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.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Jeannine D
authors.initials: JD
authors.lastname: Schneider
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Marillonnet
authors.forename: Alexandra
authors.initials: A
authors.lastname: Castilho
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.forename: Stefan
authors.initials: S
authors.lastname: Werner
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.forename: Victor
authors.initials: V
authors.lastname: Klimyuk
authors.forename: Tsafrir S
authors.initials: TS
authors.lastname: Mor
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D002091:Butyrylcholinesterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D030821:Plants, Genetically Modified / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D002091:Butyrylcholinesterase
keywords: butyrylcholinesterase; glycoengineering; plants; sialic acid; subcellular targeting