Limits...
Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana.

Schneider JD, Marillonnet S, Castilho A, Gruber C, Werner S, Mach L, Klimyuk V, Mor TS, Steinkellner H - Plant Biotechnol. J. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH
Subcellular localization of rBChE in Nicotiana benthamiana leaf epidermal cells. Expression of p20BChE (rBChE-GFP) and GnTI-CAAATS-mRFP (a fusion protein that is predominantly endoplasmic-reticulum-retained) was monitored 2 dpi by live-cell confocal laser scanning microscopy. (a) CLSM image of a cell expressing p20BChE; (b) CLSM image of GnTI-CAAATS-mRFP expressed in the same cell. Punctate structures represent Golgi bodies as frequently observed with GnTI-CAAATS-mRFP (Farid et al., 2011; Schoberer et al., 2009); (c) corresponding overlay of both images. Co-localization appears in yellow. Boxed insets (bottom left corner) show a higher magnification of the region indicated by arrowheads. A significant co-localization of the two constructs was observed. Scale bar = 40 μm for all images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4265266&req=5

fig03: Subcellular localization of rBChE in Nicotiana benthamiana leaf epidermal cells. Expression of p20BChE (rBChE-GFP) and GnTI-CAAATS-mRFP (a fusion protein that is predominantly endoplasmic-reticulum-retained) was monitored 2 dpi by live-cell confocal laser scanning microscopy. (a) CLSM image of a cell expressing p20BChE; (b) CLSM image of GnTI-CAAATS-mRFP expressed in the same cell. Punctate structures represent Golgi bodies as frequently observed with GnTI-CAAATS-mRFP (Farid et al., 2011; Schoberer et al., 2009); (c) corresponding overlay of both images. Co-localization appears in yellow. Boxed insets (bottom left corner) show a higher magnification of the region indicated by arrowheads. A significant co-localization of the two constructs was observed. Scale bar = 40 μm for all images.

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).


Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana.

Schneider JD, Marillonnet S, Castilho A, Gruber C, Werner S, Mach L, Klimyuk V, Mor TS, Steinkellner H - Plant Biotechnol. J. (2014)

Subcellular localization of rBChE in Nicotiana benthamiana leaf epidermal cells. Expression of p20BChE (rBChE-GFP) and GnTI-CAAATS-mRFP (a fusion protein that is predominantly endoplasmic-reticulum-retained) was monitored 2 dpi by live-cell confocal laser scanning microscopy. (a) CLSM image of a cell expressing p20BChE; (b) CLSM image of GnTI-CAAATS-mRFP expressed in the same cell. Punctate structures represent Golgi bodies as frequently observed with GnTI-CAAATS-mRFP (Farid et al., 2011; Schoberer et al., 2009); (c) corresponding overlay of both images. Co-localization appears in yellow. Boxed insets (bottom left corner) show a higher magnification of the region indicated by arrowheads. A significant co-localization of the two constructs was observed. Scale bar = 40 μm for all images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265266&req=5

fig03: Subcellular localization of rBChE in Nicotiana benthamiana leaf epidermal cells. Expression of p20BChE (rBChE-GFP) and GnTI-CAAATS-mRFP (a fusion protein that is predominantly endoplasmic-reticulum-retained) was monitored 2 dpi by live-cell confocal laser scanning microscopy. (a) CLSM image of a cell expressing p20BChE; (b) CLSM image of GnTI-CAAATS-mRFP expressed in the same cell. Punctate structures represent Golgi bodies as frequently observed with GnTI-CAAATS-mRFP (Farid et al., 2011; Schoberer et al., 2009); (c) corresponding overlay of both images. Co-localization appears in yellow. Boxed insets (bottom left corner) show a higher magnification of the region indicated by arrowheads. A significant co-localization of the two constructs was observed. Scale bar = 40 μm for all images.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH