Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae

Accepted Manuscript Title: Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae Author: Nena Pavlidi, Vasilis Tseliou, Maria Riga, Ralf Nauen, Thomas Van Leeuwen, Nikolaos E. Labrou, John Vontas PII: DOI: Reference:

S0048-3575(15)00010-3 http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.009 YPEST 3782

To appear in:

Pesticide Biochemistry and Physiology

Received date: Accepted date:

31-10-2014 13-1-2015

Please cite this article as: Nena Pavlidi, Vasilis Tseliou, Maria Riga, Ralf Nauen, Thomas Van Leeuwen, Nikolaos E. Labrou, John Vontas, Functional characterization of glutathione Stransferases associated with insecticide resistance in Tetranychus urticae, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae Nena Pavlidia, Vasilis Tselioua, Maria Rigaa, Ralf Nauenb, Thomas Van Leeuwenc, Nikolaos E.Labroud and John Vontase,f*

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a

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b

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c

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d

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e

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f

Department of Biology, University of Crete, 71409 Heraklion, Greece

BayerCropScience AG, RD-SMR Pest Control Biology, Alfred Nobel Str. 50, D-40789 Monheim, Germany Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1098 XH Amsterdam, The Netherlands Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 IeraOdos Street, GR11855-Athens, Greece. Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13, Heraklion Crete, Greece Laboratory of Pesticide Science, Department of Crop Science, Agricultural University of Athens, 75 IeraOdos Street, GR-11855-Athens, Greece.

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*Corresponding author. E-mail addresses: [email protected], [email protected] (J.

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Vontas).

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Highlights

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Expression of three Glutathione Transferases associated with insecticide resistance Characterization of recombinant proteins (steady state kinetics-interaction with insecticides) TuGSTd14 showed the strongest interaction with abamectin Analysis of TuGSTd14 structure by modelling

Graphical Abstract

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Abstract

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The two-spotted spider mite Tetranychus urticae is one of the most important

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agricultural pests world-wide. It is extremely polyphagous and develops resistance to

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acaricides. The overexpression of several glutathione S-transferases (GSTs) has been

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associated with insecticide resistance. Here, we functionally expressed and

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characterized three GSTs, two of the deltaclass (TuGSTd10, TuGSTd14) and one of

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mu class (TuGSTm09), which had been previously associated with striking resistance

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phenotypes against abamectin and other acaricides/insecticides, by transcriptional

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studies. Functional analysis showed that all three GSTs were capable of catalyzing the

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conjugation of both 1-chloro-2,4 dinitrobenzene (CDNB) and 1,2- dichloro-4-

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nitrobenzene(DCNB), to glutathione (GSH), as well as they exhibit GSH-dependent

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peroxidase activity towards Cumene hydroperoxide (CumOOH). The steady-state

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kinetics of the T. urticae GSTs for the GSH/CDNB conjugation reaction were

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determined and compared with other GSTs. The interaction of the three recombinant

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proteins with several acaricides and insecticides was also investigated. TuGSTd14 2 Page 2 of 22

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showed the highest affinity towards abamectin and a competitive type of inhibition,

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which suggests that the insecticide may bind to the H-site of the enzyme. The three-

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dimensional structure of the TuGSTd14 was predicted based on X-ray structures of

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delta class GSTs using molecular modeling. Structural analysis was used to identify

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key structural characteristics and to provide insights into the substrate specificity and

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the catalytic mechanism of TuGSTd14.

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Key words: Tetranychusurticae, insecticide resistance, abamectin, glutathione S-

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transferases, enzyme modelling

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Introduction

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The polyphagous spider mite Tetranychus urticae Koch is one of the most damaging

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agricultural pests in the world. Its control is best achieved by the use of

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acaricides/insecticides. However, there is a limited portfolio of active ingredients to

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tackle the problem, which results in the selection of acaricide/insecticide resistance

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[1]. The problem of insecticide resistance in T. urticae is enormous, as the species is

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among the “most resistant species” in terms of the total number of pesticides to which

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populations have become resistant (www.pesticideresistance.org).

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Genome-wide gene expression analysis in a T. urticae strain (Mar-ab), which was

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isolated from a rose greenhouse near Athens and exhibited >1600-fold resistance to

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abamectin, as well as high levels of resistance to other pesticides identified a set of

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genes that are associated with the phenotype. This included several detoxification

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genes, such as several members of the cytochrome P450 and the glutathione S-

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transferase (GST) gene families [2]. Some of these genes were also up-regulated in

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another multi-resistant strain isolated from Belgium [2]. The functional role of some

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P450s (such as the CYP392A16) present in the consensus has been already elucidated

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[3]. However, the putative role of the GSTs, which were found up-regulated in both

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multi-resistant strains, has not been studied at the protein level as yet.

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GSTs are a major family of detoxification enzymes mainly involved in phase II

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metabolism. They catalyze the conjugation of the tripeptide glutathione (GSH) to the

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electrophilic centre of xenobiotics thus increasing their water solubility and aiding

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excretion from the cell [4]. The GSTs have been involved in insecticide detoxification

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by mediating the O-dealkylation or O-dearylation of organophosphorus insecticides

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[5, 6], catalyzing the dehydrochlorination of organochlorines [7], as well as primary

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insecticide metabolism or lipid peroxidation byproducts [8]. GSTs may also

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contribute to insecticide resistance by binding insecticide molecules (such as

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pyrethroids) via a sequestration mechanism [9]. The role of GSTs in conferring

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resistance against abamectin, an active ingredient which has been widely used for the

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control of T. urticae, has not been investigated, although a strong association of

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elevated GST activity with abamectin resistance has been demonstrated in several

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occasions in this pest [10].

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Thirty one GSTs have been identified in the T. urticae genome. They belong to the

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classes delta (16 GSTs), mu (12 GSTs), omega (2 GSTs) and theta (1 GST) [11].

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Two TuGSTs of delta class, the TuGSTd10 and the TuGSTd14, and one of the mu

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class, the TuGSTm09, have been strongly associated with the abamectin/multiple

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resistance phenotype in recent microarray–based transcriptional studies [2]. Delta

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class GSTs have been involved in insecticide detoxification in insects [12], while mu

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class GSTs have been involved in detoxification of reactive oxygen species in

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mammals [13, 14].

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Here, we functionally expressed the TuGSTd10, TuGSTd14 and TuGSTm09 in

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Escherichia coli and examined their catalytic properties against model substrates, as

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well as their potential to interact with abamectin and other insecticides.

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Materials and methods Cloning, functional expression and purification of recombinant GSTs The cDNA sequences encoding for TuGSTd10 (TeturID: tetur26g02802), TuGSTd14

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(TeturID: tetur29g00220) and TuGSTm09 (TeturID: tetur05g05260), were amplified

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from Mar-ab cDNA [3]. For cDNA preparation, total RNA of adult spider mites was

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extracted using RNeasy mini kit (Qiagen, USA), treated with Turbo DNA-free

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(Ambion Life Technologies, USA) and reverse transcribed using Superscript III

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reverse transcriptase (Invitrogen Life Technologies, USA) and oligo(-dT)17 primer.

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For PCR amplification of TuGSTs,Pfu DNA polymerase (Thermo Scientific, USA)

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and specific primers (listed at Table 1) were used. PCR conditions were 95oC for 3

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min, followed by 35 cycles of 95oC for 30 sec, 61oC for 30 sec and 60oC for 30 sec.

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PCR products were cloned into pET100/D-TOPO vector (Invitrogen Life

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Technologies, USA), whichallow expression of recombinant protein with an N-

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terminal 6x His tag, following manufacturer’s instructions. Ligation reaction was used

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to transform DH5a competent cells and the resulting colonies were screened using

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cloning primers. Plasmid was extracted using NucleoSpin Plasmid (Macherey-Nagel,

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Germany) and 3 different clones were sent for sequencing. A clone of the correct

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DNA sequence was selected for downstream experiments.

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For heterologous expression of TuGSTd10 and TuGSTd14, E.coli BL21(DE3)

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competent cells, harboring corresponding plasmids were grown at 37oC in 2lt LB

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containing 100μg/ml ampicillin. The synthesis of GSTs was induced by the addition

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of 1mM isopropyl b-D-thiogalactoside (IPTG) when the absorbance at 590 nm was at

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0.7-1. Four hours after induction, cells were harvested by centrifugation at 5.000 g for

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20 min, re-suspended in sodium phosphate buffer (20 mM sodium phosphate buffer,

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40 mM imidazole, 500 mM NaCl, pH 7.4), sonicated and centrifuged in 10,000 g for

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30 min at 4oC. The supernatant was collected and the GST was purified via Ni-NTA

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chromatography (Qiagen, USA) following manufacturer’s

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TuGSTm09, BL21(DE3) competent cells harboring corresponding plasmid were

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grown at 37oC and when absorbance at 590 nm was at 0.7- 1 the induction was

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performed with 0.5 mM IPTG for 4 hours in 28oC.

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Protein concentration was determined by Bradford assay [15] and purity was judged

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by SDS-PAGE gel. In order to verify that recombinant GSTs are active, GST activity

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against 1- chloro- 2, 4 dinitrobenzene (CDNB) was measured according to the method

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described in [4].

instructions.

For

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Determination of substrate specificities for model substrates and kinetic studies

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GST activity towards 1- chloro- 2, 4 dinitrobenzene (CDNB, Sigma-Aldrich, UK) and

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1, 2- dicloro- 4- nitrobenzene (DCNB, Sigma-Aldrich, UK) was measured at 25oC

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according to the method described in [4]. Glutathione peroxidase activity was

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determined at 25oC by coupling the reduction of Cumene hydroperoxide (CumOOH,

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Sigma Aldrich, UK) by GSH to the oxidation of NADPH by oxidized glutathione

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disulfide (GSSG) with glutathione reductase according to the method described in

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[16]. Kinetic measurements were performed at 25oC in 0.1 M potassium phosphate

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buffer, pH 6.5. Initial velocities were determined in the presence of 2.475 mM GSH,

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and CDNB was used in the concentration range of 0.03 to3 mM. Alternatively, CDNB

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was used at a fixed concentration (0.99 mM) and GSH was used in the concentration

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range of 0.075 to15 mM. All the measurements were carried out in 96-well plates

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(NuncMaxiSorp, Thermo Scientific, USA) using a SpectraMaxM2e multimode

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microplate reader (Molecular Devices, Berkshire, UK). The kinetic parameters kcat

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and Km were determined by fitting the steady-state data to the Michaelis- Menten

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equation using GraFit3 software (Ericathus Software Ltd., Version 3.06).

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Enzyme – acaricides/insecticides interaction studies

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The potential interaction of TuGSTs with abamectin (98.7% purity, 80% avermectin

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B1a/ 20% avermectin B1b, Sigma-Aldrich, UK) , hexythiazox (99.9% purity, Sigma-

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Aldrich, UK), clofentezine (99.9% purity, Sigma-Aldrich, UK), bifenthrin (98.6%

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purity, Sigma-Aldrich, UK) and pyridaben (99.7% purity, Sigma-Aldrich, UK), active

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ingredients which showed reduced toxicity against the multi-resistant Mar-ab T.

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urticae strain [3] was determined by analyzing the inhibition of activity towards

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CDNB, in the presence of 0.05 mM of each acaricide/insecticide (in 5-10% final

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concentration of methanol or aceton). In the case of TuGSTd14 and its interaction

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with abamectin, for IC50 calculation, percentage inhibition of TuGSTd14 activity was

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determined at different concentrations of abamectin (in a range of 12.5 to 200 μM) in

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the presence of 10% methanol and 0.99 mM CDNB (concentration below Km). IC50

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was determined using Grafit3 software (Ericathus Software Ltd., Version 3.06).

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Dixon plot analysis was performed at 3 different concentrations of CDNB (0.1, 1 and

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2 mM), using

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presence of 10% methanol in order to determine the type of inhibition. All the

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measurements were carried out in 96-well plates (NuncMaxiSorp, Thermo Scientific,

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UK) using a SpectraMaxM2e multimode microplate reader (Molecular Devices,

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Berkshire, UK) at 25oC based on the method described in [4].

4 different concentrations of abamectin (0, 25, 50, 100 μM) in the

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Bioinformatic analysis and molecular modelling

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TuGSTd14 models were constructed using the IntFOLD2-TS method [17] as

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implemented in the IntFOLD server [18]. Disorder prediction was carried out using

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DISOclust [19]. Ligand binding site prediction was carried out using FunFOLD [20].

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Model quality assessment was carried out using ModFOLDclust2 [21]. The available

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crystal structures of GSTs that were used as templates for model construction were

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3vk9A and 4i97A (UniProt). The global model quality score was estimated 0.9443 7 Page 7 of 22

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and the p-value 4.92E-5 (probability that the model is incorrect), suggesting high

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quality of the model. For inspection of models and crystal structures the program

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PyMOL

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(http://sts.bioengr.uic.edu/castp/calculation.php) [23] was used for pocket calculations

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using as probe radius 1.4 Angstrom.

(http://www.pymol.org/,

[22])

was

used.

CastP

server

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Results and discussion

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Cloning, heterologous expression and purification of TuGSTs

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from cDNA template prepared from RNA isolated from adults of Mar-ab strain.

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Coding sequences were successfully cloned into pET100/D-TOPO vector (Invitrogen

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Life Technologies, USA). The corresponding constructs were sequenced and it was

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ensured that no errors had been introduced during PCR amplification.Induction of the

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constructs with 1mM IPTG for 4 hours at 37oC in E.coli expression cells resulted in

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good levels of protein production. The majority of recombinant TuGSTd10 and

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TuGSTd14 were found to be in the soluble fraction, however, TuGSTm09 was

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primarily expressed at the insoluble fraction (inclusion bodies). By reducing the

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concentration of IPTG to 0.5 mM and the induction temperature to 25oC we managed

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to obtain sufficient protein production sequestered in the soluble fraction. All three

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TuGSTs were purified successfully using metal chelate affinity chromatography and

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the recombinant proteins were found to be catalytically active.

The sequences encoding for TuGSTd10, TuGSTd14 and TuGSTm09 were amplified

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Substrate specificities and kinetic properties of recombinant TuGSTd10, TuGSTd14 and TuGSTm09

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Recombinant TuGSTs were assayed towards selected model substrates to investigate

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if they exhibit glutathione transferase activity (towards CDNB and DCNB) and

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glutathione peroxidase activity (towards CumOOH). The results are presented in

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Table 2. All three recombinant TuGSTs were active as they were capable of

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conjugating both CDNB and DCNB substrates to GSH. TuGSTd10 and TuGSTd14

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displayed low specific activities for CDNB, compared to other delta GSTs derived

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from insects, such as, the AgGSTd1-5 (56.44 ± 8.7 μmol/min/mg) and the AgGSTd1-

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6 (195 ± 11.9 μmol/min/mg) from Anopheles gambiae and the AdGSTd1 (174 ± 4.86 8 Page 8 of 22

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μmol/min/mg) and the AdGSTd2 (39.9- 43.3 μmol/min/mg) from Anopheles dirus

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(reviewed in [24]). The specific activity of the TuGSTd10 and TuGSTd14 for DCNB

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was also lower compared to A. gambiae GSTd1-5 (0.33 ± 0.03 μmol/min/mg),

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GSTd1-6 (0.64 ± 0,03 μmol/min/mg) and A. dirus GSTd1 (0.28 ± 0,01 μmol/min/mg)

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and GSTd2 (0.08 ± 0.01 μmol/min/mg) [24]. Peroxidase activities of the TuGSTd10

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and TuGSTd14 are comparable to the other delta GSTs ( < 0,13 μmol/min/mg for A.

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gambiae GSTd1-5, 0.98 ± 0.06 μmol/min/mg for A. gambiae GSTd1-6, 0.65 ± 0.06

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μmol/min/mg for A. dirus GSTd1 [24].

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The specific activity of the TuGSTm09 for CDNB and DCNB is lower compared to

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other mu class GSTs from the cattle tick, Boophilus annulatus (121 μmol/min/mg for

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CDNB and 29.3 μmol/min/mg for DCNB, respectively, [25]), but similar compared

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to mu class GSTs from human [26]. The peroxidase activity of the TuGSTm09 is

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lower than the respective activity of the mu tick GST (62.4 μmol/min/mg, [25]), but

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higher compared to human mu class GSTs (1.3 and 0.63 μmol/min/mg, [26].

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The steady-state kinetics was subsequently determined for all three GSTs (Table 3).

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The Km values of TuGSTd10 and TuGSTd14and TuGSTm09 for GSH

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comparable with the corresponding affinities of GSTs from another mite species, i.e.

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Sarcoptes scabiei (0.50 ± 0.10 mM for ScGSTD1-1, 0.70 ± 0.20 mM for ScGSTD2-2,

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0.3 mM for ScGSTD3-3; 0.30 mM for ScGSTM1-1 and 0.40 mM for GSTM2-2,

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[27]). The Km values for CDNB are also comparable to the ScGSTs (0.30 mM for

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ScGSTD1-1, 0.40 ± 0.10 mM for ScGSTD2-2, 1.2 mM for ScGSTD3-3, 0.30 mM for

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ScGSTM1-1 and 4.20 ± 0.10 mM for GSTM2-2, [27]).The kcat values of TuGSTd10

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and TuGSTd14 as well as their kcat/km ratio values are higher for both GSH and

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CDNB in comparison with the delta ScGSTs [27]. TuGSTm09 exhibits a notably

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higher catalytic activity, for both GSH and CDNB, compared with the two mu class

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ScGSTs previously characterized (kcat values for GSH: 0.15 min-1 for ScGSTM1-1,

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0.06 min-1 for ScGSTM2-2, and kcat values for CDNB: 0.17 ± 0.01 min-1 for

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ScGSTM1-1, 0.10 min-1 for ScGSTM2-2, [27]). The catalytic effectiveness (kcat/km)

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of TuGSTm09 was also remarkably higher, for both GSH and CDNB substrates,

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compared to the two mu class SsGSTs.

are

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Enzyme – acaricide/insecticide interaction studies 9 Page 9 of 22

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Inhibition assays were performed, in order to investigate the possible interaction of

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the three recombinant enzymes with the acaricides/insecticides that show reduced

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toxicity against Mar-ab strain (i.e. hexythiazox, pyridaben, bifenthrin, clofentezine

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and abamectin) [3]. All acaricides/insecticides were used in a 0.05 mM final

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concentration and the percentage inhibitions in the activity of TuGSTs towards

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CDNB are presented in Table 4. With the exception of bifenthrin that caused 7.27 ±

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1.70% inhibition, none of the acaricides/insecticides exhibited significant levels of

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inhibition of the TuGSTd10 activity under assay conditions, indicating that

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TuGSTd10 may not strongly interact with any of these active ingredients. Similarly,

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bifenthrin and abamectin did not cause any inhibitory effect in TuGSTm09 under

268

assay conditions but hexythiazox, pyridaben and bifenthrin caused a low inhibition of

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12.94 ± 3.74%, 11.21 ± 3.00% and 28.09 ± 4.84% respectively. Pyridaben and

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bifenthrin did not inhibit the TuGSTd14 CDNB conjugating activity, while

271

hexythiazox caused 11.21 ± 3.00% and clofentezine 28.08 ± 4.84% inhibition.

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However, the strongest interaction-inhibition among the active ingredients and the

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recombinant TuGSTs included in this study, was recorded for abamectin against

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TuGSTd14. Based on this data as well as the strong association of GSTs with

275

abamectin resistance in several studies in the past [10], we focused our subsequent

276

analysis on the TuGSTd14- abamectin interaction.

277

To evaluate the inhibitory potential, the concentration of abamectin needed to inhibit

278

the CDNB conjugating activity by half (IC50) was calculated by a dose-response

279

curve (data not shown) and was determined at 88.47 ± 7.55 μM, showing significant

280

inhibition. Dixon plot analysis with varying CDNB or abamectin concentrations was

281

performed in order to define the type of inhibition (Figure 1). The resulting three

282

linear curves intersect above the x axis proving that the inhibition is competitive and

283

the inhibition constant (Ki) was determined at 34.06 ± 0.68 µM. The Ki is almost 50-

284

fold lower than Km for CDNB (Table 2), indicating that most likely abamectin is a

285

strong inhibitor. Interestingly, the competitive type of inhibition implies that

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abamectin competes with CDNB for the same site, thus binds adjacent to the H-site of

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the enzyme.

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Overall structure of TuGSTd14

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To understand the structural and catalytic properties of TuGSTd14, the enzyme

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sequence was subjected to structural prediction using the IntFOLD2-TS method. The

293

three-dimensional structure was modeled based on X-ray structures of delta class

294

GSTs: 3vk9A and 4i97A. Each monomer of TuGSTd14 constitutes two distinct

295

domains, a smaller thioredoxin-like N-terminal domain and a larger helical C-terminal

296

domain (Figure 2A). The N-terminal small domain is an α/β structure with the folding

297

topology βαβαββα arranged in the order β2, β1, β3 and β4 with β3 anti-parallel to the

298

others, forming a regular β-sheet with a right-handed twist surrounded by three α-

299

helices. At the end of helix H3 begins a short linker that joins the N- and C-terminal

300

domains. The core of the C-terminal domain is a bundle of five helices

301

(H4H5H6H7H8). Active site solvent accessible surface (Richards' surface) for

302

TuGSTd14 has been calculated equal to 653.9Ų, suggesting that the active site (G-

303

and H-sites) is large enough (Figure 2B) to accommodate large ligands. The GSH

304

moiety is located in a polar region, formed by the beginning of helices H1, H2 and H3

305

in the N-terminal domain (Figure 2A). The SNAIL/TRAIL-like motif [28] in the N-

306

terminal domain, that is present in most GST classes and contributes polar functional

307

groups to the GSH binding site, is located in the dimer interface at amino acids

308

position 65-69 (TuGSTd14 numbering, Figure 3) with some modifications. The H-site

309

(hydrophobic ligand binding site) of GSTd14is located next to the G-site (GSH

310

binding site), exposed to the bulk solvent and is formed by hydrophobic residues

311

mainly from the C-terminal domain. The H-site exhibit a low degree of sequence

312

identity between different members of delta class and hence considered a unique

313

structure that reflects their different substrate specificity (Figure 2C, Figure 3).

314

Interestingly, positively charged residues (e.g. Lys212, His50) point to the H-site.

315

These basic residues form a positively charged region at the H-site, which presumably

316

enable the enzyme to bind negatively charged substrates. Coulombic surface analysis

317

(Figure 2D) showed that the G- and H-site in TuGSTd14 shows positive electrostatic

318

potential. This positive electrostatic potential presumably contributes to –SH

319

ionisation of GSH as well as to the binding of polar ligands.

320 321

The C-terminal domain of cytosolic GSTs contains a conserved N-capping box motif

322

(Ser/Thr-Xaa-Xaa-Asp) at the beginning of Η6 helix, consisting of a hydrogen

323

bonding interaction of the hydroxyl group of Ser/Thr with Asp [29, 30]. This N-

324

capping box, is involved in the Η6-helix formation, plays crucial structural and 11 Page 11 of 22

325

functional roles and is essential to the folding of GSTs. Interestingly, TuGSTd14

326

possessa N-capping box motif (Thr-Leu-Ala-Asp) that is located between amino acids

327

153-156 (Figure 4). This motif is conserved among all delta class GSTs.

328 329

Identification and the role of key active site residues of TuGSTd14

330 331

It is well established that the delta class GSTs possess as a catalytic residue Ser[31].

332

The Ser hydroxyl group acts as hydrogen bond donor to the thiol group of GSH,

333

contributing to stabilization of reactive thiolate anion (GS-) which is the nucleophile

334

group for the electrophilic substrate. Analysis of TuGSTd14 modeled structure shows

335

that Ser11 could be the catalytically important residue which is within hydrogen bond

336

distance with the S atom of GSH (Figure 2A, Figure 3).

337 338

Another structural feature of the delta class GSTs that contributes to catalysis is the

339

existence of a conserved electron-sharing network [32]. This electron-sharing network

340

assists the glutamyl γ-carboxylate of GSH to act as a catalytic base accepting the

341

proton from the -SH thiol group of GSH, forming an ionized GSH. It is formed from

342

two critical residues that interact with the negatively charged glutamyl carboxylate

343

group of GSH, a positively-charged residue (primarily Arg) and a negatively-charged

344

residue (Glu or Asp) stabilized by hydrogen-bonding networks with surrounding

345

residues (Ser, Thr) and/or water-mediated contacts. In the TuGSTd14, the residues

346

Gln16, Glu64, Ser65 and Asp100 appear to form the proposed electron-sharing

347

network (Figure 2E). This network of interaction appears to be a functionally

348

conserved motif that contributes to the “base-assisted deprotonation” model suggested

349

to be essential for the GSH ionization step of the catalytic mechanism [32, 33].

350 351

Conclusion

352

Our study provided further evidence and supported earlier work that GSTs are likely

353

to play a role in abamectin resistance, particularly in T. urticae [10, 34]. However

354

further studies on the metabolic fate of abamectin in resistant and susceptible spider

355

mites are warranted in order to provide functional evidence for a catalytic interaction

356

of abamectin with arthropod GSTs, resulting in conjugated metabolites, which seem

357

to be elusive so far. We cannot exclude that the observed competitive inhibition of 12 Page 12 of 22

358

CDNB conjugation by abamectin is due to its binding to regions adjacent the active

359

site, i.e. not directly interfering with the substrate recognition site..

360 361

Acknowledgments

362

Part of this research has been co-financed by the European Union (European Social

363

Fund e ESF) and Greek national funds through the Operational Program"Education

364

and Lifelong Learning" of the National Strategic Reference Framework (NSRF) e

365

Research Funding Program: THALES (projects 377301 and 380264). Investing in

366

knowledge society through the European Social Fund.The work also received funds

367

by a grant from Bayer Crop Science (to JV).

13 Page 13 of 22

368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416

References [1] T. Van Leeuwen, J. Vontas, A. Tsagkarakou, W. Dermauw, L. Tirry, Acaricide resistance mechanisms in the two-spotted spider mite Tetranychus urticae and other important Acari: A review, Insect Biochem. Mol. Biol., 40 (2010) 563-572. [2] W. Dermauw, N. Wybouw, S. Rombauts, B. Menten, J. Vontas, M. Grbic, R.M. Clark, R. Feyereisen, T. Van Leeuwen, A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae, Proc. Natl. Acad. Sci. U. S. A., 110 (2013) E113-E122. [3] M. Riga, D. Tsakireli, A. Ilias, E. Morou, A. Myridakis, E.G. Stephanou, R. Nauen, W. Dermauw, T. Van Leeuwen, M. Paine, J. Vontas, Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae, Insect Biochem. Mol. Biol., 46 (2014) 43-53. [4] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-Transferases - First Enzymatic Step in Mercapturic Acid Formation, J. Biol. Chem., 249 (1974) 7130-7139. [5] J.H.a.C. Wolf, Role of gloutathione transferases in drug resistance, in: H.K. Sies, B. (Ed.), Academic press Ltd, London, 1988, pp. 315-355. [6] M.S. Lyall, S.R. Dundas, S. Curran, G.I. Murray, Profiling markers of prognosis in colorectal cancer, Clin. Cancer Res., 12 (2006) 1184-1191. [7] A.G. Clark, N.A. Shamaan, Evidence that DDT-dehydrochlorinase from the house fly is a glutathione S-transferase, Pestic. Biochem. Physiol., 22 (1984) 249-261. [8] J.G. Vontas, G.J. Small, J. Hemingway, Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens, Biochem. J., 357 (2001) 65-72. [9] I. Kostaropoulos, A.I. Papadopoulos, A. Metaxakis, E. Boukouvala, E. PapadopoulouMourkidou, Glutathione S-transferase in the defence against pyrethroids in insects, Insect Biochem. Mol. Biol., 31 (2001) 313-319. [10] S. Konanz, R. Nauen, Purification and partial characterization of a glutathione Stransferase from the two-spotted spider mite, Tetranychus urticae, Pestic. Biochem. Physiol., 79 (2004) 49-57. [11] M. Grbic, T. Van Leeuwen, R.M. Clark, S. Rombauts, P. Rouze, V. Grbic, E.J. Osborne, W. Dermauw, C.T.N. Phuong, F. Ortego, P. Hernandez-Crespo, I. Diaz, M. Martinez, M. Navajas, E. Sucena, S. Magalhaes, L. Nagy, R.M. Pace, S. Djuranovic, G. Smagghe, M. Iga, O. Christiaens, J.A. Veenstra, J. Ewer, R.M. Villalobos, J.L. Hutter, S.D. Hudson, M. Velez, S.V. Yi, J. Zeng, A. Pires-daSilva, F. Roch, M. Cazaux, M. Navarro, V. Zhurov, G. Acevedo, A. Bjelica, J.A. Fawcett, E. Bonnet, C. Martens, G. Baele, L. Wissler, A. Sanchez-Rodriguez, L. Tirry, C. Blais, K. Demeestere, S.R. Henz, T.R. Gregory, J. Mathieu, L. Verdon, L. Farinelli, J. Schmutz, E. Lindquist, R. Feyereisen, Y. Van de Peer, The genome of Tetranychus urticae reveals herbivorous pest adaptations, Nature, 479 (2011) 487-492. [12] H. Ranson, C. Claudianos, F. Ortelli, C. Abgrall, J. Hemingway, M.V. Sharakhova, M.F. Unger, F.H. Collins, R. Feyereisen, Evolution of supergene families associated with insecticide resistance, Science, 298 (2002) 179-181. [13] J. Seguraaguilar, C. Lind, On the Mechanism of the Mn-3+-Induced Neurotoxicity of Dopamine - Prevention of Quinone-Derived Oxygen-Toxicity by Dt-Diaphorase and Superoxide-Dismutase, Chem. Biol. Interact., 72 (1989) 309-324. [14] S. Baez, J. SeguraAguilar, M. Widersten, A.S. Johansson, B. Mannervik, Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes, Biochem. J., 324 (1997) 25-28. [15] M.M. Bradford, Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding, Anal. Biochem., 72 (1976) 248-254.

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[16] T.W. Simmons, I.S. Jamall, R.A. Lockshin, Selenium-Independent Glutathione-Peroxidase Activity Associated with Glutathione S-Transferase from the Housefly, Musca-Domestica, Comp Biochem Phys B, 94 (1989) 323-327. [17] M.T. Buenavista, D.B. Roche, L.J. McGuffin, Improvement of 3D protein models using multiple templates guided by single-template model quality assessment, Bioinformatics, 28 (2012) 1851-1857. [18] D.B. Roche, M.T. Buenavista, S.J. Tetchner, L.J. McGuffin, The IntFOLD server: an integrated web resource for protein fold recognition, 3D model quality assessment, intrinsic disorder prediction, domain prediction and ligand binding site prediction, Nucleic Acids Res., 39 (2011) W171-W176. [19] L.J. McGuffin, Intrinsic disorder prediction from the analysis of multiple protein fold recognition models, Bioinformatics, 24 (2008) 1798-1804. [20] D.B. Roche, S.J. Tetchner, L.J. McGuffin, FunFOLD: an improved automated method for the prediction of ligand binding residues using 3D models of proteins, Bmc Bioinformatics, 12 (2011). [21] L.J. McGuffin, D.B. Roche, Rapid model quality assessment for protein structure predictions using the comparison of multiple models without structural alignments, Bioinformatics, 26 (2010) 182-188. [22] W.L. DeLano, The PyMOL Molecular graphics system, DeLano scientific San Carlos, CA, USA, 2002. [23] J. Dundas, Z. Ouyang, J. Tseng, A. Binkowski, Y. Turpaz, J. Liang, CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues, Nucleic Acids Res., 34 (2006) W116-W118. [24] A. Che-Mendoza, R.P. Penilla, D.A. Rodriguez, Insecticide resistance and glutathione Stransferases in mosquitoes: A review, African Journal of Biotechnology, 8 (2009) 1386-1397. [25] Y.E. Shahein, A.E.S. El-Hakim, A.M.K. Abouelella, R.R. Hamed, S.A.M. Allam, N.M. Farid, Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus, Vet. Parasitol., 152 (2008) 116-126. [26] E. Campbell, Y. Takahashi, M. Abramovitz, M. Peretz, I. Listowsky, A Distinct Human Testis and Brain Mu-Class Glutathione S-Transferase - Molecular-Cloning and Characterization of a Form Present Even in Individuals Lacking Hepatic Type Mu-Isoenzymes, J. Biol. Chem., 265 (1990) 9188-9193. [27] E. Molin, In vitro characterization of Glutathione Tranferases from Sarcoptes scabiei, Swedish University of Argicultural Sciences, 2009. [28] S.E. Pemble, A.F. Wardle, J.B. Taylor, Glutathione S-transferase class Kappa: Characterization by the cloning of rat mitochondrial GST and identification of a human homologue, Biochem. J., 319 (1996) 749-754. [29] A. Aceto, B. Dragani, S. Melino, N. Allocati, M. Masulli, C. DiIlio, R. Petruzzelli, Identification of an N-capping box that affects the alpha 6-helix propensity in glutathione Stransferase superfamily proteins: A role for an invariant aspartic residue, Biochem. J., 322 (1997) 229-234. [30] R. Cocco, G. Stenberg, B. Dragani, D.R. Principe, D. Paludi, B. Mannervik, A. Aceto, The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif, J. Biol. Chem., 276 (2001) 32177-32183. [31] A. Bocedi, R. Fabrini, A. Farrotti, L. Stella, A.J. Ketterman, J.Z. Pedersen, N. Allocati, P.C.K. Lau, S. Grosse, L.D. Eltis, A. Ruzzini, T.E. Edwards, L. Morici, E. Del Grosso, L. Guidoni, D. Bovi, M. Lo Bello, G. Federici, M.W. Parker, P.G. Board, G. Ricci, The Impact of Nitric Oxide Toxicity on the Evolution of the Glutathione Transferase Superfamily A PROPOSAL FOR AN EVOLUTIONARY DRIVING FORCE, J. Biol. Chem., 288 (2013) 24936-24947.

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[32] P. Winayanuwattikun, A.J. Ketterman, An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes, J. Biol. Chem., 280 (2005) 31776-31782. [33] I. Axarli, P. Dhavala, A.C. Papageorgiou, N.E. Labrou, Crystal structure of Glycine max glutathione transferase in complex with glutathione: investigation of the mechanism operating by the Tau class glutathione transferases, Biochem. J., 422 (2009) 247-256. [34] N. Stumpf,R. Nauen, Biochemical markers linked to abamectin resistance in Tetranychus urticae (Acari: Tetranychidae). Pestic. Biochem. Physiol. 72 (2002) 111-121. .

477 478 479

Figure legends

480

Figure 1.Dixon plot analysis for the inhibition of CDNB conjugating activity of

481

TuGSTd14 by different abamectin concentrations. Three different concentrations

482

of CDNB (0,1, 1 and 2mM) and 4 different concentrations of abamectin (0, 25, 50,

483

100μM) were used and data are mean of three replicates ± S.D. Analysis denoted a

484

competitive type of inhibition and the Ki was determined at 34,06 ± 0,68 µM.

485

Figure 2. A: Ribbon diagrams of TuGSTd14 protein model. Helices (H) are in red

486

β‐strands in yellow. The GSH analogues (S‐hexyl‐GSH) is represented in a stick and

487

colored according to atom type. The location of active site Ser residue, the G‐ and

488

H‐site as well as the C‐, and N terminal and the linker are labeled. The molecular

489

figure was created using PyMOL[22]. B. Surface analysis of S‐hexyl‐GSH binding in

490

GStd14 protein model. C: Important residues that contributes to G- and H-site

491

formation. D: Coulombic surface analysis of TuGSTd14.The analysis was carried out

492

using PyMol[22]. The Coulomb electrostatic surface shows regions of neutral (grey),

493

positive (blue) and negative (red) charge. E: Representation of the electron‐sharing

494

network of TuGSTd14. The residues Gln16, Glu64, Ser65 and Asp100 form the

495

proposed electron‐sharing network.

496

Figure 3.Sequence alignment of members of the delta class family of GSTs

497

compared with the secondary structure of TuGSTd14 model. TuGSTd14

498

numbering is shown above the alignment. Conserved areas are shown shaded. A

499

column is framed, if more than 70% of its residues are similar according to

500

physico‐chemical properties. This sequence alignment was created using the

501

following sequences (NCBI accession numbers are in parentheses): GST from 16 Page 16 of 22

502

Tetranychus urticae (AGE34481.1); GST from Tetranychus urticae (AGE34482.1);

503

GST from Panonychus citri (AFD36890.1); GST from Panonychus citri; GST from

504

Tetranychus cinnabarinus ( AGZ87455.1); GST from Tetranychus cinnabarinus

505

(AGZ87454.1); GST from Tetranychus urticae (AFQ61039.1); GST from

506

Panonychus citri ( AFD36886.1). The figure was produced using ESPript.

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529

Tables

530 531

Table 1. Primers used for the cloning of TuGSTs

532

17 Page 17 of 22

Gene

TeturID

Primer

Sequence (5’-3’)

TuGSTd10

tetur26g02802

TuGSTd10F TuGSTd10R

CACCATGTCAGTCCAATTATATCACCATAATCTTTCTC TTATGAATTTTTAGAAGCCATTGATTTGAGTAAATCTG

660

TuGSTd14

tetur29g00220

TuGSTd14F TuGSTd14R

CACCATGGTGATTGAACTGTATCAAGTTCCCA TTAAAGTTTACTTTGAAGAAAATCTCGAAATTCC

642

TuGSTm09 tetur05g05260 TuGSTm09F CACCATGGCACCAGTTATCGGTTATTGG TuGSTm09R TCAATATGGCTTTTGAATTGTGTCATTTCC

Prod. Size(bp)

684

533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

18 Page 18 of 22

550

Table 2.Substrate specificities of TuGSTd10,TuGSTd14andTuGSTm09. Specific activitya(Unitsb/mg) Substrate

TuGSTd10

TuGSTd14

TuGSTm09

1-Chloro-2,4dinitrobenzene (CDNB)

1.10 ± 0.25

0.69 ± 0.05

15.94 ± 0.70

1,2-Dichloro-4-nitrobenzene (DCNB)

0.01 ± 0.00

0.08 ± 0.01

0.09 ± 0.00

Cumene hydroperoxide (CumOOH)

0.34 ± 0.16

0.10 ± 0.01

3.34 ± 0.39

551 552 553 554 555

The values presented in table are means of three independent experiments ± S.D.aAmount of product produced per minute per mg of the total enzyme at 25oC. b One unit (U) is the amount of enzyme that catalyzes the reaction of 1 μmol of substrate per minute at 25oC.

556 557 558

19 Page 19 of 22

559

Table 3.Steady-state kinetics of TuGSTd10,TuGSTd14andTuGSTm09.

560

Kinetic parameter

TuGSTd10

TuGSTd14

TuGSTm09

Km (mM) GSH

0.33 ± 0.05

3.79 ± 0.69

2.34 ± 0.31

Km (mM) CDNB

0.84 ± 0.13

1.69 ± 0.24

0.26 ± 0.04

Kcat (min-1) GSH

1.59 ± 0.06

1.22 ± 0.10

23.4 ± 1.52

Kcat (min-1) CDNB

3.06 ± 0.23

1.78 ± 0.25

14.7 ± 0.85

Kcat/Km (mM-1· min-1) GSH

4.72

0.32

10

Kcat/Km (mM-1· min-1) CDNB

3.64

1.08

56.53

561 562 563 564

All values are means ± S.D. of three independent experiments. Results were determined by varying the concentration of GSH (0.075-15 mM) and CDNB (0.03-3 mM) at fixed concentrations of CDNB (0.99 mM) and GSH (2.47 mM) respectively.

565 566 567 568 569 570 571 572 573 574 575 576 577

20 Page 20 of 22

578

Table 4.Percentage inhibition of TuGSTd10,TuGSTd14and TuGSTm09activity

Acaricide/ Insecticide

Inhibition of enzyme activity (%) TuGSTd10 TuGSTd14 TuGSTm09

Structure

Hexythiazox

n.d.

11.21 ± 3.00

12.94 ± 4.55

Pyridaben

n.d.

n.d.

25.84 ± 14.00

Bifenthrin

7.27 ± 2.70

n.d.

n.d.

Clofentezine

n.d.

28.05 ± 4.84

27.68 ± 2.99

Abamectin

n.d.

38.46 ± 3.79

n.d.

579 580 581 582

All values are means ± S.D. of three independent experiments. Enzymes were assayed using GSH and CDNB as substrates and acaricides/insecticides were used in a 0.05mM final concentration. N.d.: not detected (under assay conditions).

583

21 Page 21 of 22

584 585 586

22 Page 22 of 22