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

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 ( BC h E ) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BC h E (r BC h E ) in N icotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co‐expression of BC h E with a novel gene‐stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of r BC h E decorated with sialylated N ‐glycans. The N ‐glycosylation profile of monomeric r BC h E secreted to the apoplast largely resembles the plasma‐derived orthologue. In contrast, r BC h E 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 r BC h E oligomers in the endoplasmic reticulum or endoplasmic‐reticulum‐derived compartments. In summary, we show the assembly of r BC h E multimers, however, also points to the need for in‐depth studies to explain the unexpected subcellular targeting of oligomeric BC h E in plants.