Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue

Abstract

Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. However, the presence and functional necessity of complex N-glycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. Here we transiently co-expressed BChE cDNA in the model plant Nicotiana benthamiana with vectors carrying the genes necessary for in planta protein sialylation. Site-specific sugar profiling of secreted recombinant BChE (rBChE) collected from the intercellular fluid revealed the presence of mono- and di-sialylated N-glycans, which largely resembles to the plasma-derived orthologue. Attempts to increase that sialylation content of rBChE by the over-expression of an additional glycosylation enzyme that generates branched N-glycans (i.e. β1,4-N-acetylglucosaminyl-transferase IV), allowed the production of rBChE decorated with tri-sialylated structures (up to 70%). Sialylated and non-sialylated plant-derived rBChE exhibited functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.

Keywords: Butyrylcholinesterase; Glycoengineering; Plants; Recombinant biopharmaceuticals; Sialic acid.

Figure 1

Expression of human BChE A, Schematic representation of the expression vector and the protein backbone of human BChE. BChE cDNA was cloned into a modified TMV-based magnICON vector (pICHα26211) resulting in pBChE. Apo: Signal peptide sequence from barley α amylase; BChE: Human butyrylcholinesterase sequence lacking the native signal peptide; LB: Left border; MP: Movement protein; PAct2: Arabidopsis thaliana actin 2 promoter; RB: Right border; Tnos: Nopaline synthase gene terminator; TVCV polymerase: Turnip vein clearing virus RNA-dependent RNA polymerase; 3’UTR: TVCV 3’-untranslated region; B and C, Monitoring of rBChE expression in N. benthamiana. Expression of BChE was analyzed over a period of time ranging from 0–12 dpi. TSP (panel C, 4 µg protein per lane) and IF-derived proteins (panel D, 1µg protein per lane) were fractionated using 10% SDS-PAGE and the proteins were detected using anti-BChE antibodies. Experiments were performed using 4 different leaves transiently expressing BChE and Western blots were done in triplicates. D, Protein backbone of human BChE. Numbers in the box (1–9) refer to the glycopeptides obtained upon trypsin digestion. Bold numbers below the box indicate the positions of the N-glycosylated asparagine residues within the amino acid sequence of the protein.

Figure 2

N-Glycan profiles of rBChE glycopeptides Gps 2 and 7 expressed in N. benthamiana wild-type (WT) and the glycosylation mutant ΔXT/FT. A, Mass spectra of IF-derived BChE. B, Mass spectra of IF-derived rBChE co-expressed with genes necessary for in planta sialylation (BChE_sia). Western blot analysis of BChE_sia (lane 1) and BChE (lane 2) are shown on the left. Protein bands used for glycan analysis are boxed. Mass spectra were generated after liquid-chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of glycopeptides (Gp) obtained upon trypsin digestion. Peaks are labeled in accordance with the ProGlycAn system (www.proglycan.com). The suffix “iso” at the end of glycan abbreviations denotes the probable presence of isomers. Major glyco-forms are shown as cartoons, for detailed description of cartoons see Supporting Information, Figure S3. Unassigned peaks are background originating from co-eluting peptides. Figure shows a representative glycoprofiling of BChE expressed in different plants out of an average of four experiments.

Figure 3

N-Glycan profiles of IF-derived rBChE Gps 2 and 7 co-expressed with genes necessary for in planta sialylation (BChE_sia) and N-acetylglucosaminyltransferase II (GnTII). WT (top panel) and ΔXT/FT (middle and bottom panels) plants were used as expression hosts. Bottom panels show the MS profile of IF-derived BChE_trisia obtained upon co-expression of genes for in planta sialyltion, GnTII, and GnTIV (glycosyltransferase responsible for branching). Mass spectra were generated after liquid-chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of glycopeptides (Gp) obtained upon trypsin digestion. The suffix “iso” at the end of glycan abbreviations denotes the probable presence of isomers. Major glyco-forms are shown as cartoons, for detailed description of cartoons see Supporting Information, Figure S3. Unassigned peaks are background originating from co-eluting peptides. Figure shows a representative glycoprofiling of BChE expressed in different plants out of an average of four experiments.

Figure 4

Evaluation of in vitro activity of plant-derived rBChE by a modified Ellman assay. Hydrolysis of butyrylthiocholine was evaluated in the presence of Ellman’s reagent in sodium phosphate buffer. Substrate conversion rates were measured at different time points. Equine serum-derived BChE served as a reference. TSP_WT: TSP of wild-type plants infiltrated with agrobacteria that do not carry the BChE cDNA; eBChE: equine BChE; BChE_WT: BChE expressed in WT plants (main glyco-form GnGnXF); BChE_ΔXT/FT: BChE expressed in ΔXT/FT plants (main glyco-form GnGn); BChE_sia: BChE co-expressed with the genes for in planta sialylation in ΔXT/FT plants (main glyco-form NaNa). All samples were analyzed in triplicates.