Title: Biochemical and toxicological properties of two acetylcholinesterases from the common bed bug, Cimex lectularius Hwang CE, Kim YH, Kwon DH, Seong KM, Choi JY, Je YH, Lee SH Ref: Pestic Biochem Physiol, 110:20, 2014 : PubMed
We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.
Title: The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae Kwon DH, Choi JY, Je YH, Lee SH Ref: Insect Biochemistry & Molecular Biology, 42:212, 2012 : PubMed
The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8 approximately 27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.
Title: Three acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus: insights into distinct physiological functions Kang JS, Lee DW, Choi JY, Je YH, Koh YH, Lee SH Ref: Molecular & Biochemical Parasitology, 175:154, 2011 : PubMed
Acetylcholinesterase (AChE) plays a key role in postsynaptic transmission in most animals. Nematodes encode multiple AChEs, implying its functional diversity. To explore physiological functions of multiple AChEs, three distinct AChEs (BxACE-1, BxACE-2, and BxACE-3) were identified and characterized from the pinewood nematode. Sequencing comparison with Torpedo AChE and Caenorhabditis elegans ACEs identified choline-binding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that BxACE-3 is more actively transcribed than BxACE-1 (2-3 times) and BxACE-2 (9-18 times) in both propagative and dispersal stages. The three BxACEs were functionally expressed using baculovirus system. Kinetic analysis of in vitro-expressed BxACEs revealed that the substrate specificity was highest in BxACE-1 whereas the catalytic efficiency was highest in BxACE-2. In inhibition assay, BxACE-3 showed the lowest inhibition rate. Taken together, it appears that both BxACE-1 and BxACE-2 play common but non-overlapping roles in synaptic transmission, whereas BxACE-3 may have non-neuronal functions. The current findings should provide valuable insights into the evolutionary process and various physiological roles of AChE.
Title: Functional analysis and molecular characterization of two acetylcholinesterases from the German cockroach, Blattella germanica Kim YH, Choi JY, Je YH, Koh YH, Lee SH Ref: Insect Molecular Biology, 19:765, 2010 : PubMed
Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. With the exceptions of paraoxon and propoxur, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulphide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagen-like tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.
Title: Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.) Lee DW, Choi JY, Kim WT, Je YH, Song JT, Chung BK, Boo KS, Koh YH Ref: Biochemical & Biophysical Research Communications, 353:591, 2007 : PubMed
Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.
