Seminars

Human butyrylcholinesterase (hBuChE EC 3.1.1.8) is a 574 amino acid, pseudo-acetylcholinesterase that serves as a bioscavenger for organophosphates (OP) like sarin. OPs are highly toxic inhibitors of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase, resulting in the accumulation of acetylcholine. The build up of acetylcholine can lead to respiratory collapse and death. Current therapies are based on elevating the serum levels of OP bioscavengers like hBuChE. The major limitation of this therapy is high cost, with plasma-derived hBuChE costing more than \1,000,000/400mg/treatment. Limitations like cost and availability necessitate an alternative expression platform capable of large scale, low-cost production of a fully active and efficacious recombinant hBuChE (rhuChE). The development of an effective rhBuChE is a pressing national security concern in terms of protecting warfighters and civilian populations from the threat of attack with OP agents. We describe the use of plant viral amplicon expression systems based on the Tobacco mosaic virus (TMV) to express rhBuChE in Nicotiana benthamiana plants using transient agroinfiltration. Human BuChE is a glycoprotein with nine potential N-glycosylation sites. The glycan structures are complex and capped with N-acetylneuraminic (sialic) acid. For this reason, the plant-made rhBuChE was characterized and all nine sites were found to be occupied by typical plant glycans. It is also known that plants are incapable of sialylating glycoproteins naturally and that sialylation is essential for the normal serum half-life of proteins. To increase the number of sialic acid residues per rhBuChE molecule, we systematically added GlcNAC, galactose and sialic acid to biantennary-termini of plant N-glycans using “one-pot” multistep enzymatic reactions (i.e., in vitro sialylation). Preliminary results indicate that approximately 11% of the apoplast-targeted rhBuChE was sialylated. Because 30% of all commercial biopharmaceuticals are glycoproteins, these results could make plant-made pharmaceuticals a viable alternative to mammalian or insect expression systems.