Amino acid substitutions conferring insecticide insensitivity in Ace-paralogous acetylcholinesterase

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Abstract

Since insecticide insensitivity of acetylcholinesterase (AChE) was, found about 40 years ago, a cause of the resistance to organophosphates in the spider mite, more than 30 insect and Acarus species have added to the instance. Based on the 3-dimensional analysis of Torpedo AChE structure and sequencing of Drosophila AChE gene (Ace), amino acid substitutions conferring the insensitivity have been found in Drosophila melanogaster. However, no amino acid substitution responsible for the AChE insensitivity had been found in insects and Acari except Brachicera flies until the second type of AChE paralogous to Ace was discovered in Schizaphis graminus and Anopheles gambiae. Sequencing of Ace-paralogous AChE cDNAs has been followed in insect species of various orders. Now, various amino acid substitutions are found and correspond to different biochemical properties of insensitive AChEs in relation to the function of substituted amino acids in the 3-dimensional structure. Existence of two AChE genes raises questions about differentiation of the two genes, site of gene expression, and function of each enzyme.

Introduction

The altered acetylcholinesterase (AChE), as well as other insecticide-target molecules, GABA receptor [1], and sodium ion channel [2], [3], is an important resistant mechanism. In the target insensitivity, it appeared that the substitution of amino acid residues occurs at the conserved positions of the structural protein in a wide range of species from different insect orders. The position of the substitution is located in the proposed important site of sodium ion channel [2], [3]. The insensitivity of AChE also accompanies amino acid substitutions and has been reviewed timely [4], [5], [6], [7], [8]. In this review, the recent progress on molecular mechanism of insensitivity to organophosphates and carbamates is described especially concerning an insect AChE family that is paralogous to Drosophila AChE (Ace).

The insect AChE gene was first sequenced in Drosophila melanogaster [9] and several amino acid substitutions conferring insecticide insensitivity were also first elucidated in this species [10] followed by Brachycera fly species, Musca domestica [11], [12], [13], Lucilia cuprina [14], and Bactrocera oleae [15]. However, there was no amino acid substitution in the Ace-orthologues of other insects whose AChE insensitivity had been well characterized [4], because genome data of D. melanogaster, a model of insects, showed that only one AChE gene exists in its genome [16]. Several years had passed without progress, when studies on the mechanism of AChE insensitivity were restarted by the discovery of the second AChE genes paralogous to Ace in insects [17], [18].

Names of insect AChE are now confused since the second AChE gene was discovered. After the discovery of the new AChE gene, two types of AChEs have been named freely by the workers. To avoid confusion, we use AO-AChE and AP-AChE for Drosophila Ace-orthologous and -paralogous gene family members, respectively, in the text.

Section snippets

Structure of AChE

Acetylcholinesterase (AChE, EC 3.1.1.7) terminates synaptic transmission at cholinergic synapses by hydrolyzing excess acetylcholine (ACh) released from the presynaptic membrane. The inhibition of AChE by organophosphate and carbamate insecticides accumulates ACh in the synaptic gap and causes desensitization of the ACh receptor, leading to a blockage of the signal transmission. Insect AChE is a homodimeric globular protein of about 150 kDa linked to membranes by its C-terminal end with a

AChEs in resistant insects and their biochemical properties

Organophosphates and carbamates have structure analogous to that of the substrate of ACh and inhibit competitively AChE at the active site. Hydrolysis of these inhibitors leads to an enzyme with phosphorylated or carbamylated active serine, and then retards the reactivation of the enzyme excessively.

The insensitivity of AChE to organophosphates was first reported in the two-spotted spider mite, Tetranychus urticae, associated with organophosphate resistance [21]. Biochemical properties of the

Discovery of Ace-paralogous AChE

Molecular studies revealed that the insensitivity of AChEs was accompanied by some amino acid replacements in Brachycera fly species. However, no insensitivity-specific mutation was successfully identified in the AO-AChE transcripts from resistant strains of Cx. pipiens [36], Cx. tritaeniorhynchus [37], M. persicae [38], A. gossypii [39], [40], N. cincticeps [41], Plutella xylostella (Terada unpublished), and Oulema oryzae (Tomita, unpublished). In Cx. tritaeniorhynchus, both the structural

AA substitution in AP-AChE conferring insensitivity

Several different positions have been pointed out for the amino acid substitution conferring insecticide insensitivity of AP-AChE. One is Gly to Ser at the oxyanion hole in Cx. pipiens [18], An. gambiae [47], and Anopheles albimanus [56], and the second is Phe to Trp in Cx. tritaeniorhynchus [37] or Ser to Phe in M. persicae [38], and A. gossypii [39], [40] at the acyl pocket as shown in Table 2. The amino acid substitution of Gly280(119)Ser (Gly280 is replaced by Ser. The number of the

Expression of two AChE genes

There have been reported several studies on the expression of AP-AChE. Northern blot analysis indicated that AP-AChE expression was 1.5-fold higher in the resistant strain of S. graminum than in the susceptible strain [54]. In Boophilus microplus, RT-PCR analysis showed that AChE1 is expressed in salivary glands and ovaries as well as synganglia [55]. Since most of the insects except Brachycera flies appeared to have two types of AChEs, it is necessary to investigate precisely the expression of

Acknowledgments

We thank Dr. DeMar Taylor, Graduate School of Life and Environmental Sciences, University of Tsukuba, for critical reading of the manuscript. We also thank Dr. T. Kozaki, Mr. H. Mizuno, Mr. S.-H. Oh, Mr. O. Komagata, and Mr. M. Terada for their assistance in arranging data.

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