Expression of biologically active human \u3ci\u3ebutyrylcholinesterase\u3c/i\u3e in the cabbage looper (\u3ci\u3eTrichoplusia ni\u3c/i\u3e)

This investigation examined the utility of three recombinant protein-expression systems (COS cells, insect cells and insect larvae) to cost-effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus-infected insect cells (Sf9 and High 5) expressed 3.5- and 8.2-fold, respectively, more active enzyme than COS-7 cells. Baculovirus-infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells ; in fact, one baculovirus-infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti-cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical-warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein-expression system for human BuChE

Evidence for Nonacetylcholinesterase Targets of Organophosphorus Nerve Agent: Supersensitivity of Acetylcholinesterase Knockout Mouse to VX Lethality

The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34–55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 µg/kg in AChE-/-, 17 µg/kg in AChE+/-, and 24 µg/kg in AChE+/- mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE