Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase

Like lower and higher eucaryotes, insects have a 1,2-mannosidases which fun ction in the processing of N-glycans. We previously cloned and characterize d an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a m ember of a family of N-glycan processing a 1,2-mannosidases (Kawar, Z., Her scovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2- mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobio logy 7, 433-443). These enzymes have similar protein sequences, require cal cium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect a 1,2-manno sidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiol ogy 10, 347-355). Now, we have examined the biosynthesis and subcellular lo calization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is ch anged to one that is highly utilized in a mammalian system. We also found t hat an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lyso somes. Finally, N-glycosylation had no influence over the substrate specifi city or subcellular localization of SfManI. (C) 2001 Elsevier Science Ltd. All rights reserved.