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

Jeannine D. Schneider, Sylvestre Marillonnet, Alexandra Castilho, Clemens Gruber, Stefan Werner, Lukas Mach, Victor Klimyuk, Tsafrir Leket-Mor, Herta Steinkellner

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. 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. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.

Original languageEnglish (US)
Pages (from-to)832-839
Number of pages8
JournalPlant Biotechnology Journal
Volume12
Issue number7
DOIs
StatePublished - 2014

Fingerprint

Butyrylcholinesterase
Nicotiana benthamiana
cholinesterase
glycosylation
Glycosylation
Tobacco
Endoplasmic Reticulum
endoplasmic reticulum
Polysaccharides
Proteins
polysaccharides
proteins
Secretory Pathway
apoplast
post-translational modification
Post Translational Protein Processing
Plantae
Genes
blood proteins
Blood Proteins

Keywords

  • Butyrylcholinesterase
  • Glycoengineering
  • Plants
  • Sialic acid
  • Subcellular targeting

ASJC Scopus subject areas

  • Plant Science
  • Biotechnology
  • Agronomy and Crop Science

Cite this

Schneider, J. D., Marillonnet, S., Castilho, A., Gruber, C., Werner, S., Mach, L., ... Steinkellner, H. (2014). Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. Plant Biotechnology Journal, 12(7), 832-839. https://doi.org/10.1111/pbi.12184

Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. / Schneider, Jeannine D.; Marillonnet, Sylvestre; Castilho, Alexandra; Gruber, Clemens; Werner, Stefan; Mach, Lukas; Klimyuk, Victor; Leket-Mor, Tsafrir; Steinkellner, Herta.

In: Plant Biotechnology Journal, Vol. 12, No. 7, 2014, p. 832-839.

Research output: Contribution to journalArticle

Schneider, JD, Marillonnet, S, Castilho, A, Gruber, C, Werner, S, Mach, L, Klimyuk, V, Leket-Mor, T & Steinkellner, H 2014, 'Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana', Plant Biotechnology Journal, vol. 12, no. 7, pp. 832-839. https://doi.org/10.1111/pbi.12184
Schneider, Jeannine D. ; Marillonnet, Sylvestre ; Castilho, Alexandra ; Gruber, Clemens ; Werner, Stefan ; Mach, Lukas ; Klimyuk, Victor ; Leket-Mor, Tsafrir ; Steinkellner, Herta. / Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana. In: Plant Biotechnology Journal. 2014 ; Vol. 12, No. 7. pp. 832-839.
@article{c34a004db0cc424bb334c3ce6eb69788,
title = "Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana",
abstract = "Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. 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. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.",
keywords = "Butyrylcholinesterase, Glycoengineering, Plants, Sialic acid, Subcellular targeting",
author = "Schneider, {Jeannine D.} and Sylvestre Marillonnet and Alexandra Castilho and Clemens Gruber and Stefan Werner and Lukas Mach and Victor Klimyuk and Tsafrir Leket-Mor and Herta Steinkellner",
year = "2014",
doi = "10.1111/pbi.12184",
language = "English (US)",
volume = "12",
pages = "832--839",
journal = "Plant Biotechnology Journal",
issn = "1467-7644",
publisher = "Wiley-Blackwell",
number = "7",

}

TY - JOUR

T1 - Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana

AU - Schneider, Jeannine D.

AU - Marillonnet, Sylvestre

AU - Castilho, Alexandra

AU - Gruber, Clemens

AU - Werner, Stefan

AU - Mach, Lukas

AU - Klimyuk, Victor

AU - Leket-Mor, Tsafrir

AU - Steinkellner, Herta

PY - 2014

Y1 - 2014

N2 - Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. 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. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.

AB - Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. 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. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.

KW - Butyrylcholinesterase

KW - Glycoengineering

KW - Plants

KW - Sialic acid

KW - Subcellular targeting

UR - http://www.scopus.com/inward/record.url?scp=84906270407&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84906270407&partnerID=8YFLogxK

U2 - 10.1111/pbi.12184

DO - 10.1111/pbi.12184

M3 - Article

C2 - 24618259

AN - SCOPUS:84906270407

VL - 12

SP - 832

EP - 839

JO - Plant Biotechnology Journal

JF - Plant Biotechnology Journal

SN - 1467-7644

IS - 7

ER -