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The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae
Insect Biochemistry and Molecular Biology 42 (2012) 212e219
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The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae Deok Ho Kwon a, Jae Young Choi b, Yeon Ho Je b, Si Hyeock Lee a, b, * a b
Research Institute for Agriculture Life Sciences, Seoul National University, Seoul 151-921, South Korea Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2011 Received in revised form 2 December 2011 Accepted 6 December 2011
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 17.8w27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophosresistant 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 6copy 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. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Tetranychus urticae Acetylcholinesterase Gene duplication Mutation Compensation Resistance
1. Introduction Point mutations in acetylcholinesterase (AChE) have been found in a wide variety of insects and mites, and these mutations confer AChE insensitivities to organophosphate (OP) and carbamate (CB) pesticides (Fournier, 2005; Van Leeuwen et al., 2010). Although these point mutations are required for survival under insecticide selection pressure, they usually cause a change in the catalytic activity of the enzyme because they are generally located near the catalytic triads and other functionally important regions (Fournier, 2005). Such resistance-associated point mutations affecting the
* Corresponding author. Department of Agricultural Biotechnology, Seoul National University, Bldg. 200, Rm# 6120, Seoul 151-742, South Korea. Fax: þ82 2 873 2319. E-mail address: [email protected] (S.H. Lee). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.12.003
catalytic efficiency of AChE, therefore, have been suggested as the primary cause of the observed fitness costs (Shi et al., 2004). When 15 mutant AChEs in Drosophila melanogaster were produced by in vitro expression and their catalytic activities and insensitivities to OP insecticides were determined, some resistance-conferring mutations caused a reduction in the catalytic activity and stability of the enzyme (Shi et al., 2004). Two mechanisms to compensate for the reduced catalytic efficiency can be suggested. One mechanism involves the introduction of counteracting point mutations. In D. melanogaster, single point mutations (I161V, F330Y and G368A) in AChE reduce the catalytic efficiency of the enzyme, but the AChE activity was recovered when these point mutations were combined with the G265A mutation that results in hyperactivity (Shi et al., 2004). The second compensation mechanism involves maintaining both the susceptible (wildtype) allele and the resistance allele containing mutations via AChE duplication. In the case of the Culex mosquito, two
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alleles of the ace-1 gene encoding AChE, one susceptible and the other resistant to insecticides, were generated by gene duplication in response to insecticide pressure (Labbe et al., 2007a). It has been proposed that the duplication was selected because the presence of the susceptible ace-1 allele reduces the fitness cost associated with the resistant allele via the generation of persistent, more adaptable heterozygotes (Labbe et al., 2007b). A recent report by Alout et al. showed that the occurrence of ace-1 duplications in Culex pipiens is high (Alout et al., 2011). Three point mutations (G228S, A391T and F439W; G119S, A280T and F331W in the amino acid numbering of Torpedo californica AChE, respectively) in AChE have been reported to confer resistance to monocrotophos, an OP acaricide, in the two-spotted spider mite Tetranychus urticae (Kwon et al., 2010b). Among these mutations, the A391T mutation was found in all of the green-type T. urticae populations, which differ slightly from the susceptible UD strain in their mitochondrial cytochrome oxidase I marker sequences (Kwon et al., 2011). A kinetic analysis of the AChEs from three T. urticae strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) revealed that the AChEs from the AD and PyriF strains have reduced catalytic efficiencies, suggesting that the resistant form of AChE is likely accompanied by a fitness cost (Kwon et al., 2010a). A comparison of the T. urticae AChE (TuAChE) gene (Tuace) copy number showed that relatively more Tuace copies are present in the resistant strains than in the susceptible strain (Kwon et al., 2010a). The relative copy numbers of Tuace in field populations correlated precisely with the levels of resistance, indicating an additional role of Tuace gene duplication in resistance. Given that gene dosage is precisely regulated in living organisms, extensive gene duplication may disrupt this balance (Veitia, 2005). In the case of Tuace duplication, however, increased AChE dosages in both the AD and PyriF strains do not appear to disrupt the overall function of AChE because the total activities of the increased amounts of AChE in these mite strains were not significantly different from that of the UD strain (which likely has only a single copy of AChE), as judged by their similar vmax and Km values. Rather, the AChE dosage in the resistant mites appears to have been increased to adjust the overall AChE activity to a level similar to that of UD. The recent analysis of T. urticae genome revealed that Tuace is the only locus encoding a functional AChE (Grbic et al., 2011), excluding the possibility of other paralogous AChE(s) contributing to AChE activity. It is currently unclear, however, whether the extensive Tuace duplication actually results in TuAChE overexpression at the protein level and, if this is the case, how such overexpression has been allowed for AChE, of which titer should be carefully regulated to maintain the homeostasis of the excitatory nervous system. The advantages and costs of ace-1 duplications in relation to OP resistance have previously been described for Culex mosquitoes from the perspective of gene dosage (Labbe et al., 2007a, 2007b). On the basis of the observation that the catalytic activity of the resistant form of AChE1 is less than 60% of the susceptible form (Bourguet et al., 1997, 1996), it was suggested that ace-1 duplication may restore the normal gene dosage, which is otherwise phenotypically reduced by the resistance mutation (Labbe et al., 2007a). In this study, the amounts of TuAChE were measured using a TuAChE-specific antibody and correlated with the Tuace copy numbers in several field populations. Then, the TuAChE variants with different mutation combinations were expressed in vitro, and their enzymatic properties were analyzed from the perspective of resistance and fitness cost. To investigate the effects of copy number and mutation on the total AChE activity, appropriate AChE variants were combined to generate the desired AChE copies and mutation combinations/frequencies, and their enzyme kinetics were determined. Finally, Tuace duplication as a compensatory
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adaptation mechanism for the catalytic efficiency reduction as a result of resistance-conferring mutations was discussed in relation to OP resistance. 2. Materials and methods 2.1. Mites T. urticae mites were collected from five host plants in the field or greenhouses between 2006 and 2008 (Supplemental Table 1). The collected strains were maintained on kidney bean plants (2 weeks old, Phaseolus vulgaris variety humilis) in the laboratory under the conditions of 25 1 C, 55 5% relative humidity and a 16:8 (L:D) photoperiod. The UD and PyriF strains were reared without any exposure to acaricides. The AD strain was selected approximately every two months with a LC50 dose of monocrotophos. 2.2. Genomic DNA extraction and gene copy number determination The genomic DNA (gDNA) from seven strains of T. urticae was extracted from 5 mg female adults using a DNeasy Blood & Tissue kit (Qiagen GmBH, Germany) according to the manufacturer’s instructions. Briefly, the mites were ground using a fabricated plastic homogenizer in tissue lysis (TL) buffer and incubated with 10 ml proteinase K (10 mg/ml) at 56 C for 1 h. The gDNA was isolated using Mini-spin columns, and the purified gDNA was stored at 20 C until use. Quantitative real-time PCR (qPCR) was employed to determine the gene copy number of Tuace as described previously (Kwon et al., 2010a). 2.3. Generation of an anti-TuAChE polyclonal antibody The rabbit anti-TuAChE1 polyclonal antibodies were produced by Ab Frontier (Seoul, Korea). The two bovine serum albumin-fused peptide fragments [RKKAYQRSLAL (amino acid position 346e356) and EDPEVSLRTKNFK (amino acid position 411e423)] were synthesized. The rabbits were immunized three times with 0.5 mg of the two synthesized peptides. The serum-specific antibodies were affinity-purified on columns using immobilized antigen peptides. The titer of the anti-TuAChE1 polyclonal antibody was approximately 2 mg/ml. 2.4. Native polyacrylamide gel electrophoresis (PAGE) and Western blotting The female mites (ca. 15 mg) were homogenized in 300 ml extraction buffer (0.1 M TriseHCl, pH 7.8) using a 2-ml glasseglass tissue grinder (Radnoti, Monrovia, CA), and the homogenate was centrifuged at 10,000 g for 15 min. Following centrifugation, the supernatant was decanted and used as the TuAChE source. The protein concentration was determined by BCA methods using bovine serum albumin as a standard. The duplicate enzyme samples (40 mg protein each) were separated by discontinuous native PAGE (5% T/2.67% C stacking and 7.5% T/2.67% C separating gels) at 120 V for 1 h and at 180 V for 3 h in a 4 C cold chamber. After electrophoresis, one gel was activity-stained for the visualization of the TuAChE band according to the method of Lewis and Shute (1966). The second gel was electro-blotted to a nylon membrane (Amersham Hybond-ECL, GE Healthcare, Piscataway, NJ) at 35 V for 1 h 10 min, and the blotted membrane was used for a Western blot analysis. The membrane was hybridized with the primary antibody (diluted 1:5000 in PBST buffer) for 2 h and washed three times with PBST buffer in 10-min intervals. The membrane was further
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hybridized with the secondary antibody (Rabbit Anti-Mouse IgG, Thermo Fisher Scientific, Rockford, IL) diluted 1:20,000 with 5% skim milk for 1 h and was then washed three times with PBST buffer in 10-min intervals. The membrane was incubated with Western blotting luminol reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and exposed to X-ray film (Agfa-Gevaert, Mortsel, Belgium). The exposure time was minimized (ca. 5 min) to obtain band intensities with a more linear range. The band intensities of the activity-stained gel and the Western blot were quantified using an EDAS 290 gel image analyzer (Eastman Kodak Company, Rochester, NY). 2.5. Expression vector construction The full-length Tuace clone (2061 bp, 687 amino acids) of the UD strain was cloned into a pGEM-T easy vector (Promega, Madison, WI, USA). A truncated Tuace cDNA fragment (649 amino acids, 1947 bp, ‘MVPMF w LINAL’) with its C-terminal hydrophobic region excluded was PCR-amplified with gene-specific primers containing restriction enzyme sites (XbaI and EcoRI) and a 6 His tag sequence (Supplemental Table 2) using an Advantage 2 Polymerase Mix (Takara Bio Inc., Shiga, Japan) under the following thermal program: (95 C for 30 s, 62 C for 30 s, 72 C for 3 min) 35 cycles. The PCR-amplified DNA fragment (TuAChE_UD) was digested with XbaI and EcoRI and inserted into the pBacPAK8 vector (approximately 5517-bp) (Clontech, Palo Alto, CA, USA) that had been predigested with the same restriction enzymes. 2.6. Site-directed mutagenesis Using the TuAChE_UD clone in the pBacPAK8 expression vector (approximately 7491-bp) as a template, site-directed mutagenesis was conducted to generate Tuace clone variants having single (SAF, GTF and GAW), double (SAW, STF and SAW) and triple (STW) mutations using appropriate primer sets according to the manufacturer’s instructions (Supplemental Table 2). First, the clones with a single point mutation (G228S, A391T and F439W) were constructed using respective primer sets, and then the clones with double point mutations (G228S þ A391T, A391T þ F439W and G228S þ F439W) were generated from the single mutation clones. Finally, a clone with triple mutations (G228S þ A391T þ F439W) was constructed using one of the double-mutation clones as a template. 2.7. TuAChE expression and purification To generate recombinant Autographa californica nucleopolyhedroviruses (AcMNPVs), the resulting transfer vectors were cotransfected with bAcGOZA DNA (Je et al., 2001) into Sf9 cells maintained in TC-100 medium (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum at 27 C. The transfection was performed using the Cellfectin (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions, and the recombinant viruses were purified by a plaque assay of the Sf9 cells as previously described (O’Reilly et al., 1992). To exclude the serumderived esterase activity, the Sf9 cells maintained in SF900-II serum-free medium (Invitrogen) were seeded onto 100-mm diameter tissue culture dishes at a density of 5 106 cells/dish, followed by incubation at 27 C for 30 min to allow for cell attachment. The attached cells were washed twice with 3 ml of incomplete TC-100 medium and inoculated with 1 ml of properly diluted viral stock. After incubation for 84 h at 27 C, the cell pellets were collected by centrifugation at 5000 g for 15 min and sonicated with a Branson Sonifier (Branson SonifierÒ, Danbury, CT) under TriseHCl (pH 7.8, 0.02 M NaCl) buffer conditions for 6 min at
4 C (Out control, 3; Duty cycle, 30%). The lysed cells were centrifuged at 12,000 g for 10 min, and the supernatant was centrifuged again at approximately. 134,000 g for 20 min at 4 C in an ULTRA 4.0 ultracentrifuge (Hanil Science Industrial, Incheon, Korea). The supernatant was used for further His-Tag purification. Approximately 6e7 ml of supernatant was loaded onto a 5-ml HisTrap HP affinity column (Amersham Biosciences Corp., Piscataway, NJ) at a flow rate of 5 ml/min using an AKTA FPLC (Amersham Bioscience UPC-900). The washing and binding fractions were discarded, and the column was eluted with 250 mM imidazolecontaining elution buffer. Each fraction was divided with a Frac920 fraction collector (Amersham Biosciences). The eluted protein was concentrated with a YM-50 filter and stored at 80 C until use. The protein concentration of the semi-purified TuAChE was estimated based on the band intensities determined both by Western blotting and Coomassie staining after SDS-PAGE. The protein concentration of the semi-purified TuAChE solution was normalized prior to the enzyme kinetic analysis. 2.8. AChE kinetic analysis and determination of the median inhibition (I50) concentrations The AChE activity was measured by Ellman’s method with a slight modification (Ellman et al., 1961). For the calculation of the kinetic constants, the enzyme (in approximately 1 ng of total protein) was incubated with 0.4 mM 5,5-dithio-bis 2-nitrobenzoic acid (DTNB, SigmaeAldrich Co.) and 0.03e1 mM acetylthiocholine iodide (ATChI) at 30 C. The AChE activity was measured at 412 nm in 20-second intervals over 10 min using a VERSAmax microplate reader (Molecular Device Inc., Sunnyvale, CA). The MichaeliseMenten constants (Km) and maximum enzyme velocities (vmax) were calculated from Lineweaver-Burk plots. The kcat value was determined by dividing the vmax value by the Km constant. The I50 concentration was determined based on the following procedures. Aliquots [2 ml for wildtype, all single mutation variants and one double mutation (A391T þ F439W) variant; 10 ml for the remaining double and triple mutation variants] of purified TuAChE were preincubated with a range of monocrotophos solutions (0.0003e10 mM) for 10 min, and the remaining activities were measured by adding 0.4 mM DTNB and 1 mM ATChI at 30 C. All of the kinetic constants and inhibition values were determined from three independent experiments. The statistical analysis was carried out with ANOVA post-hoc analysis using the SAS software package (SAS Institute, NC, USA). 2.9. Reconstitution of TuAChE To simulate the TuAChEs in the AD and PyriF strains in terms of TuAChE copy number and the frequencies of the G228S and F439W mutations, reconstitution of TuAChE was conducted by combining the appropriate TuAChE variants. For the TuAChE of the UD strain, the TuAChEGAF variant was used directly. To simulate the PyriF strain, which has ca. 50% G228S mutation frequency in two copies of TuAChE, equal amounts of the TuAChEGTF and TuAChESTF variants were combined. To simulate the AD strains possessing six copies of TuAChE with mutation frequencies of 50% for G228S and 75% for F439W, the TuAChESTF, TuAChEGTW and 25% TuAChESTW variants were combined in a ratio of 1:2:1. The concentration of each TuAChE variant was normalized to 0.48 ng/ml. The concentrations of the reconstituted TuAChEs were adjusted to 0.48, 0.96 and 2.88 ng/ ml to represent the copy numbers of 1 (UD), 2 (PyriF) and 6 (AD), respectively. The reconstituted TuAChEs were designated as TuAChEUD, TuAChEPyriF and TuAChEAD.
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3. Results 3.1. Correlation between TuAChE quantity and Tuace copy number Western blotting using anti-TuAChE antibody (TuAChE-Ab) following native PAGE revealed that the quantity of TuAChE varied notably among the different strains of T. urticae (Fig. 1A, upper gel). In contrast, the total activity of TuAChE from different strains differed only slightly, as judged by the similar band intensities in the activity-stained gel (Fig. 1A, lower gel). When the TuAChE quantities (TuAChE band intensities in the Western blot) were plotted against the corresponding Tuace copy numbers, a high level of correlation (r2 ¼ 0.914) was obtained, demonstrating that the quantity of TuAChE is proportional to the copy number (Fig. 1B). There was no apparent correlation (r2 ¼ 0.317) between the TuAChE activities (TuAChE band intensities in the activity-stained gel) and the Tuace copy numbers (data not shown), revealing that TuAChE activity is not dependent on its amount. In summary, these results demonstrated that Tuace duplication results in the overexpression of TuAChE at the protein level, but an increase in the quantity of TuAChE does not affect the total enzyme activity. 3.2. Expression of TuAChE variants To verify the authenticity and purity of the TuAChE variants that were expressed and purified in vitro, the samples were analyzed by SDS-PAGE in conjunction with Western blotting (Supplemental Fig. 1). A single TuAChE band (ca. 72.7 kD) was detected with the TuAChE-Ab in all of the TuAChE variants, as shown in the Western blot analysis (see upper gel). Even after its purification by Ni2affinity chromatography, however, the sample contained other contaminating proteins (see lower gel). Based on the band intensity
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of the protein-stained gels, the proportion of expressed TuAChE in the sample was determined to be approximately 3e11%. Therefore, the actual TuAChE concentrations in the subsequent enzyme kinetics and inhibition assays were determined by multiplying by the purity. 3.3. Enzymatic kinetic properties of the in vitro expressed TuAChE variants Eight different TuAChE variants (TuAChEGAF, TuAChESAF, TuAChEGTF, TuAChEGAW, TuAChESAW, TuAChESTF, TuAChEGTW and TuAChESTW; see the designation in Table 1), possessing the three point mutations putatively associated with AChE insensitivity either singly or in combination, were expressed in vitro, and their kinetic properties (vmax, Km and kcat) were examined (Fig. 2 and Supplemental Table 3). Upon comparison of the vmax values (Fig. 2A), the wildtype (TuAChEGAF) and TuAChEGTF exhibited similar vmax values (0.106 0.030 and 0.119 0.028 mM sec1 mg1, respectively) (Fig. 2A), suggesting that the A391T mutation does not alter the catalytic activity of TuAChE significantly. When the G228S mutation is present alone or with the A391T mutation (TuAChESAF and TuAChESTF), the vmax values decreased ca. 10.3- and 10.4-fold compared with those of TuAChEGAF and TuAChEGTF, respectively. Likewise, the presence of the F439W mutation, alone or with the A391T mutation (TuAChEGAW and TuAChEGTW), resulted in a ca. 5.2and 2.3-fold reduction in the vmax values relative to those of TuAChEGAF and TuAChEGTF, respectively. In addition, the presence of the A391T mutation did not influence the function of the G228S or the F439W mutation in the vmax determination. Interestingly, the vmax values of the TuAChESAW and TuAChESTW variants, possessing both G228S and F439W mutations together, decreased ca. 32.7- and 63.3-fold compared with the values determined for TuAChEGAF and TuAChEGTF, respectively. These results suggest that the presence of the G228S and F439W mutations together have a synergistic effect on each other and can impair the catalytic activity of TuAChE dramatically. The Km values of the eight TuAChE variants ranged from 0.08 to 0.3 mM (Fig. 2B). TuAChEGAF and TuAChEGTF exhibited relatively higher Km values (0.29 0.12 and 0.31 0.07 mM, respectively) compared with the other variants. No significant difference between the wildtype enzyme and the TuAChEGTF variant was found, indicating that the A391T mutation does not change the substrate affinity, similar to its lack of effect on the vmax value. However, the Km values of the TuAChE variants possessing the G228S mutation (TuAChESAF and TuAChESTF) decreased 5.8- and 4.0-fold compared to the values determined for TuAChEGAF and TuAChEGTF, indicating that the G228S mutation increases the substrate affinity of the enzyme. The presence of the F439W mutation, alone or with the A391T mutation (TuAChEGAW and TuAChEGTW), also reduced the Km values ca. 1.7- and 1.9-fold, Table 1 Description of alleles about each mutant TuAChE.
Fig. 1. Western blotting (upper gel) and activity staining (lower gel) of the TuAChE samples from seven strains of T. urticae following native PAGE (A). The correlation between the TuAChE quantity (band intensity from the Western blot) and the corresponding Tuace copy number (B).
TuAChE variant designationa
Mutation
Remarks
TuAChEGAF TuAChESAF TuAChEGAW TuAChESAW TuAChEGTF TuAChESTF TuAChEGTW TuAChESTW
G228 þ A391 þ F439 G228S þ A391 þ F439 G228 þ A391 þ F439W G228S þ A391 þ F439W G228 þ A391T þ F439 G228S þ A391T þ F439 G228 þ A391T þ F439W G228S þ A391T þ F439W
Wildtype Single mutation Single mutation Double mutation Single mutation Double mutation Double mutation Triple mutation
a Bolded and underlined characters represent the amino acids at the mutation site.
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(TuAChESAF and TuAChESTF) exhibited 1.7- and 2.7-fold decreased kcat values, respectively. When the F439W mutation was introduced into the wildtype background (TuAChEGAW), the catalytic efficiency decreased 2.6-fold. Interestingly, when the F439W mutation is present together with the A391T mutation, as in the TuAChEGTW variant, the catalytic efficiency was not significantly different from that of the wildtype enzyme (p ¼ 0.521), suggesting that the A391T mutation may counteract the effects of the F439W mutation to restore the normal AChE function. In the TuAChE variants possessing both the G228S and F439W mutations together (TuAChESAW and TuAChESTW), the kcat values decreased 17.6- and 29.0-fold compared to the values determined for TuAChEGAF and TuAChEGTF. These findings suggest that both the G228S and F439W mutations dramatically reduce the catalytic efficiency of TuAChE in a synergistic manner. 3.4. Monocrotophos inhibition properties of in vitro expressed TuAChE variants The I50 concentrations of TuAChEGAF and TuAChEGTF were 0.025 0.006 and 0.016 0.005 mM, respectively (Fig. 3 and Supplemental Table 3). Although TuAChEGTF showed a slightly higher (1.6 fold) sensitivity to monocrotophos compared to that of the wildtype TuAChEGAF, this difference was statistically insignificant (p ¼ 0.108). This finding suggests that, in contrast to our expectations, the A391T mutation is not associated with the AChE insensitivity (Kwon et al., 2010b). The TuAChESAF and TuAChESTF variants possessing the G228S mutation exhibited 16.1- and 26.1fold reduced sensitivity compared with those of TuAChEGAF and TuAChEGTF, respectively, as judged by their I50 values. When the F439W mutation was present, the I50 values increased 60.7- and 99.4-fold in the TuAChEGAW and TuAChEGTW variants, respectively. These findings further suggest that the effects of the F439W mutation on AChE insensitivity are approximately 3.8-fold larger than those of the G228S mutation. Intriguingly, the monocrotophos sensitivities of the TuAChESAW and TuAChESTW variants, possessing double mutations simultaneously, were remarkably reduced, showing 1406- and 1163-fold increases in their I50 values. These findings demonstrate that both the G228S and F439W mutations confer a reduced sensitivity to monocrotophos in a synergistic manner, and the effects of F439W are greater than those of G228S.
Fig. 2. The kinetic properties of eight in vitro expressed TuAChE variants with various combinations of three mutations (G228S, A391T and F439W). vmax constant (A), Km constant (B) and catalytic efficiency (kcat) (C). The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
respectively. When both the G228S and F439W mutations existed together, the TuAChESAW and TuAChESTW variants exhibited 1.9and 2.2-fold lower Km values, respectively. These findings suggest that all of the mutations except for A391T have a tendency to increase the substrate affinity of the resulting enzymes. The TuAChEGAF and TuAChEGTF variants exhibited the highest catalytic efficiency (kcat) values (0.40 0.09 and 0.38 0.05, respectively) (Fig. 2C). The presence of the A391T mutation did not affect the catalytic efficiency, as no significant difference between the wildtype enzyme and the TuAChEGTF variant was observed. The TuAChE variants possessing the single point mutation of G228S
Fig. 3. The median inhibition concentration (I50) values of eight in vitro expressed TuAChE variants having various combinations of three mutations (G228S, A391T and F439W). The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
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3.5. Kinetic and inhibition properties of reconstituted TuAChE Because the TuAChE variant possessing the A391T mutation (TuAChEGTF) did not differ from the wildtype enzyme (TuAChEGAF), the reconstitution of TuAChE was conducted using the TuAChEGTF variant as a wildtype enzyme. The vmax values of TuAChEPyriF and TuAChEAD were 1.5-fold lower than those of TuAChEUD, but the differences were not significant (p ¼ 0.196) (Fig. 4A). Larger Km values (1.7 fold), when compared with TuAChEUD, were observed in TuAChEPyriF and TuAChEAD, suggesting their slightly reduced substrate affinity, although these values were not significantly different (p ¼ 0.241). Both TuAChEPyriF and TuAChEAD have 2.7-fold lower kcat values than TuAChEUD, revealing that reconstituted TuAChEs have reduced, but still comparable, catalytic efficiencies
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compared to that of wildtype TuAChE. This slightly reduced level of catalytic efficiency coincided well with the slightly reduced total activity of AChE observed in the crude enzyme preparation of the AD strain (Kwon et al., 2010a). However, the kcat values of TuAChEPyriF and TuAChEAD showed no significant differences, suggesting that both TuAChEs representing the PyriF and AD strains maintain identical levels of activity despite their difference in copy number. In the inhibition assay, the I50 value of TuAChEAD was 158.7- and 84.5-fold higher than those of TuAChEUD and TuAChEPyriF, respectively (Fig. 5). This insensitivity level of TuAChEAD was comparable to that observed previously with the AChE preparation extracted from the AD strain (i.e., ca. 90.6- and 41.9-fold increased insensitivity compared to the UD and PyriF strains) (Kwon et al., 2010b). TuAChEPyriF exhibited a 1.9-fold lower I50 value than TuAChEUD, suggesting a slightly increased sensitivity to monocrotophos, but the difference was not significant (p ¼ 0.062). 4. Discussion 4.1. Overexpression of TuAChE by gene duplication According to our previous report, the transcription of Tuace was proportional to the level of duplication at a ratio of 1:1 in the three strains examined, indicating that all duplicated copies of Tuace are actively transcribed (Kwon et al., 2010a). The present study confirmed that Tuace duplication results in the overexpression of TuAChE at the protein level. A cross-comparison of several mite strains demonstrated that the quantity of TuAChE was directly proportional to the Tuace copy numbers (Fig. 1). Regardless of TuAChE quantity, however, the overall AChE activity was nearly identical each other, indicating that the extent of Tuace duplication and the total TuAChE activity are precisely maintained at a level similar to that of the wildtype (susceptible) T. urticae. 4.2. Roles of TuAChE mutations in resistance and fitness cost It was hypothesized previously that the A391T mutation may confer a basal level of resistance based on its 100% frequency in all field populations examined (Kwon et al., 2010b). However, the current findings demonstrate that the A391T mutation does not affect either the substrate kinetics or the monocrotophos inhibition profiles when this mutation exists alone. Intriguingly, however,
Fig. 4. The kinetic properties of the reconstituted TuAChE variants to simulate the UD (one copy with no mutation), PyriF (two copies with the mutation frequencies of 100% A391T and 50% G228S) and AD (six copies with the mutation frequencies of 50% G228S, 100% A391T and 75% F439W) strains. The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
Fig. 5. The median inhibition concentration (I50) values of the reconstituted TuAChE variants to simulate the UD (one copy with no mutation), PyriF (two copies with the frequency of 50% G228S) and AD (six copies with the mutation frequencies of 50% G228S, 100% A391T and 75% F439W) strains. The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
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when the A391T mutation was present together with the F439W mutation, it could restore the decreased vmax value by F439W mutation approximately 2.5 fold (Fig. 2A), thereby resulting in no significant reduction in the net catalytic efficiency (Fig. 2C) without affecting the level of AChE insensitivity caused by the F439W mutation (Fig. 2D). These findings suggest that the A391T mutation counteracts the negative influence of the F439W mutation on the catalytic efficiency reduction and compensates for the possible fitness cost caused by the F439W mutation. Because the A391T mutation is always found in all field populations of T. urticae possessing relatively high frequencies of the F439W mutation, it may have evolved to accompany the F439W mutation to reduce the fitness cost of this latter mutation. Both the G228S and F439W mutations were confirmed to be responsible for AChE insensitivity to monocrotophos (Fig. 3), but these two mutations simultaneously resulted in the reduction of the enzyme’s catalytic efficiency (Fig. 2AeC). The G228 residue forms the oxyanion hole of AChE together with the G227 and A339 residues (Johnson and Moore, 2006). The hydrophobic side chains of these amino acid residues in the oxyanion hole form hydrogen bonds with the carbonyl oxygen of the ligand, thereby stabilizing the tetrahedral transition state of the substrate (Zhang et al., 2002). Therefore, the G228S substitution likely interferes with the acylation process in particular, contributing to the reduction of catalytic efficiency toward the acetylcholine (ACh) substrate. This notion is supported by the finding that the catalytic efficiency reduction caused by the G228S mutation was solely attributed to the decrease of the vmax value. The same mutation at the position corresponding to G228 was also observed in other insects, including Culex and Anopheles mosquitoes, and was determined to be responsible for OP and CB resistance (Weill et al., 2003; Weill et al., 2004), suggesting that G228S is commonly exploited as a resistanceconferring but fitness-costing mutation in arthropods. Moreover, a carboxylesterase mutation (G151D) at the position corresponding to G228 was determined to reduce the catalytic activity toward bnaphthyl acetate substrate but increase the OP hydrolysis activity (Cui et al., 2011), confirming that the G228 residue in the oxyanion hole has an evolutionary conserved role in the substrate and inhibitor kinetics of esterases, including carboxylesterase and AChE. The F439W mutation is located near the anionic subsite, which is involved in the binding of the quaternary trimethylammonium choline moiety of ACh through p-cation interactions, thereby positioning the ester linkage at the acylation site in the esteratic subsite (Harel et al., 1993). The substitution of phenylalanine with tryptophan likely disturbs the normal p-cation interactions of F439, which causes the abnormal positioning of inhibitors and substrates at the esteratic subsite and likely results in the reduction of both the inhibitor sensitivity and the catalytic efficiency of the enzyme. As seen from our kinetic analysis, the F439W mutation caused not only a dramatic increase in the monocrotophos insensitivity but also a decrease in the enzyme’s catalytic efficiency. The effects of the F439W mutation on the insensitivity and catalytic efficiency of AChE were significantly larger than those of the G228S mutation. The F439W mutation resulted in 3.8-fold increase in the AChE insensitivity compared to the G228S mutation, showing that it plays a larger role in resistance. On the other hand, the extent of catalytic efficiency reduction caused by the F439W mutation was approximately 1.5-fold greater than that of the G228S mutation, likely resulting in a larger degree of fitness cost. When the G228S and F439W mutations were present together, their effects on both the inhibitor insensitivity and substrate catalytic efficiency increased drastically. This finding demonstrates that the two mutations work in a synergistic manner, thus boosting the resistance but decreasing the fitness advantage simultaneously.
4.3. Tuace duplication as an adaptive compensation for fitness cost It is apparent that any TuAChE having either a G228S or F439F single mutation or double mutations are very inefficient in substrate hydrolysis. As expected, no T. urticae population having a single copy of Tuace with any of these mutations has been found to date. Instead, duplicated copies (2w6 copies) of Tuace with mutation frequencies in the range of approximately 50w80% are present in all T. urticae populations that are resistant to monocrotophos via the AChE insensitivity mechanism (Kwon et al., 2010a). This close connection between the presence of mutations and the multiple copies of TuAChE suggests that the duplication of Tuace has evolved as a compensating mechanism for the fitness cost attributable to the accumulation of mutations to restore a normal AChE activity level. To demonstrate this concept, we reconstituted TuAChEs that mimic the PyriF and AD strains (Figs. 4 and 5). The reconstituted six copies of the TuAChEs exhibited a level of catalytic efficiency that is comparable to that of the wildtype TuAChE, while still retaining a relatively high level of monocrotophos insensitivity (approximately 159 fold). This result demonstrates that the overexpression of both resistant and susceptible TuAChEs not only rescues the catalytically inefficient AChE encoded by the resistant alleles having mutations, but also confers resistance to monocrotophos. The duplication of Tuace also appears to contribute to the adaptive evolutionary process in T. urticae by allowing the accumulation of more mutations within multiple copies of the AChE gene, which, in turn, would increase the probability for positive selection to occur under continuing chemical pressure. In summary, T. urticae appears to employ two strategies to overcome the fitness costs caused by resistance-conferring mutations. As seen in the case of the A391T mutation that is observed in all field populations of T. urticae, one strategy is to introduce a counteracting mutation that can mitigate the negative impacts of a resistance-conferring mutation on the substrate kinetics, thereby restoring the normal level of catalytic efficiency. The other strategy is the extensive duplication of Tuace, through which greater amounts of the enzyme, including both catalytically efficient and inefficient enzymes, are generated to compensate for the normal level of enzyme activity. Based on these adaptive mechanisms, the monocrotophos resistance mediated by the target site insensitivity mechanism in T. urticae appears to have evolved rapidly through a combination of mutation and extensive gene duplication within a relatively short period of time.
Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008134), Rural Development Administration, Republic of Korea. We thank CE Hwang for her technical assistance in the protein purifications.
Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.ibmb.2011.12.003.
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Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb
The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae Deok Ho Kwon a, Jae Young Choi b, Yeon Ho Je b, Si Hyeock Lee a, b, * a b
Research Institute for Agriculture Life Sciences, Seoul National University, Seoul 151-921, South Korea Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2011 Received in revised form 2 December 2011 Accepted 6 December 2011
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 17.8w27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophosresistant 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 6copy 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. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Tetranychus urticae Acetylcholinesterase Gene duplication Mutation Compensation Resistance
1. Introduction Point mutations in acetylcholinesterase (AChE) have been found in a wide variety of insects and mites, and these mutations confer AChE insensitivities to organophosphate (OP) and carbamate (CB) pesticides (Fournier, 2005; Van Leeuwen et al., 2010). Although these point mutations are required for survival under insecticide selection pressure, they usually cause a change in the catalytic activity of the enzyme because they are generally located near the catalytic triads and other functionally important regions (Fournier, 2005). Such resistance-associated point mutations affecting the
* Corresponding author. Department of Agricultural Biotechnology, Seoul National University, Bldg. 200, Rm# 6120, Seoul 151-742, South Korea. Fax: þ82 2 873 2319. E-mail address: [email protected] (S.H. Lee). 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.12.003
catalytic efficiency of AChE, therefore, have been suggested as the primary cause of the observed fitness costs (Shi et al., 2004). When 15 mutant AChEs in Drosophila melanogaster were produced by in vitro expression and their catalytic activities and insensitivities to OP insecticides were determined, some resistance-conferring mutations caused a reduction in the catalytic activity and stability of the enzyme (Shi et al., 2004). Two mechanisms to compensate for the reduced catalytic efficiency can be suggested. One mechanism involves the introduction of counteracting point mutations. In D. melanogaster, single point mutations (I161V, F330Y and G368A) in AChE reduce the catalytic efficiency of the enzyme, but the AChE activity was recovered when these point mutations were combined with the G265A mutation that results in hyperactivity (Shi et al., 2004). The second compensation mechanism involves maintaining both the susceptible (wildtype) allele and the resistance allele containing mutations via AChE duplication. In the case of the Culex mosquito, two
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alleles of the ace-1 gene encoding AChE, one susceptible and the other resistant to insecticides, were generated by gene duplication in response to insecticide pressure (Labbe et al., 2007a). It has been proposed that the duplication was selected because the presence of the susceptible ace-1 allele reduces the fitness cost associated with the resistant allele via the generation of persistent, more adaptable heterozygotes (Labbe et al., 2007b). A recent report by Alout et al. showed that the occurrence of ace-1 duplications in Culex pipiens is high (Alout et al., 2011). Three point mutations (G228S, A391T and F439W; G119S, A280T and F331W in the amino acid numbering of Torpedo californica AChE, respectively) in AChE have been reported to confer resistance to monocrotophos, an OP acaricide, in the two-spotted spider mite Tetranychus urticae (Kwon et al., 2010b). Among these mutations, the A391T mutation was found in all of the green-type T. urticae populations, which differ slightly from the susceptible UD strain in their mitochondrial cytochrome oxidase I marker sequences (Kwon et al., 2011). A kinetic analysis of the AChEs from three T. urticae strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) revealed that the AChEs from the AD and PyriF strains have reduced catalytic efficiencies, suggesting that the resistant form of AChE is likely accompanied by a fitness cost (Kwon et al., 2010a). A comparison of the T. urticae AChE (TuAChE) gene (Tuace) copy number showed that relatively more Tuace copies are present in the resistant strains than in the susceptible strain (Kwon et al., 2010a). The relative copy numbers of Tuace in field populations correlated precisely with the levels of resistance, indicating an additional role of Tuace gene duplication in resistance. Given that gene dosage is precisely regulated in living organisms, extensive gene duplication may disrupt this balance (Veitia, 2005). In the case of Tuace duplication, however, increased AChE dosages in both the AD and PyriF strains do not appear to disrupt the overall function of AChE because the total activities of the increased amounts of AChE in these mite strains were not significantly different from that of the UD strain (which likely has only a single copy of AChE), as judged by their similar vmax and Km values. Rather, the AChE dosage in the resistant mites appears to have been increased to adjust the overall AChE activity to a level similar to that of UD. The recent analysis of T. urticae genome revealed that Tuace is the only locus encoding a functional AChE (Grbic et al., 2011), excluding the possibility of other paralogous AChE(s) contributing to AChE activity. It is currently unclear, however, whether the extensive Tuace duplication actually results in TuAChE overexpression at the protein level and, if this is the case, how such overexpression has been allowed for AChE, of which titer should be carefully regulated to maintain the homeostasis of the excitatory nervous system. The advantages and costs of ace-1 duplications in relation to OP resistance have previously been described for Culex mosquitoes from the perspective of gene dosage (Labbe et al., 2007a, 2007b). On the basis of the observation that the catalytic activity of the resistant form of AChE1 is less than 60% of the susceptible form (Bourguet et al., 1997, 1996), it was suggested that ace-1 duplication may restore the normal gene dosage, which is otherwise phenotypically reduced by the resistance mutation (Labbe et al., 2007a). In this study, the amounts of TuAChE were measured using a TuAChE-specific antibody and correlated with the Tuace copy numbers in several field populations. Then, the TuAChE variants with different mutation combinations were expressed in vitro, and their enzymatic properties were analyzed from the perspective of resistance and fitness cost. To investigate the effects of copy number and mutation on the total AChE activity, appropriate AChE variants were combined to generate the desired AChE copies and mutation combinations/frequencies, and their enzyme kinetics were determined. Finally, Tuace duplication as a compensatory
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adaptation mechanism for the catalytic efficiency reduction as a result of resistance-conferring mutations was discussed in relation to OP resistance. 2. Materials and methods 2.1. Mites T. urticae mites were collected from five host plants in the field or greenhouses between 2006 and 2008 (Supplemental Table 1). The collected strains were maintained on kidney bean plants (2 weeks old, Phaseolus vulgaris variety humilis) in the laboratory under the conditions of 25 1 C, 55 5% relative humidity and a 16:8 (L:D) photoperiod. The UD and PyriF strains were reared without any exposure to acaricides. The AD strain was selected approximately every two months with a LC50 dose of monocrotophos. 2.2. Genomic DNA extraction and gene copy number determination The genomic DNA (gDNA) from seven strains of T. urticae was extracted from 5 mg female adults using a DNeasy Blood & Tissue kit (Qiagen GmBH, Germany) according to the manufacturer’s instructions. Briefly, the mites were ground using a fabricated plastic homogenizer in tissue lysis (TL) buffer and incubated with 10 ml proteinase K (10 mg/ml) at 56 C for 1 h. The gDNA was isolated using Mini-spin columns, and the purified gDNA was stored at 20 C until use. Quantitative real-time PCR (qPCR) was employed to determine the gene copy number of Tuace as described previously (Kwon et al., 2010a). 2.3. Generation of an anti-TuAChE polyclonal antibody The rabbit anti-TuAChE1 polyclonal antibodies were produced by Ab Frontier (Seoul, Korea). The two bovine serum albumin-fused peptide fragments [RKKAYQRSLAL (amino acid position 346e356) and EDPEVSLRTKNFK (amino acid position 411e423)] were synthesized. The rabbits were immunized three times with 0.5 mg of the two synthesized peptides. The serum-specific antibodies were affinity-purified on columns using immobilized antigen peptides. The titer of the anti-TuAChE1 polyclonal antibody was approximately 2 mg/ml. 2.4. Native polyacrylamide gel electrophoresis (PAGE) and Western blotting The female mites (ca. 15 mg) were homogenized in 300 ml extraction buffer (0.1 M TriseHCl, pH 7.8) using a 2-ml glasseglass tissue grinder (Radnoti, Monrovia, CA), and the homogenate was centrifuged at 10,000 g for 15 min. Following centrifugation, the supernatant was decanted and used as the TuAChE source. The protein concentration was determined by BCA methods using bovine serum albumin as a standard. The duplicate enzyme samples (40 mg protein each) were separated by discontinuous native PAGE (5% T/2.67% C stacking and 7.5% T/2.67% C separating gels) at 120 V for 1 h and at 180 V for 3 h in a 4 C cold chamber. After electrophoresis, one gel was activity-stained for the visualization of the TuAChE band according to the method of Lewis and Shute (1966). The second gel was electro-blotted to a nylon membrane (Amersham Hybond-ECL, GE Healthcare, Piscataway, NJ) at 35 V for 1 h 10 min, and the blotted membrane was used for a Western blot analysis. The membrane was hybridized with the primary antibody (diluted 1:5000 in PBST buffer) for 2 h and washed three times with PBST buffer in 10-min intervals. The membrane was further
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hybridized with the secondary antibody (Rabbit Anti-Mouse IgG, Thermo Fisher Scientific, Rockford, IL) diluted 1:20,000 with 5% skim milk for 1 h and was then washed three times with PBST buffer in 10-min intervals. The membrane was incubated with Western blotting luminol reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and exposed to X-ray film (Agfa-Gevaert, Mortsel, Belgium). The exposure time was minimized (ca. 5 min) to obtain band intensities with a more linear range. The band intensities of the activity-stained gel and the Western blot were quantified using an EDAS 290 gel image analyzer (Eastman Kodak Company, Rochester, NY). 2.5. Expression vector construction The full-length Tuace clone (2061 bp, 687 amino acids) of the UD strain was cloned into a pGEM-T easy vector (Promega, Madison, WI, USA). A truncated Tuace cDNA fragment (649 amino acids, 1947 bp, ‘MVPMF w LINAL’) with its C-terminal hydrophobic region excluded was PCR-amplified with gene-specific primers containing restriction enzyme sites (XbaI and EcoRI) and a 6 His tag sequence (Supplemental Table 2) using an Advantage 2 Polymerase Mix (Takara Bio Inc., Shiga, Japan) under the following thermal program: (95 C for 30 s, 62 C for 30 s, 72 C for 3 min) 35 cycles. The PCR-amplified DNA fragment (TuAChE_UD) was digested with XbaI and EcoRI and inserted into the pBacPAK8 vector (approximately 5517-bp) (Clontech, Palo Alto, CA, USA) that had been predigested with the same restriction enzymes. 2.6. Site-directed mutagenesis Using the TuAChE_UD clone in the pBacPAK8 expression vector (approximately 7491-bp) as a template, site-directed mutagenesis was conducted to generate Tuace clone variants having single (SAF, GTF and GAW), double (SAW, STF and SAW) and triple (STW) mutations using appropriate primer sets according to the manufacturer’s instructions (Supplemental Table 2). First, the clones with a single point mutation (G228S, A391T and F439W) were constructed using respective primer sets, and then the clones with double point mutations (G228S þ A391T, A391T þ F439W and G228S þ F439W) were generated from the single mutation clones. Finally, a clone with triple mutations (G228S þ A391T þ F439W) was constructed using one of the double-mutation clones as a template. 2.7. TuAChE expression and purification To generate recombinant Autographa californica nucleopolyhedroviruses (AcMNPVs), the resulting transfer vectors were cotransfected with bAcGOZA DNA (Je et al., 2001) into Sf9 cells maintained in TC-100 medium (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum at 27 C. The transfection was performed using the Cellfectin (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions, and the recombinant viruses were purified by a plaque assay of the Sf9 cells as previously described (O’Reilly et al., 1992). To exclude the serumderived esterase activity, the Sf9 cells maintained in SF900-II serum-free medium (Invitrogen) were seeded onto 100-mm diameter tissue culture dishes at a density of 5 106 cells/dish, followed by incubation at 27 C for 30 min to allow for cell attachment. The attached cells were washed twice with 3 ml of incomplete TC-100 medium and inoculated with 1 ml of properly diluted viral stock. After incubation for 84 h at 27 C, the cell pellets were collected by centrifugation at 5000 g for 15 min and sonicated with a Branson Sonifier (Branson SonifierÒ, Danbury, CT) under TriseHCl (pH 7.8, 0.02 M NaCl) buffer conditions for 6 min at
4 C (Out control, 3; Duty cycle, 30%). The lysed cells were centrifuged at 12,000 g for 10 min, and the supernatant was centrifuged again at approximately. 134,000 g for 20 min at 4 C in an ULTRA 4.0 ultracentrifuge (Hanil Science Industrial, Incheon, Korea). The supernatant was used for further His-Tag purification. Approximately 6e7 ml of supernatant was loaded onto a 5-ml HisTrap HP affinity column (Amersham Biosciences Corp., Piscataway, NJ) at a flow rate of 5 ml/min using an AKTA FPLC (Amersham Bioscience UPC-900). The washing and binding fractions were discarded, and the column was eluted with 250 mM imidazolecontaining elution buffer. Each fraction was divided with a Frac920 fraction collector (Amersham Biosciences). The eluted protein was concentrated with a YM-50 filter and stored at 80 C until use. The protein concentration of the semi-purified TuAChE was estimated based on the band intensities determined both by Western blotting and Coomassie staining after SDS-PAGE. The protein concentration of the semi-purified TuAChE solution was normalized prior to the enzyme kinetic analysis. 2.8. AChE kinetic analysis and determination of the median inhibition (I50) concentrations The AChE activity was measured by Ellman’s method with a slight modification (Ellman et al., 1961). For the calculation of the kinetic constants, the enzyme (in approximately 1 ng of total protein) was incubated with 0.4 mM 5,5-dithio-bis 2-nitrobenzoic acid (DTNB, SigmaeAldrich Co.) and 0.03e1 mM acetylthiocholine iodide (ATChI) at 30 C. The AChE activity was measured at 412 nm in 20-second intervals over 10 min using a VERSAmax microplate reader (Molecular Device Inc., Sunnyvale, CA). The MichaeliseMenten constants (Km) and maximum enzyme velocities (vmax) were calculated from Lineweaver-Burk plots. The kcat value was determined by dividing the vmax value by the Km constant. The I50 concentration was determined based on the following procedures. Aliquots [2 ml for wildtype, all single mutation variants and one double mutation (A391T þ F439W) variant; 10 ml for the remaining double and triple mutation variants] of purified TuAChE were preincubated with a range of monocrotophos solutions (0.0003e10 mM) for 10 min, and the remaining activities were measured by adding 0.4 mM DTNB and 1 mM ATChI at 30 C. All of the kinetic constants and inhibition values were determined from three independent experiments. The statistical analysis was carried out with ANOVA post-hoc analysis using the SAS software package (SAS Institute, NC, USA). 2.9. Reconstitution of TuAChE To simulate the TuAChEs in the AD and PyriF strains in terms of TuAChE copy number and the frequencies of the G228S and F439W mutations, reconstitution of TuAChE was conducted by combining the appropriate TuAChE variants. For the TuAChE of the UD strain, the TuAChEGAF variant was used directly. To simulate the PyriF strain, which has ca. 50% G228S mutation frequency in two copies of TuAChE, equal amounts of the TuAChEGTF and TuAChESTF variants were combined. To simulate the AD strains possessing six copies of TuAChE with mutation frequencies of 50% for G228S and 75% for F439W, the TuAChESTF, TuAChEGTW and 25% TuAChESTW variants were combined in a ratio of 1:2:1. The concentration of each TuAChE variant was normalized to 0.48 ng/ml. The concentrations of the reconstituted TuAChEs were adjusted to 0.48, 0.96 and 2.88 ng/ ml to represent the copy numbers of 1 (UD), 2 (PyriF) and 6 (AD), respectively. The reconstituted TuAChEs were designated as TuAChEUD, TuAChEPyriF and TuAChEAD.
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3. Results 3.1. Correlation between TuAChE quantity and Tuace copy number Western blotting using anti-TuAChE antibody (TuAChE-Ab) following native PAGE revealed that the quantity of TuAChE varied notably among the different strains of T. urticae (Fig. 1A, upper gel). In contrast, the total activity of TuAChE from different strains differed only slightly, as judged by the similar band intensities in the activity-stained gel (Fig. 1A, lower gel). When the TuAChE quantities (TuAChE band intensities in the Western blot) were plotted against the corresponding Tuace copy numbers, a high level of correlation (r2 ¼ 0.914) was obtained, demonstrating that the quantity of TuAChE is proportional to the copy number (Fig. 1B). There was no apparent correlation (r2 ¼ 0.317) between the TuAChE activities (TuAChE band intensities in the activity-stained gel) and the Tuace copy numbers (data not shown), revealing that TuAChE activity is not dependent on its amount. In summary, these results demonstrated that Tuace duplication results in the overexpression of TuAChE at the protein level, but an increase in the quantity of TuAChE does not affect the total enzyme activity. 3.2. Expression of TuAChE variants To verify the authenticity and purity of the TuAChE variants that were expressed and purified in vitro, the samples were analyzed by SDS-PAGE in conjunction with Western blotting (Supplemental Fig. 1). A single TuAChE band (ca. 72.7 kD) was detected with the TuAChE-Ab in all of the TuAChE variants, as shown in the Western blot analysis (see upper gel). Even after its purification by Ni2affinity chromatography, however, the sample contained other contaminating proteins (see lower gel). Based on the band intensity
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of the protein-stained gels, the proportion of expressed TuAChE in the sample was determined to be approximately 3e11%. Therefore, the actual TuAChE concentrations in the subsequent enzyme kinetics and inhibition assays were determined by multiplying by the purity. 3.3. Enzymatic kinetic properties of the in vitro expressed TuAChE variants Eight different TuAChE variants (TuAChEGAF, TuAChESAF, TuAChEGTF, TuAChEGAW, TuAChESAW, TuAChESTF, TuAChEGTW and TuAChESTW; see the designation in Table 1), possessing the three point mutations putatively associated with AChE insensitivity either singly or in combination, were expressed in vitro, and their kinetic properties (vmax, Km and kcat) were examined (Fig. 2 and Supplemental Table 3). Upon comparison of the vmax values (Fig. 2A), the wildtype (TuAChEGAF) and TuAChEGTF exhibited similar vmax values (0.106 0.030 and 0.119 0.028 mM sec1 mg1, respectively) (Fig. 2A), suggesting that the A391T mutation does not alter the catalytic activity of TuAChE significantly. When the G228S mutation is present alone or with the A391T mutation (TuAChESAF and TuAChESTF), the vmax values decreased ca. 10.3- and 10.4-fold compared with those of TuAChEGAF and TuAChEGTF, respectively. Likewise, the presence of the F439W mutation, alone or with the A391T mutation (TuAChEGAW and TuAChEGTW), resulted in a ca. 5.2and 2.3-fold reduction in the vmax values relative to those of TuAChEGAF and TuAChEGTF, respectively. In addition, the presence of the A391T mutation did not influence the function of the G228S or the F439W mutation in the vmax determination. Interestingly, the vmax values of the TuAChESAW and TuAChESTW variants, possessing both G228S and F439W mutations together, decreased ca. 32.7- and 63.3-fold compared with the values determined for TuAChEGAF and TuAChEGTF, respectively. These results suggest that the presence of the G228S and F439W mutations together have a synergistic effect on each other and can impair the catalytic activity of TuAChE dramatically. The Km values of the eight TuAChE variants ranged from 0.08 to 0.3 mM (Fig. 2B). TuAChEGAF and TuAChEGTF exhibited relatively higher Km values (0.29 0.12 and 0.31 0.07 mM, respectively) compared with the other variants. No significant difference between the wildtype enzyme and the TuAChEGTF variant was found, indicating that the A391T mutation does not change the substrate affinity, similar to its lack of effect on the vmax value. However, the Km values of the TuAChE variants possessing the G228S mutation (TuAChESAF and TuAChESTF) decreased 5.8- and 4.0-fold compared to the values determined for TuAChEGAF and TuAChEGTF, indicating that the G228S mutation increases the substrate affinity of the enzyme. The presence of the F439W mutation, alone or with the A391T mutation (TuAChEGAW and TuAChEGTW), also reduced the Km values ca. 1.7- and 1.9-fold, Table 1 Description of alleles about each mutant TuAChE.
Fig. 1. Western blotting (upper gel) and activity staining (lower gel) of the TuAChE samples from seven strains of T. urticae following native PAGE (A). The correlation between the TuAChE quantity (band intensity from the Western blot) and the corresponding Tuace copy number (B).
TuAChE variant designationa
Mutation
Remarks
TuAChEGAF TuAChESAF TuAChEGAW TuAChESAW TuAChEGTF TuAChESTF TuAChEGTW TuAChESTW
G228 þ A391 þ F439 G228S þ A391 þ F439 G228 þ A391 þ F439W G228S þ A391 þ F439W G228 þ A391T þ F439 G228S þ A391T þ F439 G228 þ A391T þ F439W G228S þ A391T þ F439W
Wildtype Single mutation Single mutation Double mutation Single mutation Double mutation Double mutation Triple mutation
a Bolded and underlined characters represent the amino acids at the mutation site.
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(TuAChESAF and TuAChESTF) exhibited 1.7- and 2.7-fold decreased kcat values, respectively. When the F439W mutation was introduced into the wildtype background (TuAChEGAW), the catalytic efficiency decreased 2.6-fold. Interestingly, when the F439W mutation is present together with the A391T mutation, as in the TuAChEGTW variant, the catalytic efficiency was not significantly different from that of the wildtype enzyme (p ¼ 0.521), suggesting that the A391T mutation may counteract the effects of the F439W mutation to restore the normal AChE function. In the TuAChE variants possessing both the G228S and F439W mutations together (TuAChESAW and TuAChESTW), the kcat values decreased 17.6- and 29.0-fold compared to the values determined for TuAChEGAF and TuAChEGTF. These findings suggest that both the G228S and F439W mutations dramatically reduce the catalytic efficiency of TuAChE in a synergistic manner. 3.4. Monocrotophos inhibition properties of in vitro expressed TuAChE variants The I50 concentrations of TuAChEGAF and TuAChEGTF were 0.025 0.006 and 0.016 0.005 mM, respectively (Fig. 3 and Supplemental Table 3). Although TuAChEGTF showed a slightly higher (1.6 fold) sensitivity to monocrotophos compared to that of the wildtype TuAChEGAF, this difference was statistically insignificant (p ¼ 0.108). This finding suggests that, in contrast to our expectations, the A391T mutation is not associated with the AChE insensitivity (Kwon et al., 2010b). The TuAChESAF and TuAChESTF variants possessing the G228S mutation exhibited 16.1- and 26.1fold reduced sensitivity compared with those of TuAChEGAF and TuAChEGTF, respectively, as judged by their I50 values. When the F439W mutation was present, the I50 values increased 60.7- and 99.4-fold in the TuAChEGAW and TuAChEGTW variants, respectively. These findings further suggest that the effects of the F439W mutation on AChE insensitivity are approximately 3.8-fold larger than those of the G228S mutation. Intriguingly, the monocrotophos sensitivities of the TuAChESAW and TuAChESTW variants, possessing double mutations simultaneously, were remarkably reduced, showing 1406- and 1163-fold increases in their I50 values. These findings demonstrate that both the G228S and F439W mutations confer a reduced sensitivity to monocrotophos in a synergistic manner, and the effects of F439W are greater than those of G228S.
Fig. 2. The kinetic properties of eight in vitro expressed TuAChE variants with various combinations of three mutations (G228S, A391T and F439W). vmax constant (A), Km constant (B) and catalytic efficiency (kcat) (C). The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
respectively. When both the G228S and F439W mutations existed together, the TuAChESAW and TuAChESTW variants exhibited 1.9and 2.2-fold lower Km values, respectively. These findings suggest that all of the mutations except for A391T have a tendency to increase the substrate affinity of the resulting enzymes. The TuAChEGAF and TuAChEGTF variants exhibited the highest catalytic efficiency (kcat) values (0.40 0.09 and 0.38 0.05, respectively) (Fig. 2C). The presence of the A391T mutation did not affect the catalytic efficiency, as no significant difference between the wildtype enzyme and the TuAChEGTF variant was observed. The TuAChE variants possessing the single point mutation of G228S
Fig. 3. The median inhibition concentration (I50) values of eight in vitro expressed TuAChE variants having various combinations of three mutations (G228S, A391T and F439W). The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
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3.5. Kinetic and inhibition properties of reconstituted TuAChE Because the TuAChE variant possessing the A391T mutation (TuAChEGTF) did not differ from the wildtype enzyme (TuAChEGAF), the reconstitution of TuAChE was conducted using the TuAChEGTF variant as a wildtype enzyme. The vmax values of TuAChEPyriF and TuAChEAD were 1.5-fold lower than those of TuAChEUD, but the differences were not significant (p ¼ 0.196) (Fig. 4A). Larger Km values (1.7 fold), when compared with TuAChEUD, were observed in TuAChEPyriF and TuAChEAD, suggesting their slightly reduced substrate affinity, although these values were not significantly different (p ¼ 0.241). Both TuAChEPyriF and TuAChEAD have 2.7-fold lower kcat values than TuAChEUD, revealing that reconstituted TuAChEs have reduced, but still comparable, catalytic efficiencies
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compared to that of wildtype TuAChE. This slightly reduced level of catalytic efficiency coincided well with the slightly reduced total activity of AChE observed in the crude enzyme preparation of the AD strain (Kwon et al., 2010a). However, the kcat values of TuAChEPyriF and TuAChEAD showed no significant differences, suggesting that both TuAChEs representing the PyriF and AD strains maintain identical levels of activity despite their difference in copy number. In the inhibition assay, the I50 value of TuAChEAD was 158.7- and 84.5-fold higher than those of TuAChEUD and TuAChEPyriF, respectively (Fig. 5). This insensitivity level of TuAChEAD was comparable to that observed previously with the AChE preparation extracted from the AD strain (i.e., ca. 90.6- and 41.9-fold increased insensitivity compared to the UD and PyriF strains) (Kwon et al., 2010b). TuAChEPyriF exhibited a 1.9-fold lower I50 value than TuAChEUD, suggesting a slightly increased sensitivity to monocrotophos, but the difference was not significant (p ¼ 0.062). 4. Discussion 4.1. Overexpression of TuAChE by gene duplication According to our previous report, the transcription of Tuace was proportional to the level of duplication at a ratio of 1:1 in the three strains examined, indicating that all duplicated copies of Tuace are actively transcribed (Kwon et al., 2010a). The present study confirmed that Tuace duplication results in the overexpression of TuAChE at the protein level. A cross-comparison of several mite strains demonstrated that the quantity of TuAChE was directly proportional to the Tuace copy numbers (Fig. 1). Regardless of TuAChE quantity, however, the overall AChE activity was nearly identical each other, indicating that the extent of Tuace duplication and the total TuAChE activity are precisely maintained at a level similar to that of the wildtype (susceptible) T. urticae. 4.2. Roles of TuAChE mutations in resistance and fitness cost It was hypothesized previously that the A391T mutation may confer a basal level of resistance based on its 100% frequency in all field populations examined (Kwon et al., 2010b). However, the current findings demonstrate that the A391T mutation does not affect either the substrate kinetics or the monocrotophos inhibition profiles when this mutation exists alone. Intriguingly, however,
Fig. 4. The kinetic properties of the reconstituted TuAChE variants to simulate the UD (one copy with no mutation), PyriF (two copies with the mutation frequencies of 100% A391T and 50% G228S) and AD (six copies with the mutation frequencies of 50% G228S, 100% A391T and 75% F439W) strains. The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
Fig. 5. The median inhibition concentration (I50) values of the reconstituted TuAChE variants to simulate the UD (one copy with no mutation), PyriF (two copies with the frequency of 50% G228S) and AD (six copies with the mutation frequencies of 50% G228S, 100% A391T and 75% F439W) strains. The different letters on top of the bars indicate statistically significant differences (p < 0.05; ANOVA post-hoc analysis).
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when the A391T mutation was present together with the F439W mutation, it could restore the decreased vmax value by F439W mutation approximately 2.5 fold (Fig. 2A), thereby resulting in no significant reduction in the net catalytic efficiency (Fig. 2C) without affecting the level of AChE insensitivity caused by the F439W mutation (Fig. 2D). These findings suggest that the A391T mutation counteracts the negative influence of the F439W mutation on the catalytic efficiency reduction and compensates for the possible fitness cost caused by the F439W mutation. Because the A391T mutation is always found in all field populations of T. urticae possessing relatively high frequencies of the F439W mutation, it may have evolved to accompany the F439W mutation to reduce the fitness cost of this latter mutation. Both the G228S and F439W mutations were confirmed to be responsible for AChE insensitivity to monocrotophos (Fig. 3), but these two mutations simultaneously resulted in the reduction of the enzyme’s catalytic efficiency (Fig. 2AeC). The G228 residue forms the oxyanion hole of AChE together with the G227 and A339 residues (Johnson and Moore, 2006). The hydrophobic side chains of these amino acid residues in the oxyanion hole form hydrogen bonds with the carbonyl oxygen of the ligand, thereby stabilizing the tetrahedral transition state of the substrate (Zhang et al., 2002). Therefore, the G228S substitution likely interferes with the acylation process in particular, contributing to the reduction of catalytic efficiency toward the acetylcholine (ACh) substrate. This notion is supported by the finding that the catalytic efficiency reduction caused by the G228S mutation was solely attributed to the decrease of the vmax value. The same mutation at the position corresponding to G228 was also observed in other insects, including Culex and Anopheles mosquitoes, and was determined to be responsible for OP and CB resistance (Weill et al., 2003; Weill et al., 2004), suggesting that G228S is commonly exploited as a resistanceconferring but fitness-costing mutation in arthropods. Moreover, a carboxylesterase mutation (G151D) at the position corresponding to G228 was determined to reduce the catalytic activity toward bnaphthyl acetate substrate but increase the OP hydrolysis activity (Cui et al., 2011), confirming that the G228 residue in the oxyanion hole has an evolutionary conserved role in the substrate and inhibitor kinetics of esterases, including carboxylesterase and AChE. The F439W mutation is located near the anionic subsite, which is involved in the binding of the quaternary trimethylammonium choline moiety of ACh through p-cation interactions, thereby positioning the ester linkage at the acylation site in the esteratic subsite (Harel et al., 1993). The substitution of phenylalanine with tryptophan likely disturbs the normal p-cation interactions of F439, which causes the abnormal positioning of inhibitors and substrates at the esteratic subsite and likely results in the reduction of both the inhibitor sensitivity and the catalytic efficiency of the enzyme. As seen from our kinetic analysis, the F439W mutation caused not only a dramatic increase in the monocrotophos insensitivity but also a decrease in the enzyme’s catalytic efficiency. The effects of the F439W mutation on the insensitivity and catalytic efficiency of AChE were significantly larger than those of the G228S mutation. The F439W mutation resulted in 3.8-fold increase in the AChE insensitivity compared to the G228S mutation, showing that it plays a larger role in resistance. On the other hand, the extent of catalytic efficiency reduction caused by the F439W mutation was approximately 1.5-fold greater than that of the G228S mutation, likely resulting in a larger degree of fitness cost. When the G228S and F439W mutations were present together, their effects on both the inhibitor insensitivity and substrate catalytic efficiency increased drastically. This finding demonstrates that the two mutations work in a synergistic manner, thus boosting the resistance but decreasing the fitness advantage simultaneously.
4.3. Tuace duplication as an adaptive compensation for fitness cost It is apparent that any TuAChE having either a G228S or F439F single mutation or double mutations are very inefficient in substrate hydrolysis. As expected, no T. urticae population having a single copy of Tuace with any of these mutations has been found to date. Instead, duplicated copies (2w6 copies) of Tuace with mutation frequencies in the range of approximately 50w80% are present in all T. urticae populations that are resistant to monocrotophos via the AChE insensitivity mechanism (Kwon et al., 2010a). This close connection between the presence of mutations and the multiple copies of TuAChE suggests that the duplication of Tuace has evolved as a compensating mechanism for the fitness cost attributable to the accumulation of mutations to restore a normal AChE activity level. To demonstrate this concept, we reconstituted TuAChEs that mimic the PyriF and AD strains (Figs. 4 and 5). The reconstituted six copies of the TuAChEs exhibited a level of catalytic efficiency that is comparable to that of the wildtype TuAChE, while still retaining a relatively high level of monocrotophos insensitivity (approximately 159 fold). This result demonstrates that the overexpression of both resistant and susceptible TuAChEs not only rescues the catalytically inefficient AChE encoded by the resistant alleles having mutations, but also confers resistance to monocrotophos. The duplication of Tuace also appears to contribute to the adaptive evolutionary process in T. urticae by allowing the accumulation of more mutations within multiple copies of the AChE gene, which, in turn, would increase the probability for positive selection to occur under continuing chemical pressure. In summary, T. urticae appears to employ two strategies to overcome the fitness costs caused by resistance-conferring mutations. As seen in the case of the A391T mutation that is observed in all field populations of T. urticae, one strategy is to introduce a counteracting mutation that can mitigate the negative impacts of a resistance-conferring mutation on the substrate kinetics, thereby restoring the normal level of catalytic efficiency. The other strategy is the extensive duplication of Tuace, through which greater amounts of the enzyme, including both catalytically efficient and inefficient enzymes, are generated to compensate for the normal level of enzyme activity. Based on these adaptive mechanisms, the monocrotophos resistance mediated by the target site insensitivity mechanism in T. urticae appears to have evolved rapidly through a combination of mutation and extensive gene duplication within a relatively short period of time.
Acknowledgments This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008134), Rural Development Administration, Republic of Korea. We thank CE Hwang for her technical assistance in the protein purifications.
Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.ibmb.2011.12.003.
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