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. 2013 Mar 8;14(3):5482-500.
doi: 10.3390/ijms14035482.

Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

Rashid Ahmed Khan  1 , Ji Yuan Liu, Maryam Rashid, Dun Wang, Ya Lin Zhang
Affiliations

Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

Rashid Ahmed Khan et al. Int J Mol Sci. .

Abstract

Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity.

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Figures

Figure 1
Figure 1
Mean percentage mortalities of H. armigera. Third-instar larvae were subjected to bioassay. Error bars show ± SD among three replications. Number of third-instar larvae tested per treatment (n = 72).
Figure 2
Figure 2
SDS-PAGE analysis of fusion protein. (A) Lane M, Protein weight marker; Lane1, DE-3; Lane 2, DE-3 + pET28a; Lane 3, DE-3 + pET28a-HaGST (without IPTG); Lane 4–6, expression level of the soluble fusion protein at 1–6 h; (B) Purified soluble rHaGST. Lane M, Protein weight marker; Lane 1, Purified rHaGST by Ni2+-nitrilotriacetate (NTA) column; Lane 2, non purified protein; (C) Immunoblotting of rHaGST with 6× His mouse monoclonal primary antibody. Lane M, Molecular weight marker; Lane 1, rHaGST detected by peroxidase conjugated goat anti-mouse IgG secondary antibody.
Figure 3
Figure 3
Specific activity of the GSTs in larval midgut of H. armigera subjected to sub-lethal dose of cantharidin using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) secondary substrate. The activity of the GSTs was measured at 340 nm both in treated and control samples.
Figure 4
Figure 4
Lineweaver-Burk plot of the GSTs activity in crude enzyme extract. (A) Specific activity of GSTs with and without cantharidin. IC50 value of cantharidin for the GSTs using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) secondary substrate; (B) IC50 value was obtained using a plot of percent activities vs. varying concentrations of cantharidin. S-Hexylglutathione (GTX) was used as positive control.
Figure 5
Figure 5
Lineweaver-Burk plot of the purified soluble rHaGST. (A) Specific activity of GSTs with and without cantharidin. IC50 value of cantharidin for GSTs using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) secondary substrate; (B) IC50 value was obtained using a plot of percent activities vs. varying concentrations of cantharidin. S-Hexylglutathione (GTX) was used as positive control.
Figure 6
Figure 6
The real-time q-PCR analysis of the HaGST mRNA transcript at different time intervals from 24 to 96 h. Target gene expression was normalized by comparing ΔΔCT to an untreated control. Error bars show standard deviation using three replications.
Figure 7
Figure 7
Sequence alignment results. The target protein HaGST and template protein, IPN9. Deep green color indicates conserved residues in both the sequences. Red bands show α helices while blue arrows show β sheets.
Figure 8
Figure 8
The ribbon representation of the 3-D model of the HaGST. Blue and red colors represent a chain trace from the N-terminus to C-terminus, respectively. α helices and β sheets are represented by H and B, respectively. The active site is located in the cleft formed at the interface of H8, H3 and between loops of H2, B3.
Figure 9
Figure 9
The binding model of GTX with the HaGST. Red dotted lines show hydrogen bonding among the corresponding atoms of amino acid residues of the active site and GTX.
Figure 10
Figure 10
The interaction diagram of the insect delta-class HaGST from Helicoverpa armigera with its inhibitor, GTX.
Figure 11
Figure 11
Binding mode of the cantharidin-HaGST complex. Red dotted lines show hydrogen bonding between the amino acid residues of the active site and atoms of cantharidin. The O atom of GLY10 and NH atom of ALA12 interact with the cantharidin O3 atom, simultaneously by hydrogen bonding. The OH atom of TRY116 also interacts with the O3 atom of cantharidin. A OH atom of TRY108 forming a hydrogen bond with O1 of cantharidin plays an important role in binding affinity based on its lowest inter-atomic distance.
Figure 12
Figure 12
The Interaction diagram of the delta class HaGST with cantharidin. ALA12 and TRY108 are mainly responsible for making hydrogen bonds with the O atoms of cantharidin.
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