Biochemical characterization of two distinct acetylcholinesterases possessing almost identical catalytic activity in the damselfly Vestalis gracilis

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2). It has been recently reported that only one AChE (either AChE1 or AChE2) has been selected as the main synaptic enzyme and it varies with different insect lineages (Kim et al., 2012, Kim and Lee, 2013). Interestingly, however, both AChE1 and AChE2 are almost equally active in a damselfly species, providing a unique example of the incomplete specialization of one AChE function after duplication, where, consequently, both AChE1 and AChE2 likely play a similar role in synaptic transmission. In this study, therefore, we investigated the tissue distribution patterns and the molecular and inhibitory properties of two AChEs (i.e., VgAChE1 and VgAChE2) from the Vestalis gracilis damselfly as a model species possessing two AChEs that are equally active. VgAChEs exhibited almost identical catalytic activity and were expressed in the central nervous system (CNS). The most predominant molecular form of both VgAChEs was a disulfide-bridged dimer, which is associated with the cell membrane via a glycosylphosphatidylinositol anchor. In an inhibition assay, however, VgAChE1 and VgAChE2 exhibited different sensitivities to organophosphate and carbamate insecticides depending on the structure of the inhibitors. These findings suggest that both VgAChEs have neuronal functions. In addition, soluble monomeric and cleaved molecular forms were detected in both the CNS and peripheral nervous system tissues by an AChE2-specific antibody, implying that VgAChE2 probably shares both neuronal and non-neuronal physiological functions in V. gracilis. Our results support the notion that both VgAChEs, paralogous of each other, are involved in synaptic transmission, with VgAChE2 being in the early stage of acquiring non-neuronal functions.     

Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, V_max and K_m, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in K_i ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.     

Functional study on the mutations in the silkworm (Bombyx mori) acetylcholinesterase type 1 gene (ace1) and its recombinant proteins

The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1’s characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 μM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 μM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE’s roles in pesticide resistance.     

A monoclonal antibody against synaptic AChE: a useful tool for detecting apoptotic cells

The classical function of acetylcholinesterase (AChE) is to terminate synaptic transmission at cholinergic synapses by rapidly hydrolyzing the neurotransmitter acetylcholine (ACh). Non-classical functions of AChE involve accelerating the assembly of Abeta peptide into amyloid fibrils and participating in haematopoiesis and neurite growth. Although numerous antibodies have been raised against AChE, many researchers have questioned their reliability to identify the AChE in situ, especially with the regard to its non-classical roles. Researchers attended the Ninth International Meeting on Cholinesterase raised this question by showing different Western blot patterns of AChE detected by different Abs. Producing more effective and reliable Abs for measuring AChE in vivo or in situ has become an important issue in many scientific fields. In this paper, we introduce a monoclonal antibody raised against synaptic AChE that we identified by Western blot assays, immunofluorescent staining and immunoprecipitation of AChE, and mass spectrometry. Our results strongly demonstrate the specificity of our monoclonal antibody to recognize synaptic AChE; hence our antibody can be used as an effective tool to study the various functions of AChE. Since the apoptosis-related AChE was its synaptic form, our antibody can be used as a tool to detect apoptotic cells.     

Altered GPI modification of insect AChE improves tolerance to organophosphate insecticides

The olive fruit fly Bactrocera oleae is the most destructive and intractable pest of olives. The management of B. oleae has been based on the use of organophosphate (OP) insecticides, a practice that induced resistance. OP-resistance in the olive fly was previously shown to be associated with two mutations in the acetylcholinesterase (AChE) enzyme that, apparently, hinder the entrance of the OP into the active site. The search for additional mutations in the ace gene that encodes AChE revealed a short deletion of three glutamines (??3Q) from a stretch of five glutamines, in the C-terminal peptide that is normally cleaved and substituted by a GPI anchor. We verified that AChEs from B. oleae and other Dipterans are actually GPI-anchored, although this is not predicted by the “big-PI” algorithm. The ??3Q mutation shortens the unusually long hydrophilic spacer that follows the predicted GPI attachment site and may thus improve the efficiency of GPI anchor addition. We expressed the wild type B. oleae AChE, the natural mutant ??3Q and a constructed mutant lacking all 5 consecutive glutamines (??5Q) in COS cells and compared their kinetic properties. All constructs presented identical K_m and k_cat values, in agreement with the fact that the mutations did not affect the catalytic domain of the enzyme. In contrast, the mutants produced higher AChE activity, suggesting that a higher proportion of the precursor protein becomes GPI-anchored. An increase in the number of GPI-anchored molecules in the synaptic cleft may reduce the sensitivity to insecticides.     

Pharmacogenetic Regulation of Acetylcholinesterase Activity in Drosophila Reveals the Regulatory Mechanisms of AChE Inhibitors in Synaptic Plasticity

We conducted experiments in Drosophila to investigate the consequences of altered acetylcholinesterase (AChE) activity in the nervous system. In ace hypomorphic mutant larvae, the amount of ace mRNA and the activity of AChE both in vivo and in vitro were significantly reduced compared with those of controls. Reduced Ace in Drosophila larvae resulted in significant down-regulation of branch length and the number of boutons in Type 1 glutamatergic neuromuscular junctions (NMJs). These defects in ace hypomorphic mutant larvae were suppressed when Musca domestica AChE was transgenically expressed. Because AChE inhibitors are utilized for medications for Alzheimer’s disease, we investigated whether pharmacological inhibition of AChE activity induced any synaptic defects. We found that controls exposed to a sublethal dose of DDVP phenocopied the synaptic structural defects of the ace hypomorphic mutant. These results suggest that down-regulation of AChE activity, regardless of whether it is due to genetic or pharmacological manipulations, results in altered synaptic architecture. Our study suggests that exposure to AChE inhibitors for 6–12 months may induce altered synaptic architectures in human brains with Alzheimer’s diseases, similar to those reported here. These changes may underlie or contribute to the loss of efficacy of AChE inhibitors after prolonged treatment.

     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Identification and Molecular Characterization of Two Acetylcholinesterases from the Salmon Louse,Lepeophtheirus salmonis

Acetylcholinesterase (AChE) is an important enzyme in cholinergic synapses. Most arthropods have two genes (ace1 and ace2), but only one encodes the predominant synaptic AChE, the main target for organophosphates. Resistance towards organophosphates is widespread in the marine arthropod Lepeophtheirus salmonis. To understand this trait, it is essential to characterize the gene(s) coding for AChE(s). The full length cDNA sequences encoding two AChEs in L. salmonis were molecularly characterized in this study. The two ace genes were highly similar (83.5% similarity at protein level). Alignment to the L. salmonis genome revealed that both genes were located close to each other (separated by just 26.4 kbp on the L. salmonis genome), resulting from a recent gene duplication. Both proteins had all the typical features of functional AChE and clustered together with AChE-type 1 proteins in other species, an observation that has not been described in other arthropods. We therefore concluded the presence of two versions of ace1 gene in L. salmonis, named ace1a and ace1b. Ace1a was predominantly expressed in different developmental stages compared to ace1b and was possibly active in the cephalothorax, indicating that ace1a is more likely to play the major role in cholinergic synaptic transmission. The study is essential to understand the role of AChEs in resistance against organophosphates in L. salmonis.     

Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.     

Which acetylcholinesterase functions as the main catalytic enzyme in the Class Insecta?

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2; encoded by ace1 and ace2 genes, respectively). Between the two AChEs, AChE1 has been proposed as a major catalytic enzyme based on its higher expression level and frequently observed point mutations associated with insecticide resistance. To investigate the evolutionary distribution of AChE1 and AChE2, we determined which AChE had a central catalytic function in several insect species across 18 orders. The main catalytic activity in heads was determined by native polyacrylamide gel electrophoresis in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Of the 100 insect species examined, 67 species showed higher AChE1 activity; thus, AChE1 was considered as the main catalytic enzyme. In the remaining 33 species, ranging from Palaeoptera to Hymenoptera, however, AChE2 was predominantly expressed as the main catalytic enzyme. These findings challenge the common notion that AChE1 is the only main catalytic enzyme in insects with the exception of Cyclorrhapha, and further demonstrate that the specialization of AChE2 as the main enzyme or the replacement of AChE1 function with AChE2 were rather common events, having multiple independent origins during insect evolution. It was hypothesized that the generation of multiple AChE2 isoforms by alternative splicing allowed the loss of ace1 during the process of functional replacement of AChE1 with AChE2 in Cyclorrhapha. However, the presence of AChE2 as the main catalytic enzyme in higher social Hymenoptera provides a case for the functional replacement of AChE1 with AChE2 without the loss of ace1. The current study will provide valuable insights into the evolution of AChE: which AChE has been specialized as the main catalytic enzyme and to become the main target for insecticides in different insect species.     

无脊椎动物乙酰胆碱酯酶研究进展

乙酰胆碱酯酶(AChE)是生物体中一种十分重要的神经递质水解酶,也是有机磷和氨基甲酸酯类杀虫剂的作用靶标。AChE在不同生物中的性质显著不同,如编码基因个数、序列保守性、表达分布及生理功能等。作为杀虫剂的主要作用靶标之一,AChE不但可以通过单个点突变引起昆虫抗药性,还能够通过多个点突变联合作用、靶标表达量变化及基因复制等方式引起抗药性并且改变昆虫的适合度代价。本文主要从AChE的基因类型、分子进化、蛋白结构、生理功能、与昆虫的抗药性关系、同一物种中不同AChE的性质等6个方面对昆虫纲、蛛形纲和线虫等无脊椎动物AChE的研究进展作一综述。     

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP‐ or CB‐resistant strains have been generated. In this study, wild‐type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21‐fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.     

Alterations in both acetylcholinesterase activity and synaptic scaffolding protein localization in the nervous system of Drosophila presenilin mutants

Presenilins are one of two types of critical genetic factors in familial Alzheimer's disease, and they regulate various cellular functions such as intracellular Ca²⁺ homeostasis, the endoplasmic reticulum (ER) stress response, apoptosis, and synaptic transmission. We utilized Drosophila presenilin (psn) mutants as a model for studying the role of this gene in regulating acetylcholinesterase activity (AChE) and synaptic plasticity. Several lines of biochemical evidence indicated that AChE activity in a functionally null psn mutant (psn^B3) was significantly reduced. In addition, we also found that psn^B3 mutant neuromuscular junctions (NMJs) had smaller synaptic boutons and altered localization of Discs large, a synaptic scaffolding protein at the synaptic terminals compared to wild-type controls. These phenotypic defects were completely rescued in transgenic lines expressing the long form of wild-type Psn under an endogenous psn promoter cassette (PEPC-Psn^WT;psn^B3 lines). Taken together, these results indicate that Psn is important for regulating AChE activity, the size of synaptic boutons, and the localization of DLG at synaptic terminals.     

乙酰胆碱酯酶的非经典功能及其与A1zheimer's疾病的关系

乙酰胆碱酯酶（acetylcholinesterase,AChE)是主要存在于神经系统的一种水解酶,其经典功能是水解神经递质乙酰胆碱,从而终止神经冲动的传递。但是近年来,研究者发现许多证据表明它具有“非经典”的新功能,引起了人们的关注。除了水解神经递质乙酰胆碱的经典功能外,AChE对神经细胞的分化、迁移,突触的形成,造血系细胞和肿瘤细胞的增殖与分化调控也有作用。最近的研究结果显示：AChE可能在细胞凋亡过程中起重要作用,这对于认识Alzheimer‘s疾病（AD）的发病机理又有新的进步。     

Subcellular localization of overexpressed maize AChE gene in rice plant

The ACh-mediated system consisting of acetylcholine (ACh), acetylcholine receptor (AChR) and acetylcholinesterase (AChE) is fundamental for nervous system function in animals and insects. Although plants lack a nervous system, both ACh and ACh-hydrolyzing activity have been widely recognized in the plant kingdom. The function of the plant ACh-mediated system is still unclear, despite more than 30 years of research. To understand ACh-mediated systems in plants, we previously purified maize AChE and cloned the corresponding gene from maize seedlings (Plant Physiology). In a recent paper in Planta, we also purified and cloned AChE from the legume plant siratro (Macroptilium atropurpureum). In comparison with electric eel AChE, both plant AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase. On the other hand, based on Pfam protein family analysis, both plant AChEs contain a consensus sequence of the lipase GDSL family, while the animal AChEs possess a distinct alpha/beta-hydrolase fold superfamily sequence, but no lipase GDSL sequence. Thus, neither plant AChE belongs to the well-known AChE family, which is distributed throughout the animal kingdom. To address the possible physiological roles of plant AChEs, we herein report our data from the immunological analysis of the overexpressed maize AChE gene in plants.Key words: acetylcholinesterase activity, maize AChE gene, overexpression, rice, subcellular localizationIn the animal ACh-mediated system, ACh serves to propagate an electrical stimulus across the synaptic junction. At the presynaptic neuron end, an electrical impulse triggers the release of ACh, which accumulates in vesicles into the synaptic cleft via exocytosis. ACh then binds to an ACh receptor (AChR) on the postsynaptic neuron surface, and the ACh-AChR binding induces subsequent impulses to the postsynaptic neuron. Finally, ACh, which is released again by the receptor into the synaptic cleft, is rapidly degraded by acetylcholinesterase (AChE; E.C.3.1.1.7).^1,2 ACh and AChE,^3,4,5 and choline acetyltransferase activity that takes part during synthesis of ACh^6,7 have been recognized in plants. AChR has not been identified in plant cells so far. However, so-called “ACh-binding sites” were detected in membrane fractions from some bean seedlings^8,9 and evidence was also detected in plant organelles, such as chloroplasts¹⁰ and tonoplasts¹¹ using pharmacological methods.Concerning the function of the ACh-mediated system in plants, Momonoki^12,13 has proposed that it results in an asymmetric distribution of hormones and substances due to gravity stimuli, as well as changes in ACh content, AChE activity and Ca²⁺ level in response to heat stress. However, all these phenomena have been investigated using indirect techniques. Thus, to understand the plant ACh-mediated system, we purified AChEs and cloned the AChE genes from maize¹⁴ and siratro¹⁵ seedlings. The maize AChE was found to exist as two types of 88-kDa homodimers, which in turn consisted of disulfide-linked and noncovalently-linked 42- to 44-kDa subunits.¹⁴ The siratro AChE might exist as a disulfide-linked 125-kDa homotrimer consisting of 41- or 42-kDa subunits.¹⁵ The plant AChEs apparently from various quaternary structures, depending on the plant species, similar to animal AChEs. Furthermore, maize and siratro AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase, compared with electric eel AChE.¹⁵In this addendum, we report our recent immunohistochemical study using an antibody against maize AChE. In order to overexpress the maize AChE gene in rice plants, we constructed a plasmid for the sense expression of the AChE gene by cloning it into the pT7 Blue vector. The maize AChE gene¹⁴ was introduced behind the maize ubiquitin 1 promoter (Ubi) in the p2K-1⁺ plant expression vector. The Ubi::maize AChE and control (p2K-1⁺ only) plasmid were introduced into Agrobacterium tumefaciens EHA 101, which was transformed into rice (Nihonbare) via Agrobacterium-mediated transformation methods.¹⁶ The maize AChE transgenic plants exhibited approximately 650-fold higher AChE activity than was observed in the control plants but no phenotypic changes between transgenic and control plants. The subcellular localization of AChE was observed by immunofluorescence in paraffin-embedded leaf and stem tissues of transgenic rice plants. The maize AChE protein was detected in extracellular spaces in the leaf and stem of the plants (Fig. 1). Therefore, plant AChEs may function in the extracellular space, similar to some isoforms of animal AChE.^2,17Open in a separate windowFigure 1Subcellular localization of maize AChE in leaf and stem of transgenic rice. (A) Leaf cross-section of transgenic rice; (B) leaf cross-section of control; (C) stem cross-section of transgenic rice; (D) stem cross-section of control. Each section was probed with maize AChE antibody and then visualized with Alexa Fluor 488-conjugated secondary antibody. Control indicates rice plants transfected with p2K-1⁺ vector only. Arrowheads indicate localization of maize AChE.Most of the AChE activity in the root was associated with cell wall materials.¹⁸ The computer-assisted cellular sorting prediction program TargetP presumed that our purified maize AChE¹⁴ is targeted to the secretory pathway via the endoplasmic reticulum. Furthermore, the SOSUI program (http://sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), which discriminates between membrane and soluble proteins, showed that the maize AChE does not contain any likely transmembrane helical regions, which are features of proteins that associate with the lipid bilayers of the cell membrane. These findings suggested that the maize AChE might be localized at the cell wall. However, in an earlier work,¹³ we speculated that AChE is localized at the plasmodesmatal cell-to-cell interface and that it plays a role in regulation of the plasmodesmatal channel as a constituent of the ACh-mediated system. We improved our hypothesis of the role of the ACh-mediated system in a paper in Plant Physiol.¹⁴The results based on fluorescence-immunohistochemistry in transgenic rice plants reported in this paper confirmed that the maize AChE is localized at the cell wall. Here we propose again our hypothesis of an ACh-mediated system including this new finding; the system might be localized to the extracellular region around the plasmodesmatal channel and might conduct cell-to-cell trafficking by channel gating regulation. Adjoining cells in plant tissues are interconnected via plasmodesmata, which allow the trafficking of low-molecular-mass materials across the cell wall between two cells. According to a recent model,¹⁹ transport of these substances could be regulated by the opening and/or closing of conductive channels to prevent infection by pathogens and to selectively control trafficking through the plasmodesmata. Furthermore, it has been speculated that morphoregulatory proteins around the plasmodesmata could be involved in channel regulation.²⁰ Therefore, the ACh-mediated system might regulate the opening and/or closing of channels by interactions with morphoregulatory proteins at the cell wall matrix surrounding the plasmodesmata. Further research will be required to clarify the precise physiological roles of plant AChEs.     

Characterization of trimeric acetylcholinesterase from a legume plant, <Emphasis Type="Italic">Macroptilium atropurpureum</Emphasis> Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41–42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs. Kosuke Yamamoto and Yoshie S. Momonoki contributed equally to this work.     

Cloning and characterization of acetylcholinesterase 1 genes from insecticide-resistant field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae)

The psocid, Liposcelis paeta Pearman, is an increasingly important polyphagous pest of stored products worldwide. Intensive use of organophosphorous insecticides for pest control has facilitated resistance development in psocids in China. Three insecticide-resistant field populations of L. paeta were collected from Nanyang city of Henan Province (NY), and Wuzhou (WZ) and Hezhou (HZ) cities of Guangxi Province, China. Previous studies have shown that psocids have different susceptibilities to insecticides. In addition, their AChE susceptibilities to paraoxon-ethyl and demeton-S-methyl also differed from each other. Acetylcholinesterase 1, which is one of the major targets for organophosphate insecticides, has been fully cloned and sequenced from these populations of L. paeta. Comparison of both nucleotide and deduced amino acid sequences revealed nucleotide polymorphisms among L. paeta ace 1 genes from different populations, but none of these polymorphisms correspond to the active sites in AChE 1 from other insects. The results of comparative quantitative real-time PCR indicated that the relative expression level of HZ ace 1 gene was the highest among three populations, which was 1.20 and 1.02-fold higher than those of NY and WZ populations, respectively. This may due to an epigenetic inheritance phenomenon, which allows organisms to respond to a particular environment through changes in gene expression.