N-Glycan processing by a lepidopteran insect alpha1,2-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 alpha1,2-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 Man(9)GlcNAc(2)to Man(6)Glc-NAc(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 alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate.
Kawar
Z
Z
Department of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944, USA.
Romero
P A
PA
Herscovics
A
A
Jarvis
D L
DL
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D018411:Spodoptera / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose; D000117:Acetylglucosamine