Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath.
Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.
Evron
Tama
T
School of Life Sciences and Biodesign Inst, P.O. Box 874501, Arizona State University, Tempe, AZ 85287-4501, USA.
Geyer
Brian C
BC
Cherni
Irene
I
Muralidharan
Mrinalini
M
Kilbourne
Jacquelyn
J
Fletcher
Samuel P
SP
Soreq
Hermona
H
Mor
Tsafrir S
TS
D000110:Acetylcholinesterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000633:toxicity*; D010261:Paraoxon / Q000633:toxicity*; D030821:Plants, Genetically Modified; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D015996:Survival Rate; D014018:Tissue Distribution / Q000187:drug effects; D014026:Nicotiana / Q000235:genetics*
D007306:Insecticides; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D000110:Acetylcholinesterase; D010261:Paraoxon