Glycobiology, 2000, Vol. 10, No. 4 347-355
© 2000 Oxford University Press
N-Glycan processing by a lepidopteran insect 
1,2-mannosidase
2Department of Molecular Biology, University of Wyoming, Laramie, WY 82071–3944, USA and 3McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada
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 
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 SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man9GlcNAc2 to Man6GlcNAc2 isomer C, then more slowly converts Man6GlcNAc2 isomer C to Man5GlcNAc2. The slow step in the processing of Man9GlcNAc2 to Man5GlcNAc2 by SfManI is removal of the 1,2-linked mannose on the middle arm of Man9GlcNAc2. In this respect, SfManI is similar to mammalian 1,2-mannosidases IA and IB. However, additional HPLC and 1H-NMR analyses demonstrated that SfManI converts Man9GlcNAc2 to Man5GlcNAc2 primarily through Man7GlcNAc2 isomer C, the archetypal Man9GlcNAc2 missing the lower arm 1,2-linked mannose residues. In this respect, SfManI differs from mammalian 1,2-mannosidases IA and IB, and is the first 1,2-mannosidase directly shown to produce Man7GlcNAc2 isomer C as a major processing intermediate.
1 To whom correspondence should be addressed
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