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Interaction affinity of Delta and Epsilon class glutathione-s-transferases (GSTs) to bind with DDT for detoxification and conferring resistance in Anopheles gambiae, a malaria vector.

INTRODUCTION

Insecticide resistance is a growing problem in the control of disease spreading insect vectors. The basic mechanisms underlying resistance to commonly used insecticides such as organochlorines, organophosphates, carbamates and synthetic pyrethroids are well-known. Resistance mechanisms are broadly of two types, target site insensitiveness and enzyme-based detoxification (1). In mosquitoes, detoxification is performed by a group of enzymes such as esterases, oxidases and glutathione-stransferases (GSTs). Detoxification of insecticides occurs when enhanced levels of these enzymes prevent the insecticide from reaching its site of action. GSTs are the major family of detoxification enzymes which get activated in the presence of reduced glutathione (GSH) and bind to DDT (Dichlorodiphenyl-trichloroethane) molecules and convert them into a non-toxic lipophilic compound DDE (Dichlorodiphenyl-ethane) (2). Many resistant insects have elevated level of GSTs and such over expression of this enzyme is considered to be responsible for the development of resistance to organochlorine insecticides including DDT, hexachlorocyclohexane (HCH) and cyclodiene (3). Recently, elevated level of GST activity for DDT detoxification has been confirmed in Anopheles culicifacies and An. annularis (4).

Insect GSTs are classified into six classes, viz. Delta, Epsilon, Zeta, Sigma, Omega, and Theta by comparative analysis of Drosophila melanogaster and An. gambiae genomes (5-8). The Delta and Epsilon class GSTs, which are encoded by multiple genes, have been implicated in detoxification, particularly in conferring resistance to organochlorine group of insecticides (9-12). In An. gambiae, an important African malarial vector, the increased rate of DDT dehydrochlorination in the resistant vector strain was associated with quantitative increase of GST enzymes that included Delta and Epsilon classes (13). GSTs consist of a specific-glutathione (GSH)-binding site (G-site) next to a non-specific electrophilic ligand-binding site (H-site). The high level of diversity in this region is responsible for the differences in substrate specificities (14-15).

Based on the studies carried out so far in An. gambiae, Delta class comprises of 12 different genes (D1-D12), out of these D1 gene gives rise to four different transcripts, D7 and D11 genes produce two different transcripts each and each one of the remaining genes (except D9) gives rise to a single transcript and no information is available for D9 gene in the existing databases for An. gambiae. Epsilon class encompasses eight different genes (E1-E8); each gives rise to a single transcript. All the 16 transcripts of Delta class GSTs, except D7-2 and eight transcripts of Epsilon class GSTs have modbase structure deposited in the Swiss-Prot database. Further, structure of one of the transcripts of D1 gene, agGST1-6, was proved experimentally (16) and its affinity with S-hexyl glutathione (GTX), a hexyl glutathione was understood from crystallographic studies. Similarly, the structure of E2 GST was confirmed by X-ray crystallographic analysis (17).

Therefore, the Delta and Epsilon class GSTs produce a total of 24 transcripts that result in the production of corresponding enzyme proteins. However, to detect the development of resistance by mosquitoes to DDT, the conventional biochemical assay estimates the elevated level of total GSTs. Hence, it would be more reliable if the specific class of GST enzyme which has the highest molecular interaction affinity with DDT is estimated and used for screening of field mosquito population for development of DDT resistance.

Therefore, a comparative study was undertaken by docking of Delta and Epsilon class GSTs in their bioconfirmative form (GSTs and reduced GSH [glutathione] complex) with DDT using bioinformatics tools. This information would be useful to improve the biochemical analysis of the specific class GST enzyme that would further lead to design of a more specific molecular tool to determine the expression status of the particular type of GST enzyme.

MATERIAL & METHODS

The computational models of eight Epsilon (E1-E8) and 15 Delta class GSTs (D1-D8 and D10-D12) (except D9 and D7-2), that are reported to be responsible for DDT resistance in An. gambiae were retrieved from the Swiss-Prot database (http://www.expasy.ch/sprot/). The Delta class GST, D9 was not considered in the present study as its corresponding gene has no information in the data bank. But, for D7-2, while there was no structural model deposited in the Swiss-Prot database, its sequence details are available. Hence, the D7-2 GST protein that consists of 191 amino acid residues, with an ID of A0NDD6 was subjected for modeling. The NCBI, BLAST was used to select a template for the D7-2 query sequence based on the highest sequence identity. As a result, the sequence of An. dirus B with a PDB ID of 1R5A having an identity of 95% was chosen as the template and a homology model of D7-2 was developed using MODELLER. The stereo chemical quality of the model was determined by the Structure Analysis and Verification Server (http://nihserver.mbi.ucla.edu/SAVS/). The other modeled proteins of Epsilon and Delta class GSTs retrieved from the database were also reassessed for their quality using the same server (PROCHECK analysis). The modeled D7-2 was examined by superimposing it on the template 1R5A in Chimera Visualizer (Fig. 1a). Subsequently, to establish the correctness of the model, its RMSD details (Fig. 1b) were obtained using the Superpose online server tool (http:/wishart.biology.ualberta.ca/SuperPose/) (18).

CASTp (http://sts-fw.bioengr.uic.edu/castp/) was used to find out the surface pockets of the retrieved as well as the modeled proteins and Q-site Finder (http://www.bioinformatics.leeds.ac.uk/qsitefinder/help.html), an online tool and a new method of ligand binding site prediction, was used to predict the active sites of the proteins. Additionally, Fuzzy-Oil-Drop (http://www.bioinformatics.cm-uj.krakow.pl/activesite/) was also used to confirm the active sites predicted by the CASTp and Q-site Finder. The G-site (an active site for reduced GSH) and H-site (an active site for DDT) of GST enzymes were predicted using the three online servers. Since, reduced glutathione is necessary for the activation of GST, its chemical structure was taken from the PUBCHEM database (http://pubchem.ncbi.nlm.nih.gov/). Similarly, the chemical structure of DDT was also retrieved from the PUBCHEM database and their corresponding PDB files were obtained by uploading the smiles notations of the compounds to the online molecular format converter (http://www.molecular-networks.com/online_demos/corina). The transcript models that were validated using PROCHECK analysis were initially docked with GSH (Fig. 2) and the resulted (GSH + GST) (Fig. 3) complex was subsequently docked with the ligand, DDT using AutoDock to rank the transcript showing the highest interaction affinity. Labeled molecular level close up of the hydrogen bonds between GSH and GSTs has been shown in Fig. 4 and hydrophobic patch was established by DDT interaction with GST at position PHE 117 (Fig. 5).

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[FIGURE 2 OMITTED]

RESULTS & DISCUSSION

This current in-silico analysis was carried out based on the observation made in a wet lab study that confirmed the involvement of GSTs in conferring DDT resistance in the malaria vectors, An. culicifacies and An. Annularis (4). There was a 3-fold increase in GST activity in DDT-resistant An. annularis compared with its susceptible population. In An. culicifacies, the median GST activity (71.8 [micro]mol/min/mg) was almost the same as estimated in the DDT-resistant An. annularis (74.6 [micro]mol/min/mg), suggesting that the GST activity estimated in An. culicifacies could be an elevated level for detoxification of DDT. Furthermore, the GST activity in DDT-resistant An. culicifacies and An. annularis was significantly higher than that in the DDT-susceptible An. fluviatilis, which had a GST activity of 20 [micro]mol/min/[mg.sup.4].

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

In total, structural models are available for 24 GSTs, eight of Epsilon class and 16 of Delta class. Among these, 23 were the retrieved ones and the remaining one was developed during the current study. The overall quality factor of the model developed for D7-2 was 92.4% as obtained from the Ramachandran plot (Fig. 6). The quality factor for all the retrieved models was around 90% as revalidated using PROCHECK analysis (Table 1).

Finding out the active site residues of GST showing the highest affinity for GSH interaction and subsequently of GST + GSH complex for DDT interaction is an important step to carry out the docking process. In an earlier crystallographic study, one of the transcripts of Delta class D1 gene (D1-1) was examined in relation to GSH binding with GST and, based on formation of more number of hydrogen bonds, identified ILE 52 as the active site residue showing the highest affinity for GSH binding (16). This residue is present in the G-site of the D1-1 transcript. The G-site consists of 19 amino acid residues (LEU 6, SER 9, ALA 10, PRO 11, LEU 33, MET 34, HIS 38, HIS 50, ILE 52, GLU 64, SER 65, ARG 66, TYR 105, PHE 108, TYR 113, ILE 116, PHE 117, PHE 203, and PHE 207) and out of these, ILE 52 showed the highest interaction affinity with GSH (15). Hence, ILE 52 was considered in the current study as the suitable active site for carrying out the docking of GSH with the GSTs of both the Epsilon and Delta classes.

The crystallographic study with An. dirus confirmed that either PHE (117 and 203) or TYR (105 and 113) residues of the bioactively conformed GST form the hydrophobic pathway for binding with DDT (19), and out of these four residues, according to the docking results obtained in the current study, PHE 117 was found to show a higher interaction with DDT. Further, the studies with An. gambiae have illustrated that the H-site, which is meant for DDT (ligand) binding, composed of 12 amino acid residues, including those four residues that were related to DDT binding in the study with An. dirus (19). So PHE and TYR could be the residues involved in DDT binding in the case of An. gambiae also. Thus, involvement of these two residues, with PHE showing a higher interaction, in DDT binding with GSH + GST complex is clearly confirmed. Based on this information, PHE 117 could be considered as the suitable active site residue for DDT interaction. However, literature evidence did not explain about the position of ILE 52 (G-site residue) and PHE 117 (H-site residue), i.e. whether both are located in the same pocket or not. Our results from CASTp, Q-SITE FINDER and Fuzzy Oil Drop analysis confirmed that both the G-site residue (ILE 52) (Fig. 7a) and the H-site residue (PHE 117) (Fig. 7b) are present in the same pocket, but at different sites. The residues PHE and ILE are, respectively at position 117 and 52 in both D1-1 and D1-3. Apart from these two transcripts, in all others, PHE and ILE are present at different positions not at 52 and 117; these two positions are occupied by other amino acid residues. It has been reported that all the 24 Epsilon and Delta class GSTs have been implicated in the process of detoxification of DDT8. The pocket prediction analysis done in the current study also confirmed the presence of PHE and ILE in the same pocket in all the 24 computational GST models. Therefore, though present at different positions in different transcripts, considering their presence within the same pocket, the two residues, PHE and ILE should be considered as the key residues involved in DDT cleavage and hence, significance was given to the residues and not to the position they occupy. Further, the pocket having both PHE and ILE residues was taken as the active site for the interaction of GSH + GST with DDT.

[FIGURE 7A OMITTED]

[FIGURE 7B OMITTED]

The energy budget obtained as the output from the docking of the 24 models of Epsilon and Delta class GSTs with DDT in the presence of reduced GSH is given in (Table 2). Out of the 24 modeled GSTs, Delta 6 had shown the lowest binding energy followed by Delta 4 and Epsilon 2, indicating their highest degree of affinity with DDT in order. Therefore, these three proteins could be considered to be the major factor for the detoxification of DDT.

CONCLUSION

Based on the degree of interaction affinities, the modeled GST from D6 transcript, which showed the highest affinity with DDT, is likely to get over-expressed on mosquito exposure to the insecticide. Following further confirmation through wet lab studies, it would be possible to design a specific molecular assay to determine the expression level of such high affinity transcript(s) and this could be used for detection/monitoring of resistance more reliably among the field vector populations.

ACKNOWLEDGEMENTS

The investigators wish to thank Dr P. Jambulingam, Director and Dr S. Sabesan, Chief, Division of Human Resource Development, Vector Control Research Centre, Puducherry for the facilities and the support provided. The investigators are also thankful to Dr K. Tyagesan, Principal, AVC College, Mayiladuturai, Tamil Nadu for reviewing the manuscript. Mrs Sundarambal, Library and Information Officer, Mrs T. Sumathy and Mr V.K. Suresh Kumar, Vector Control Research Centre, are also gratefully acknowledged for their help during the course of the study.

REFERENCES

(1.) Brogdon WG, McAllister JC. Insecticide resistance and vector control. Emerg Infect Dis 1998; 4: 605-13.

(2.) Clark AG, Shamaan NA. Evidence that DDT-dehydrochlorinase from the house fly is a glutathione S-transferase. Pestic Biochem Physiol 1984; 22: 249-61.

(3.) Hayes JD, Wolf CR. Role of glutathione transferase in drug resistance. In: Sies H, Ketterer B, editors. Glutathione conjugation: Mechanisms and biological significance. New York: Academic Press 1998; p. 315-55.

(4.) Gunasekaran K, Muthukumaravel S, Sahu SS, Vijayakumar T, Jambulingam P. Glutathione-s-transferases (GSTs) activity in Indian vectors of malaria: A defense mechanism against DDT. J Med Entomol 2011; 48 (3): 561-9.

(5.) Fournier D, Bride JM, Poire M, Berge JB, Plapp FW Jr. Insect glutathione-s-transferases: Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides. J Biol Chem 1992; 267: 1840-5.

(6.) Syvanen M, Zhou ZH, Wang JY. Glutathione transferase gene family from the house fly Musca domestica. Mol Gen Genet 1994; 245: 25-31.

(7.) Ranson H, Claudianos C, Ortelli F, Abgrell C, Hemingway J, Sharakhova MV, et al. Evolution of supergene families associated with insecticide resistance. Science 2002; 298: 179-81.

(8.) Ding Y, Ortelli F, Rossiter LC, Hemingway J, Ranson H. The Anopheles gambiae glutathione transferase supergene family annotation, phylogeny and expression profiles. BMC Genomics 2003; 4: 35-50.

(9.) Hemingway J. The molecular basis of two contrasting metabolic mechanisms of insecticide resistance. Insect Biochem Mole 2002; 30: 1009-15.

(10.) Prapanthadara L, Promtet N, Koottathep S, Somboon P, Ketterman AJ. Isoenzymes of glutathione-s-transferases from the mosquito Anopheles dirus species B: The purification, partial characterization and interaction with various insecticides. Insect Biochem Mol Biol 2000; 30: 395-403.

(11.) Ranson H, Rossiter L, Ortelli F, Jensen B, Wang X, Roth CW, et al. Identification of a novel class of insect glutathione-s-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochem J 2001; 359: 295-304.

(12.) Ortelli F, Rossiter LC, Vontas J, Ranson H, Hemingway J. Heterologous expression of four glutathione transferase genes genetically linked to a major insecticide-resistance locus from the malaria vector Anopheles gambiae. Biochem J 2003; 373: 957-63.

(13.) Ding Y, Hawkes N, Meredith J, Eggleston P, Hemingway J, Ranson H. Characterization of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress. Biochem J 2005; 387: 879-88.

(14.) Mannervik B, Danielson UH. Glutathione transferases structure and catalytic activity. Crit Rev Biochem 1988; 23(3): 283-337.

(15.) Ranson H, Collins F, Hemingway J. The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class glutathione-s-transferase family. Proc Natl Acad Sci USA 1998; 95: 14284-9.

(16.) Chen L, Hall PR, Zhou XE, Ranson H, Hemingway J, Meehan EJ. Structure of an insect delta-class glutathione-s-transferase from DDT-resistant strain of the malaria vector Anopheles gambiae. ActaCrystallogr D BiolCrystallogr 2003; 59: 2211-7.

(17.) Wang Y, Hemingway J, Ranson H, Meehan EJ, Chen L. Structures of an insect Epsilon-class glutathione-s-transferase from the malaria vector Anopheles gambiae: Evidence for high DDT-detoxifying activity. J Struct Biol 2008; 164 (2): 228-35.

(18.) Rajarshi M, Gary H, Van Domselaar, Haiyan Z, David SW. SuperPose: A simple server for sophisticated structural superposition. Nucleic Acids Res 2004; 1: 32.

(19.) Oakley AJ, Ketterman A, Wilce MCJ. Structural biology and its applications to the health sciences. Croatian Med J 2001; 42(4): 375-8.

V. Aravindan [1], S. Muthukumaravel [2] & K. Gunasekaran [2]

[1] School of Chemical and Biotechnology, SASTRA University, Thanjavur; (2) Vector Control Research Centre (ICMR), Indira Nagar, Puducherry, India

Correspondence to: Mr S. Muthukumaravel, Division of Vector Biology and Control, Vector Control Research Centre (ICMR), Indira Nagar, Puducherry-605 006, India.

E mail: kumaravelmuthuvel@yahoo.com

Received: 6 March 2013 Accepted in revised form: 6 January 2014
Table 1. Quality factor for Delta and Epsilon classes GSTs
as validated by PROCHECK analysis

Transcripts of         Query            Template sequence
GST genes            sequence
(An. gambiae)         length      Length/ PDB ID    Organism
                    (residues)

Delta class GSTs

  D1-1                  209          209/1pn9      An. gambiae
  D1-2                  216          218/1r5a      An. dirus B
  D1-3                  209          209/1pn9      An. gambiae
  D1-4                  219          219/1jlw      An. dirus B
  D2                    209          209/1pn9      An. gambiae
  D3                    210          210/1v2a      An. dirus B
  D4                    212          210/1v2a      An. dirus B
  D5                    216          210/1v2a      An. dirus B
  D6                    222          210/1v2a      An. dirus B
  D7-1                  218          218/1r5a      An. dirus B
  D7-2                  191          218/1r5a      An. dirus B
  D8                    223          209/1pn9      An. gambiae
  D10                   211          210/1v2a      An. dirus B
  D11-1                 214          209/1jlv      An. dirus B
  D11-2                 217          209/1jlv      An. dirus B
  D12                   211          209/1v2a      An. dirus B

Epsilon class GSTs

  E1                    224          218/1r5a      An. dirus B
  E2                    221          209/1pn9      An. gambiae
  E3                    223          209/1pn9      An. gambiae
  E4                    225          209/1pn9      An. gambiae
  E5                    230          209/1pn9      An. gambiae
  E6                    227          218/1r5a      An. dirus B
  E7                    225          209/1pn9      An. gambiae
  E8                    217          218/1r5a      An. dirus B

Transcripts of      Sequence    Swiss-Prot     Residues in
GST genes           identity     Model ID     most favoured
(An. gambiae)          (%)                   region (quality
                                               factor) (%)

Delta class GSTs

  D1-1                 100        Q93113           96.2
  D1-2                 43         Q7PH26           92.4
  D1-3                 84         Q93112           95.1
  D1-4                 84         O77462            93
  D2                   52         Q94999            94
  D3                   86         Q7PQ95           94.1
  D4                   62         Q5TT03           94.7
  D5                   60         Q7QB59           92.1
  D6                   42         Q7PQB4           92.2
  D7-1                 95        O76483 *          93.1
  D7-2                 95         A0NDD6           92.4
  D8                   46         Q5TTE5           92.5
  D10                  56         Q7QA79           91.9
  D11-1                56         Q7PQ57           94.7
  D11-2                47         Q5TT04           89.5
  D12                  41         Q7QA80           94.1

Epsilon class GSTs

  E1                   32         Q7PVS5           90.6
  E2                   41         Q9GPL8           91.8
  E3                   36         Q7PVS2           93.5
  E4                   39         Q7PVS7           95.7
  E5                   41         Q7PVS8           92.8
  E6                   27         A0NG89           89.1
  E7                   36         Q7PVS4           96.2
  E8                   27         Q8WQJ5           93.1

* The primary accession code: AF 251478.

Table 2. Energy budget output from the docking of GST with
DDT bound with reduced GSH

GST class/      Binding energy    DDT interactive
transcripts       (kcal/mol)          residue

Delta class

  D1-1          -9.19             PHE 117
  D1-2          -6.42             PHE 210
  D1-3          -7.37             PHE 117
  D1-4          -7.82             PHE 123
  D2            -6.38             PHE119
  D3            -6.60             PHE194
  D4            -32.60            PHE97
  D5            -9.00             PHE157
  D6            -34.47            PHE105
  D7-1          -8.02             PHE115
  D7-2          -8.17             PHE115
  D8            -8.78             PHE3
  D10           -7.68             PHE196
  D11-1         +1.44e+05         PHE108
  D11-2         +3.04e+05         PHE211
  D12           -7.61             PHE181

Epsilon class

  E1            -8.18             PHE 111
  E2            -10.35            PHE 108
  E3            -6.31             PHE 117
  E4            -10.07            PHE 113
  E5            -7.52             PHE 112
  E6            -7.94             PHE 109
  E7            -7.02             PHE 110
  E8            -7.72             PHE 119

Fig. 1b: RMSD details of the superimposed model D7-2 on the
template, 1R5A.

SuperPose used the sequence alignment to guide the supersposition

Local RMSD

               Alpha carbons    Back bone    Heavy     All
RMSD                0.14          0.19        0.76     0.76
Atoms               190            760        1532     1532
                 Structure                  Residues
               1R5A chain 'A'                2-191
                    PDBB                     2-191

Global RMSD

               Alpha carbons    Back bone    Heavy     All
RMSD                0.14          0.19        0.76     0.76
Atoms               190            760        1532     1532
                 Structure                  Residues
               1R5A chain 'A'                2-191
                    PDBB                     2-191

SuperPose output images

WebMol

MolScript superposition image

Difference distance matrix

SuperPose output text files

Sequence alignment

Difference distance matrix

Superposition (PDB)

RMSD report

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Author:Aravindan, V.; Muthukumaravel, S.; Gunasekaran, K.
Publication:Journal of Vector Borne Diseases
Article Type:Report
Date:Mar 1, 2014
Words:3373
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