N-Glycan processing by a lepidopteran insect α1,2-mannosidase

Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha 1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI, A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column, The purified SflManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses 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)GlcNAc(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 demonstrated 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 the 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-mannosidase directly shown to produce Man(7)GlcNAc(2) isomer C as a major processing intermediate.