Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage

Insect Biochemistry and Molecular Biology 32 (2002) 765–775 www.elsevier.com/locate/ibmb

Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage J.-R. Gao 1, S. Kambhampati, K.Y. Zhu

*

Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506-4004, USA Received 16 August 2001; received in revised form 27 September 2001; accepted 19 October 2001

Abstract An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from a greenbug (Schizaphis graminum (Rondani)) cDNA library. The complete cDNA (3283 bp) contains a 2028-bp open reading frame encoding 676 amino acid residues. The putative AChE preproenzyme has a 17 amino acid signal peptide, a 78 amino acid activation peptide and a mature enzyme of 581 amino acid residues. The first nine amino acid residues (YTSDDPLII) that were determined by sequencing the N-terminus of a 72-kDa AChE purified from the greenbug matched the nine residues deduced from the cDNA. The key amino acid residues, including the three residues Ser206 (200 in Torpedo), Glu332 (327) and His446 (440) forming a catalytic triad, three pairs of cysteine putatively forming intrachain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site gorge of the Torpedo AChE, are conserved. However, Ser336 (Phe331) in the greenbug substituted an aromatic amino acid residue that is conserved in all other known AChEs. Northern blot analysis of mRNA revealed a 3.7-kb transcript, and Southern blot analysis suggested a single copy of this gene in the greenbug. The deduced amino acid sequence is most similar to AChE1 of the nematodes Caenorhabditis briggsae and C. elegans with 43% identity. Phylogenetic analysis showed that the greenbug AChE formed a cluster with those of nematodes, a squid and ticks, and grouped out of the insect cluster. This result suggests that the cloned gene evolved from a different duplicate gene lineage of insect AChEs.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Acetylcholinesterase; Amino acid sequence; cDNA cloning; Greenbug; Phylogenetic tree

1. Introduction Acetylcholinesterase (AChE, EC 3.1.1.7) is an essential enzyme at cholinergic synapses in all animals, as well as at neuromuscular junctions in nematodes and vertebrates (Eldefrawi, 1985; Chalfie and White, 1988; Soreq and Zakut, 1993). It terminates neurotransmission by rapidly hydrolyzing the neurotransmitter acetylcholine. The enzyme is the primary target site of some pharmaceuticals, as well as organophosphate (OP) and carbamate insecticides (Matsumura, 1985; Soreq and Zakut, 1993; Harel et al., 2000). Biochemical characterizations of AChE have been * Corresponding author. Tel.: +1-785-532-4721; fax: +1-785-5326232. E-mail address: [email protected] (K.Y. Zhu). 1 Current address: Department of Entomology, Fernald Hall, University of Massachusetts, Amherst, MA 01003, USA.

carried out in more than 20 insect species (Gao et al., 1998). Molecular analysis of its gene structures, however, has been completed in only a few species including fruit fly (Drosophila melanogaster; Hall and Spierer, 1986), malaria mosquito (Anopheles stephensi; Hall and Malcolm, 1991), yellow fever mosquito (Aedes aegypti; Anthony et al., 1995), Colorado potato beetle (Leptinotarsa decemlineata; Zhu and Clark, 1995), housefly (Musca domestica; Huang et al., 1997), green rice leafhopper (Nephotettix cincticeps; Tomita et al., 2000), and Australian sheep blow fly (Lucilia cuprina; Chen et al., 2001). Recently, the three-dimensional structure of Drosophila AChE has been determined (Harel et al., 2000). It showed similar folding and closely overlapped active sites to those of a vertebrate (Torpedo californica) AChE, despite their relatively low amino acid sequence identity (36%). The greenbug, Schizaphis graminum (Rondani), is an aphid species that often causes serious damage to sor-

0965-1748/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 1 ) 0 0 1 5 9 - X

766

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

ghum, wheat and other small grains in the world (Metcalf and Metcalf, 1993). Recently, we have successfully purified and characterized an AChE from this important insect (Gao and Zhu, 2001). The purified enzyme showed typical features of an AChE, including higher efficiency in hydrolyzing acetylthiocholine (ATC) than butyrylthiocholine (BTC) and high sensitivity to inhibition by eserine and BW284C51, but less sensitivity to ethopropazine. It also possessed some unique biochemical properties such as the lack of affinity to the ligand 3-(carboxyphenyl)ethyldimethyl ammonium, which has been widely used for purification of AChE from other insects (Zhu and Clark, 1994), and 20- to 200-fold higher substrate-inhibition thresholds for ATC and acetyl-(β-methyl)thiocholine (AβMTC) than AChE from other insect species (Gao and Zhu, 2001). In an attempt to further elucidate the uniqueness and importance of the greenbug AChE, we have cloned and characterized a greenbug AChE cDNA. In this paper, we report: (1) the AChE cDNA nucleotide sequence and its deduced amino acid sequence; (2) primary structures of the cDNA-deduced AChE; (3) characteristics of the greenbug AChE; and (4) the phylogenetic relationship of the greenbug AChE to those from other animal species. To our knowledge, this is the first study to characterize molecular properties of AChE in an aphid species. The study is expected to help us to understand the molecular properties of insect AChEs and potentially elucidate molecular mechanisms of altered AChE involved in OP-resistance in the greenbug.

cations. Briefly, 0.5 g of greenbugs of mixed developmental stages was pulverized in liquid nitrogen. After the nitrogen had dissipated, the sample was mixed with 10 ml of extraction buffer (100 mM Tris, 1% SDS, 100 mM EDTA and 0.2 mg/ml proteinase K) and incubated for 90 min at 65 °C. The DNA was extracted with phenol/chloroform and precipitated with ethanol. The precipitated DNA was dissolved in 10 ml of TE buffer (pH 8.0) containing 200 µg/ml RNase A, and incubated at 37 °C for 1 h. The DNA was then extracted with phenol/chloroform, precipitated with ethanol and washed with 70% ethanol once. The DNA was redissolved in TE buffer (pH 8.0) and used for Southern blot analysis. 2.3. Preparations of AChE cDNA probes A 278-bp homologous AChE cDNA probe was generated using semi-nested PCR and degenerate primers, as described by Zhu et al. (2000). The sequences of the forward, nested forward, and reverse primers are 5⬘GGAATTCGA(AG)ATGTGGAA(TC)CC(TCAG)AA (TC), 5⬘GGAATTCTGGAT(TCA)TA(TC)GGIGG(TC AG)GG, and 5⬘GGAATTCCIGCI(GC)(AT)(TC)TC (TCAG)CC(AG)AA, respectively. The PCR-generated probe was used to screen a greenbug cDNA library and later in the Northern blot analysis of the greenbug mRNA. A 1.7-kb cDNA probe, prepared from a cloned AChE cDNA from a greenbug cDNA library by EcoR I digestion, was used in Northern and Southern blot analyses. All the probes were labeled with α-[32P] dATP using a random primers DNA labeling system (Life Technologies).

2. Materials and methods 2.4. Construction of a cDNA library 2.1. Insects An organophosphate-susceptible clone (OSS) of the greenbug was isolated from an OSS strain and cultured on sorghum stalks under the same conditions as described by Zhu and Gao (1999). The OSS clone of the greenbug was mass reared on sorghum plants in a growth chamber at 25 °C with a photoperiod of 16L:8D (Gao and Zhu, 2000). Greenbugs collected from sorghum stalks and whole sorghum plants were used for molecular studies and purification of AChE, respectively. 2.2. RNA and DNA preparations Total RNA was isolated from mixed developmental stages of the greenbug using Trizol reagent (Life Technologies, Gaithersburg, MD). Next, mRNA was purified using MessageMaker Reagent Assembly (Life Technologies) and used for both cDNA synthesis and Northern blot analysis. Genomic DNA was isolated from the greenbug by the method of Sambrook et al. (1989) with slight modifi-

A greenbug cDNA library was constructed using the ZAP-cDNA synthesis kit and the ZAP-cDNA Gigapack III gold cloning kit according to the manufacturer’s instruction (Stratagene, La Jolla, CA). The doublestranded cDNA was synthesized from 5 µg of mRNA. The cDNA was blunt-ended with 5 units of Pfu DNA polymerase, and ligated to the EcoR I adaptors. After phosphorylation of the EcoR I ends, the adaptor-ligated cDNA was digested with Xho I. The cDNA was then size-fractionated through a Sepharose CL-2B gel filtration column, and 100 ng of cDNA was ligated into 1 µg of the Lambda Uni-ZAP XR vector. The ligated DNA was packaged with the Gigapack III gold packaging extract. 2.5. cDNA library screening and DNA sequencing The recombinant clones were grown on agar plates of XL1-Blue MRF⬘ strain of bacterial cells (Stratagene) and transferred onto nitrocellulose membranes (Osmonics, Minnetonka, MN), based on the method of Sambrook et

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

767

al. (1989). The 278-bp homologous PCR-generated probe was used to hybridize the nitrocellulose membranes in aqueous solution, according to Sambrook et al. (1989). The cDNA insert within the Lambda Uni-ZAP XR vector was excised in vivo using the ExAssist helper phage (Stratagene) and recirculated to generate subclones in the pBluescript SK phagemid vector. The cDNA sequence was determined for both strands using an ABI PRISM 3700 DNA analyzer (Foster City, CA) at the Sequencing and Genotyping Facility of Kansas State University, Manhattan, KS.

genetic analysis. Protein distances were calculated with PROTDIST using the Dayhoff PAM matrix and phylogenetic trees were constructed with the aligned sequences by the neighbor-joining (NJ) method (Saitou and Nei, 1987). AChE1 of Caenorhabditis elegans was used as the outgroup for the tree of insect AChEs. Bootstrap values were calculated with SEQBOOT program on 1000 replications of the initial alignment. The tree was drawn by the program TreeView (Page, 1996).

2.6. Southern and Northern blot analyses

AChE was purified from frozen greenbugs using sequential steps of ammonium sulfate precipitation, Sephadex G-150 gel filtration and procainamide-based affinity chromatography as described previously (Gao and Zhu, 2001). Purified AChE samples were subjected to reducing SDS–PAGE (9% separating gel with a 4% stacking gel) after being concentrated with Microcon 30 (Millipore, Bedford, MA). The proteins then were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane (Millipore) using Xcell II Mini-Cell (Invitrogen/NOVEX, San Diego, CA). The N-terminal amino acid residues of the most abundant protein (72 kDa) were determined from the PVDF-blotted samples using automated Edman degradation on a 473A Applied Biosystems Sequencer (Perkin-Elmer, Foster City, CA) at the Biotechnology Core Facilities of Kansas State University, Manhattan, KS.

For Southern blot analysis, genomic DNA was digested with each of five restriction enzymes (EcoR I, EcoR V, Hind III, Kpn I and Pst I) at 37 °C overnight, and separated on a 0.7% agarose gel (10 µg/lane). The DNA in the gel was depurinated with 0.2 N HCl for 10 min, rinsed in distilled water, denatured with 0.4 N NaOH containing 5×SSC for 30 min. The DNA was then transferred to a nylon membrane in the denaturation solution and neutralized in 0.5 M Tris–HCl (pH 7.2), 5×SSC for 15 min. The blot was prehybridized for 1 h at 65 °C in a buffer containing 6×SSC, 5×Denhardt’s solution, 0.5% SDS, 5% dextran sulfate and 0.2 mg/ml sheared herring sperm DNA. Hybridization with [32P]labeled 1.7-kb AChE cDNA probe was performed overnight in the above buffer at 65 °C. The blot was washed in 2×SSC, 0.5% SDS twice at room temperature then in 0.1×SSC, 0.5% SDS four times at 65 °C. The blot was exposed to Kodak X-Omat AR film for 3 h at ⫺80°C with an intensifying screen. Northern blot analysis was performed using the NorthernMax kit according to the manufacturer’s instruction (Ambion, Austin, TX). Briefly, mRNA (9.5 µg/lane) was separated on a 1% agarose gel and transferred to a nylon membrane. The blot was hybridized overnight at 42 °C with a [32P]-labeled 1.7-kb AChE cDNA probe. After being washed, the blot was exposed to Kodak X-Omat AR film for 2 h at ⫺80 °C with an intensifying screen. 2.7. Analyses of cDNA and its deduced amino acid sequences All analyses were carried out with the PHYLIP software package (Felsenstein, 1993) except for the extraction of the conserved sequence blocks. Alignment of amino acid sequences was performed with the Clustal X (version 1.8) (Jeanmougin et al., 1998). Highly conserved blocks of the sequences were first extracted by Block Maker (Henikoff et al., 1995) using blocks from motif option and then revised manually based on the sequence alignment by Clustal X. A total of 250 amino acid residues from each sequence was used for phylo-

2.8. Purification and N-terminal determination of AChE

3. Results and discussion 3.1. AChE cDNA and deduced amino acid sequences Four AChE cDNA clones were isolated from 4×105 recombinant clones from a greenbug cDNA library when a 278-bp DNA fragment amplified by semi-nested PCR from greenbug cDNA was used as a homologous probe. Sequencing of these cDNAs at both ends suggested that these clones were identical. Since we used an amplified cDNA library for screening, it is likely that these clones resulted from the cDNA library amplification. For complete sequence analysis, we sequenced both strands of one clone designated as AceII4. The complete cDNA sequence of the clone comprises 3283 bp, including an open reading frame of 2028 bp beginning with the methionine codon ATG at the position of 257 bp (Fig. 1). This start codon is rather obvious because of the stop codon TAG 45 bp upstream of it and the better Kozak’s context (Kozak, 1986) with a purine adenine in position ⫺3 and guanine in position +4 of the start codon. The stop codon TGA is located at the position of 2285–2287 bp and is followed by a non-translated region of 976 bp including a poly(A) tail of 23 bp. The 3⬘ untranslated

768

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

Fig. 1. Nucleotide and deduced amino acid sequences of AChE cDNA from S. graminum. The amino acid sequence is numbered from the start of the mature protein. The signal peptide cleavage site is marked by a vertical arrow. The N-terminal sequence of the mature protein determined by protein sequencing is boxed. Six potential N-linked glycosylation sites are underlined with dots. The stop codon upstream of the coding region is italicized and the stop codon at the end of the coding region is marked with an asterisk. A putative polyadenylation signal (AATAAA) in the 3⬘-untranslated sequence is underlined. This sequence was deposited in the GenBank (accession number: AF321574).

region possesses a typical polyadenylation signal (AATAAA) at 15 bp upstream of the poly(A) tail. The open reading frame (2028 bp) of the cloned AChE cDNA encodes a putative preproenzyme of 676 amino acid residues. To reveal the first amino acid residue of the mature AChE, we determined the first nine amino acid residues of the N-terminus of a 72-kDa AChE pur-

ified from the greenbug (Gao and Zhu, 2001). The Nterminal sequence (YTSDDPLII) of the enzyme matched the nine amino acid residues deduced from the cDNA (Fig. 1). Thus, the putative preproenzyme possibly consists of a signal peptide of 17 amino acid residues predicted by SignalP (Nielsen et al., 1997), an activation peptide of 58 and a mature enzyme of 581 experimen-

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

Fig 1.

769

(Continued)

tally determined by the N-terminal sequence. Although we cannot completely rule out the possibility of a proteolytic cleavage of the activation peptide during the enzyme preparation, the experimentally determined first amino acid residue appears to be the start site of the mature AChE because the location of this start site of

the mature enzyme is very close to those of mature AChEs of other animals including Torpedo, rabbit, bovine, human and fruit fly (Fig. 2). The cloned AChE cDNA contains two EcoR I internal restriction sites, one in the non-coding region (position 222–223) and the other in the coding region (1775–

770

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

Fig. 2. Alignment of S. graminum (S.g.), T. californica (T.c.) and D. melanogaster (D.m.) AChE sequences. Numbering of the amino acid sequences is from the N-terminus of mature proteins (residues in bold). Identical amino acids are indicated by asterisks and conservative substitutions by dots. The residues forming catalytic triads are depicted with solid diamonds. Cysteine residues involved in intrachain disulfide bonds are boxed and their disulfide bridges are indicated by connecting lines. The positions of aromatic residues lining the active site gorge in Torpedo AChE are marked with arrows. The cholinesterase signature sequence is underlined. The serine in greenbug AChE that may result in increased substrate inhibition thresholds is circled.

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

1776). It also contains one Hind III internal restriction site in the C-terminal non-coding region (position 3181– 3182). However, the cDNA does not contain any restriction sites for the enzymes EcoR V, Kpn I and Pst I. When the 1.7-kb cDNA probe was used to hybridize the EcoR I digested fragments, the blot showed strong hybridization to approximately 8.4- and 6.9-kb fragments but weak hybridization to a 5.9-kb fragment (Fig. 3). In EcoR V, Hind III and Pst I digested DNA, strong hybridization was seen to 6.9-, 6.9- and 9.0-kb fragments, respectively. In Kpn I digested DNA, however, weak hybridization was seen to the 12.7-, 7.8-, 5.5-, 4.4and 1.1-kb fragments. The multiple hybridization fragments can be explained by the presence of introns in the genomic organization of AChE gene. Nine introns have been reported in the AChE gene of D. melanogaster

771

(Fournier et al., 1989) and A. stephensi (Hall and Malcolm, 1991) and eight introns in C. elegans (Arpagaus et al., 1994). Because our probe corresponds to the region containing seven exons of AChE gene of D. melanogaster and A. stephensi, multiple bands would be expected. Our Southern blot analysis suggested that there is only a single copy of cloned AChE gene in the greenbug. Northern blot analysis using either the 278-bp or the 1.7kb cDNA probe revealed a distinctive 3.7-kb transcript (Fig. 4). This transcript size matches well with the 3.3kb of the greenbug AChE cDNA and is similar to the transcript sizes of 4.5- to 4.8-kb of D. melanogaster (Hall and Spierer, 1986). 3.2. Characteristics of the cDNA-deduced AChE The cDNA-deduced amino acid sequence of the greenbug AChE was compared with other sequences in GenBank with BLASTP. The greenbug AChE sequence exhibits all the common features for an AChE including:

Fig. 3. Southern blot analysis of greenbug genomic DNA. Ten µg of genomic DNA was digested with each of five restriction enzymes, EcoR I, EcoR V, Hind III, Kpn I and Pst I, overnight at 37 °C and separated on 0.7% agarose gel. The blot was hybridized with a [32P]labeled 1.7-kb cDNA probe. Sizes of standards are indicated on the left.

Fig. 4. Northern blot analysis of mRNA purified from the greenbug. mRNA (9.5 µg per lane) was separated on 1% agarose gel. The blot was hybridized with a [32P]-labeled 1.7-kb cDNA probe. Sizes of standards are indicated on the left.

772

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

(1) conserved active site triad, S206 (S200 in Torpedo), E332 (327) and H446 (440); (2) a choline binding site W91 (84 in Torpedo); (3) three pairs of cysteines putatively forming intrachain disulfide bonding (C74–C101, C260–C273 and C408–C530); (4) a cysteine forming interchain disulfide bonding (C553); (5) 10 conserved aromatic amino acid residues out of 14 aromatic residues lining the catalytic gorge of the Torpedo AChE (Fig. 2); and (6) the sequence FGESAG, flanking S206, conserved in all cholinesterases. In addition, the greenbug AChE contains a typical invertebrate acyl pocket (Sutherland et al., 1997) that contains only one conserved aromatic site F295 (290 in Torpedo). The other conserved aromatic site in vertebrate acyl pocket was replaced by a C293 (288 in Torpedo) in the greenbug AChE. Unexpectedly, both the identity and similarity of the cDNA-deduced greenbug AChE are lower relative to those of other insects (D. melanogaster, A. stephensi or L. decemlineata) (35–37% identity and 52–55% similarity) than to those of nematode and vertebrate species (Fig. 5). Specifically, the cDNA-deduced greenbug AChE sequence is most similar to an AChE (Ace1) of the nematodes C. elegans and C. briggsae, both with 43% identity and 61% similarity, followed by AChE of vertebrates, Bungarus fasciatus, Torpedo marmorata and T. californica with 40–43% identity and 59– 60% similarity. The greenbug AChE also exhibits 39– 40% identity and 56–57% similarity to the AChE of mouse, rat, rabbit, bovine and human. The calculated molecular mass and isoelectric point of the mature AChE was 65.4 kDa and 6.0, respectively. These values are relatively smaller than 72 kDa and 7.3 determined by SDS–PAGE and isoelectric focusing, respectively (Gao and Zhu, 2001). The deduced AChE sequence exhibits six potential N-glycosylation sites (Fig. 1), suggesting that this protein may be heavily glycosylated. The glycosylation probably affected the accuracy of molecular mass and isoelectric point determinations as demonstrated in the butyrylcholinesterase of chick (Treskatis et al., 1992) and esterases of rice planthoppers (Sakata and Miyata, 1994; Small and Hemingway, 2000). 3.3. AChE structure–function relationships Purified AChE from the greenbug showed 20- to 200fold higher substrate inhibition thresholds than AChE from other insect species for acetylthiocholine (Gao and Zhu, 2001). Substrate inhibition is associated with peripheral anionic sites as demonstrated by site-directed mutagenesis (Shafferman et al., 1992). Mutagenesis of D74N, D74G, D74K and W286A in human AChE generated enzymes with either completely or partially lost substrate inhibition at high concentrations. However, these residues are conserved in the greenbug AChE.

Thus, increased substrate inhibition thresholds in the greenbug AChE are caused by other structural differences. By examining the amino acid sequence similarity among 30 aligned sequences of 21 animal species (Fig. 5A), we found that the position for serine 336 in the greenbug AChE is substituted either for phenylalanine or for tryptophan in all known AChEs from other animal species. Shafferman et al. (1992) proposed that allosteric changes initiated at the exterior could produce an inhibitory effect by a physical blockage of the active center through a conformational change. The serine replacement for an aromatic residue in the greenbug AChE may fail to prevent or interfere with the entry of substrate to an unoccupied active site or may fail to reduce the efficiency of the deacylation of the acyl–enzyme complex due to the small size of its side chain. Therefore, this serine may be responsible for the partial loss of substrate inhibition at high substrate concentration (Gao and Zhu, 2001). The generation of an AChE with an extremely high substrate inhibition constant by mutagenesis of F338G (336 in greenbug) of mouse AChE (Radic et al., 1993) supports this hypothesis. It has also been found that AChEs from aphids and thrips are highly sensitive to sulfhydryl reagents, such as 5,5⬘-dithio-bis(2-nitrobenzoic acid) (DTNB) (Zahavi et al., 1972; Manulis et al., 1981; Brestkin et al., 1985; Novozhilov et al., 1989). The inhibitory effect of sulfhydryl reagents has been hypothesized as being due to the presence of free cysteine residues that react covalently with the sulfhydryl reagents, blocking hydrolysis of the substrate (Smissaert, 1976). By examining the amino acid sequence similarity among 30 aligned sequences of 21 animal species (Fig. 5A), we found two free cysteines (C67 and C293) in greenbug AChE (Fig. 2). These cysteines may react with sulfhydryl reagents and therefore inhibit enzyme activity (Steinberg et al., 1990). 3.4. Phylogenetic relationship of the greenbug AChE to other AChEs Phylogenetic trees were generated, using the neighbor-joining method, from the highly conserved regions of greenbug AChE and 29 other AChE amino acid sequences available in GenBank. The unrooted distance tree (Fig. 5A) consisted of four major clusters. However, the greenbug AChE gene was grouped out of the insect cluster. The divergence of greenbug AChE from insects can probably be explained by gene duplication (Page and Holmes, 1998, pp. 346). In nematodes C. elegans and C. briggsae, multiple AChE genes have been sequenced (Grauso et al., 1998). The duplicate genes form their own cluster in the unrooted distance tree (Fig. 5A). The duplication of the AChE gene in insects has been proposed in Culex pipiens based on insecticide inhibition profile and biochemi-

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

773

Fig. 5. Phylogenetic trees of deduced AChE amino acid sequences constructed by the neighbor-joining method. (A) Unrooted phylogenetic tree of 30 AChE genes from animals. (B) Rooted phylogenetic tree of eight insect AChE genes using the AChE1 gene of C. elegans as the outgroup. The trees were constructed based on highly conserved sequence blocks extracted by Block Maker with little manual modifications. The name is made up of a species abbreviation (first letter of the genus followed by the first three letters of the specific name). The bootstrap values with 1000 trials are indicated on branches. Sequences used: Aaeg: Aedes aegypti (the yellow fever mosquito), GenBank accession No. S66236; Aste: Anopheles stephensi (the malaria mosquito), P56161; Bdec: Boophilus decoloratus (tick), CAA06980; Bfas: Bungarus fasciatus (snake), AAC59905; Bflo: Branchiostoma floridae (amphioxus), 1: U74380, 2: U74381; Bmic: Boophilus microplus (the southern cattle tick), 1: CAA11702, 2: AAC18857; Btau: Bos taurus (bovine), S10712; Cbri: Caenorhabditis briggsae (nematode), 1: Q27459, 2: AF030037, 3: AF159504, 4: AF159505; Cele: Caenorhabditis elegans (nematode), 1: P38433, 2: T37255, 3: T42399, 4: AAC14017; Dmel: Drosophila melanogaster (fruit fly), CAA29326; Hsap: Homo sapiens (human), P22303; Lcup: Lucilia cuprina (sheep blow fly), AAC02779; Ldec: Leptinotarsa decemlineata (Colorado potato beetle), Q27677; Lopa: Loligo opalescens (squid), AAD15886; Mdom: Musca domestica (housefly), JE0150 (modified to Cooper strain, Huang et al., 1997); Mmus: Mus musculus (mouse), P21836; Ncin: Nephotettix cincticeps (the green rice leafhopper), AAF65202; Ocun: Oryctolagus cuniculus (rabbit), S48724; Rapp: Rhipicephalus appendiculatus (tick), CAA06981; Rnor: Rattus norvegicus (Rat), P37136; Sgra: Schizaphis graminum (greenbug), AF321574; Tcal: Torpedo californica (electric ray), P04058.

cal characterization (Bourguet et al., 1996). One gene (Ace.1) encodes a propoxur-insensitive enzyme and the other (Ace.2) encodes a sensitive enzyme that is sexlinked (Malcolm et al., 1998). However, the presently sequenced mosquito AChEs are all sex-linked even though resistance is not sex-linked (Vaughan et al., 1997). Duplication of the AChE gene was also suggested by sequence analysis of the AChE gene of Nephotettix cincticeps (Tomita et al., 2000) and Boophilus microplus (Baxter and Barker, 1998; Hernandez et al., 1999) because no mutations in AChE were detected from an organophosphate-resistant strain of these species as compared with their corresponding susceptible strains. Although the vast bulk of known insect AChE sequences

may not represent the cholinergic enzyme, the fact that all previously sequenced insect AChE genes cluster together suggests that they evolved from a single gene lineage. In contrast, the cloned greenbug AChE gene, grouping out of the insect cluster, probably represents a paralogous gene lineage, a primitive lineage, to that of previously known insect AChE genes (Fig. 5B). Further studies to identify multiple AChE genes and clarify their functions may provide new insights into the biological roles of different AChEs and facilitate our understanding of molecular mechanisms of insensitive AChE in conferring organophosphate and/or carbamate insecticide resistance in insects.

774

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

Acknowledgements We thank Michael R. Kanost for his valuable suggestions on DNA and amino acid sequence analyses, Brenda Oppert for her critical review of an earlier draft of this manuscript, and Qifa Han, Haobo Jiang, Xiaoqiang Yu, Jeffery Clark, Paul Smith and Sharon Starkey for their technical assistance. This study was supported by the Kansas Agricultural Experiment Station and the US Department of Agriculture. This manuscript is Contribution No. 02-26-J from the Kansas Agricultural Experiment Station, Kansas State University. Greenbug voucher specimens (No. 083) were deposited in the Museum of Entomological and Prairie Arthropod Research, Kansas State University, Manhattan, KS 66506.

References Anthony, N., Rocheleau, T., Mocelin, G., Lee, H.J., ffrench-Constant, R., 1995. Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti. FEBS Lett. 368, 461–465. Arpagaus, M., Fedon, Y., Cousin, X., Chatonnet, A., Berge, J.-B., Fournier, D. et al., 1994. cDNA sequence, gene structure, and in vitro expression of ace 1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. J. Biol. Chem. 369, 9957–9965. Baxter, G.D., Barker, S.C., 1998. Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterization and role in organophosphate resistance. Insect Biochem. Mol. Biol. 28, 581–589. Bourguet, D., Raymond, M., Bisset, J., Pasteur, N., Arpagaus, M., 1996. Duplication of the Ace 1 locus in Culex pipiens mosquitoes from the Caribbean. Biochem. Genet. 34, 351–362. Brestkin, A.P., Maizel, E.B., Moralev, S.N., Novozhilov, K.V., Sazonova, I.N., 1985. Cholinesterases of aphids. I. Isolation, partial purification and some properties of cholinesterases from spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 15, 309–314. Chalfie, M., White, J., 1988. The nervous system. In: Wood, W.B., The Community of C. Elegans Researchers (Eds.), The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 337–391. Chen, Z., Newcomb, R., Forbes, E., McKenzie, J., Batterham, P., 2001. The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly Lucilia cuprina. Insect Biochem. Mol. Biol. 31, 805–816. Eldefrawi, A.T., 1985. Acetylcholinesterase and anticholinesterases. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 12. Pergamon Press, New York, pp. 115–130. Felsenstein, J., 1993. PHYLIP (phylogeny inference package) version 3.5c. Distributed by the author, Department of Genetics, University of Washington, Seattle. Fournier, D., Karch, F., Bride, J.-M., Hall, L.M.C., Berge, J.-B., Spierer, P., 1989. Drosophila melanogaster acetylcholinesterase gene structure, evolution and mutations. J. Mol. Biol. 210, 15–22. Gao, J.-R., Zhu, K.Y., 2000. Comparative toxicity of selected organophosphate insecticides against resistant and susceptible clones of the greenbug, Schizaphis graminum (Homoptera: Aphididae). J. Agric. Food Chem. 48, 4717–4722. Gao, J.-R., Zhu, K.Y., 2001. An acetylcholinesterase purified from the

greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics. Insect Biochem. Mol. Biol. 31, 1095–1104. Gao, J.-R., Rao, J.V., Wilde, G.E., Zhu, K.Y., 1998. Purification and kinetic analysis of acetylcholinesterase from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Arch. Insect Biochem. Physiol. 39, 118–125. Grauso, M., Culetto, E., Combes, D., Fedon, Y., Toutant, J.-P., Arpagaus, M., 1998. Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. FEBS Lett. 424, 279–284. Hall, L.M.C., Malcolm, C.A., 1991. The acetylcholinesterase gene of Anopheles stephensi. Cell. Mol. Neurobiol. 11, 131–141. Hall, L.M.C., Spierer, P., 1986. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5⬘ leader. EMBO J. 5, 2949–2954. Harel, M., Kryger, G., Rosenberry, T.L., Mallender, W.D., Lewis, T., Fletcher, R.J. et al., 2000. Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors. Protein Sci. 9, 1063–1072. Henikoff, S., Henikoff, J.G., Alford, W.J., Pietrokovski, S., 1995. Automated construction and graphical presentation of protein blocks from unaligned sequences. Gene 163, GC17–26. Hernandez, R., He, H., Chen, A.C., Ivie, G.W., George, J.E., Wagner, G.G., 1999. Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae). J. Med. Entomol. 36, 764–770. Huang, Y., Qiao, C., Williamson, M.S., Devonshire, A.L., 1997. Characterization of the acetylcholinesterase gene from insecticideresistant houseflies (Musca domestica). Chinese J. Biotechnol. 13, 177–183. Jeanmougin, F., Thompson, J.D., Gouy, M., Higgins, D.G., Gibson, T.J., 1998. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23, 403–405. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. Malcolm, C.A., Bourguet, D., Ascolillo, A., Rooker, S.J., Garvey, C.F., Hall, L.M.C. et al., 1998. A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens. Insect Mol. Biol. 7, 107–120. Manulis, S., Ishaaya, I., Perry, A., 1981. Acetylcholinesterase of Aphis citricola: properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds. Pestic. Biochem. Physiol. 15, 267–274. Matsumura, F., 1985. Toxicology of Insecticides, 2nd ed. Plenum Press, New York. Metcalf, R.L., Metcalf, R.A., 1993. Destructive and Useful Insects: Their Habits and Control, 5th edn. McGraw-Hill, New York. Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Novozhilov, K.V., Brestkin, A.P., Khovanskikh, A.E., Maizel, E.B., Moralev, S.N., Nikanorova, E.V. et al., 1989. Cholinesterases of aphids. III. Sensitivity of acetylcholinesterases to several inhibitors as a possible phylogenetic character. Insect Biochem. 19, 15–18. Page, R.M.W., 1996. TREEVIEW: an application to display phylogenetic tree on personal computers. Comput. Appl. BioSci. 12, 357– 358. Page, R.D.M., Holmes, E.C., 1998. Molecular Evolution: A Phylogenetic Approach. Blackwell Science, Oxford, UK. Radic, Z., Pickering, N.A., Vellom, D.C., Camp, S., Taylor, P., 1993. Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitor. Biochemistry 32, 12074–12084. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

J.-R. Gao et al. / Insect Biochemistry and Molecular Biology 32 (2002) 765–775

Sakata, K., Miyata, T., 1994. Biochemical characterization of carboxylesterase in the small brown planthopper Laodelphax striatellus (Falle´ n). Pestic. Biochem. Physiol. 50, 247–256. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York. Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M. et al., 1992. Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center. EMBO J. 11, 3561–3568. Small, G.J., Hemingway, J., 2000. Differential glycosylation produces heterogeneity in elevated esterases associated with insecticide resistance in the brown planthopper Nilaparvata lugens Sta˚ l. Insect Biochem. Mol. Biol. 30, 443–453. Smissaert, H.R., 1976. Reaction of a critical sulfhydryl group of the acetylcholinesterase from aphids Myzus persicae. Pestic. Biochem. Physiol. 6, 215–222. Soreq, H., Zakut, H., 1993. Human Cholinesterases and Anticholinesterases. Academic Press, San Diego. Steinberg, N., Roth, E., Silman, I., 1990. Torpedo acetylcholinesterase is inactivated by thiol reagents. Biochem. Int. 21, 1043–1050. Sutherland, D., McClellan, J.S., Milner, D., Soong, W., Axon, N., Sanders, M. et al., 1997. Two cholinesterase activities and genes are present in amphioxus. J. Exp. Zool. 277, 213–229. Tomita, T., Hidoh, O., Kono, Y., 2000. Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps. Insect Biochem. Mol. Biol. 30, 325–333.

775

Treskatis, S., Ebert, C., Layer, P.G., 1992. Butyrylcholinesterase from chicken brain is smaller than that from serum: its purification, glycosylation, and membrane association. J. Neurochem. 58, 2236– 2247. Vaughan, A., Rocheleau, T., ffrench-Constant, R., 1997. Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity. Exp. Parasitol. 87, 237–244. Zahavi, M., Tahori, A.S., Klimer, F., 1972. An acetylcholinesterase sensitive to sulfhydryl inhibitors. Biochem. Biophys. Acta 276, 577–583. Zhu, K.Y., Clark, J.M., 1994. Purification and characterization of acetylcholinesterase from the Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem. Mol. Biol. 24, 453–461. Zhu, K.Y., Clark, J.M., 1995. Cloning and sequencing of a cDNA encoding acetylcholinesterase in Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem. Mol. Biol. 25, 1129– 1138. Zhu, K.Y., Gao, J.-R., 1999. Increased activity associated with reduced sensitivity of acetylcholinesterase in organophosphate-resistant greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Sci. 55, 11–17. Zhu, K.Y., Gao, J.-R., Starkey, S.R., 2000. Organophosphate resistance mediated by alterations of acetylcholinesterase in a resistant clone of the greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Biochem. Physiol. 68, 138–147.