Substrate Specificities and Intracellular Distributions of Three N-glycan Processing Enzymes Functioning at a Key Branch Point in the Insect N-Glycosylation Pathway

Abstract

Background: Insect cells have a branched protein N-glycosylation pathway. Results: Substrate specificities and intracellular distributions of three insect N-glycan processing enzymes were determined. Conclusion: Sf-GNT-I, Sf-GNT-II, and Sf-FDL function around the branch point and determine the net outcome of the insect protein N-glycosylation pathway. Significance: Deeper understanding of the insect protein N-glycosylation pathway.

A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency

“…The remaining pool of paucimannose-type structures might reflect the presence of a processing beta- N -acetylglucosaminidase in Sf9 cells (Altmann et al, 1995; Geisler et al, 2008), which competes with MGAT1 and MGAT2 to drive the production of paucimannose-type structures (Geisler and Jarvis, 2012b; Wagner et al, 1996a). An oft-stated goal of insect cell glycoengineering has been to reduce or eliminate this competing activity, as this might be another way to increase the efficiency of human-type N -glycan processing.…”

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology

“…Finally, we should note that the new S2R+ cell derivatives produced in this study will serve as excellent platforms enabling more efficient and efficacious bottom-up insect cell glycoengineering efforts. The production of a subpopulation of paucimannosidic N-glycans, perhaps by α-mannosidase III, could be addressed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 by editing the α-mannosidase III gene and/or overexpressing the endogenous Mgat1 gene, as the latter gene product can "rescue" paucimannosidic N-glycans and set the stage for their further elongation 41,60 . Combined with the addition of selected heterologous N-glycan elongation functions, the absence of the antagonistic trimming activity at the key N-glycan processing branchpoint in these cells should yield derivatives that can produce recombinant glycoproteins with efficiently processed, perhaps homogeneous, chemically pre-defined oligosaccharide side-chain structures.…”

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter

“…Subcellular Localization-Sf9 and Drosophila S2 cells were transfected with expression plasmids encoding RFP-tagged S. frugiperda MGAT1 (44) and GFP-tagged WT or nac 1 mutant GFR proteins (pAcP(ϩ)DmGFR-WT-GFP or pAcP(ϩ)DmGFR-nac-GFP), plated on concanavalin A-coated dishes, and photographed essentially as described before (44). An Olympus FSX100 microscope was used at ϫ80 magnification, and the manufacturer's FSX-BSW version 03.01 software was used for image capture at 1360 ϫ 1024 pixels.…”

“…To distinguish between these possibilities, we expressed GFP-tagged forms of the WT and nac 1 GFRs in Drosophila S2 cells as well as in Sf9 cells, which had been used for the in vitro GDP-fucose transport assays. We used RFP-tagged insect N-acetylglucosaminyltransferase I (MGAT1) as a Golgi marker because this enzyme acts immediately upstream of HRP epitope synthesis by producing the FucTA acceptor substrate (44,(51)(52)(53). The red and green fluorescence patterns observed in these experiments each had punctate, cytoplasmic distributions typical of the multiple Golgi apparatuses found in lepidopteran insect cells (Fig.…”