Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana.
Bottom Line: Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers.In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans.In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures.
Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.Show MeSH
Mentions: A GFP-tagged BChE fusion protein was generated (rBChE-GFP, p20BChE; Figure 1a) to monitor the subcellular localization of recombinant BChE. The integrity of rBChE-GFP was confirmed by immunoblot using antibodies against GFP to exclude artefactual staining derived from free GFP (Figure S3). Nicotiana benthamiana leaf epidermal cells expressing rBChE-GFP were examined by live-cell confocal laser scanning microscopy (CLSM) at 2 dpi. A reticulate staining pattern typical for the cortical ER network was visible in many cells (Figure 3, panel A). Co-localization of rBChE-GFP with a predominantly ER-retained mRFP fusion protein (GnTI-CAAATS-mRFP, Schoberer et al., 2009) strongly suggested at least partial retention of rBChE-GFP in the ER (Figure 3). The results agree with the data obtained by N-glycosylation analyses of FLAGBChE (and FLAGBChEsia) purified from TSP (Figure S2).
Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.