Expression of Ace-paralogous acetylcholinesterase of Culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system

In Ace paralogous acetylcholinesterase (AP-AChE) of Culex tritaeniorhynchus, an amino acid substitution, Phe455Trp, is accompanied by the insecticide insensitivity. To confirm the responsibility of the substitution to the insensitivity, AP-AChE cDNA with and without a Phe455Trp substitution and Ace orthologous AChE (AO-AChE) cDNA were expressed in a baculovirus-insect cell system and the biochemical properties of AChEs (AP-CxTI, AP-CxTS, and AO-CxT, respectively) determined. AP-CxTI which has the same level of affinity to ACh, the natural substrate, showed a drastic decline in affinity to the artificial substrates composed of a longer moiety. The sensitivity of AP-CxTI to inhibitors was extremely reduced when compared with AP-CxTS. The insensitivity to tested organophosphates was greater than to monomethyl carbamates. AO-AChE showed similar substrate specificity and a slightly higher sensitivity to inhibitors when compared with AP-CxTS. Taking the position of Phe455Trp in the acyl pocket of the active site into account, the dimensions of the acyl pocket appear to became smaller by the substitution and insensitive to inhibitors.