Glycobiology, 2002, Vol. 12, No. 6 369-377
© 2002 Oxford University Press
Biosynthesis and processing of Spodoptera frugiperda 
-mannosidase III
3 Department of Molecular Biology, University of Wyoming, P.O. Box 3944, Laramie, WY 82071-3944, USA
We previously cloned a lepidopteran insect cell cDNA that encodes a class II 
-mannosidase that is localized in the Golgi apparatus but is cobalt-dependent, has a neutral pH optimum, hydrolyzes Man5GlcNAc2 to Man3GlcNAc2, and cannot hydrolyze GlcNAcMan5GlcNAc2. This enzyme was designated SfManIII to distinguish it from Golgi -mannosidase II and indicate its derivation from the fall armyworm Spodoptera frugiperda. In the present study, we prepared a polyclonal antibody and used it to study the biosynthesis and processing of SfManIII. The results showed that Sf9 cells produce at least three different forms of SfManIII. SfManIII is initially synthesized as a precursor glycoprotein, which is slowly converted to two smaller end products with at least some endoglycosidase H-resistant N-glycans. The smallest form of SfManIII is the only one of these two products that accumulates in the extracellular fraction. Tunicamycin blocked the production of SfManIII activity and the secretion of SfManIII protein and activity. Castanospermine blocked production of the larger SfManIII product, retarded production of the smaller, increased intracellular SfManIII activity, and decreased extracellular SfManIII activity. Together, these results indicate that SfManIII is initially synthesized as a high-mannose glycoprotein precursor, its N-glycans are trimmed as it is transported to the Golgi apparatus, and a subpopulation, which appears to be proteolytically cleaved, is secreted in enzymatically active form. N-glycosylation is required for the production of active SfManIII, and N-glycosylation and N-glycan trimming are both required for the efficient secretion of an active form of this protein.
1 Present affiliation: Ambion, Inc., Austin, TX
2 To whom correspondence should be addressed; E-mail: dljarvis{at}uwyo.edu
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