man(7)GlcNAc(2) isomer C as a major proc essing intermediate.