Expression and characterization of recombinant Juvenile Hormone Epoxide Hydrolase of Spilarctia obliqua, a major pest in Agri-Sericulture  

Swetha Kumari K1 , Mamatha Dadala Mary2 , Kalpana Sriramadasu1 , Beulah Dadala3
1. Research Fellows, Dept. of Sericulture, Sri Padmavati Women’s University, Tirupati, India
2. Associate Professor, Dept. of Sericulture, Sri Padmavati Women’s University, Tirupati, India
3. Intern Student, University Preparatory Academy, San Jose, California, United States of America
Author    Correspondence author
Molecular Entomology, 2015, Vol. 6, No. 1   doi: 10.5376/me.2015.06.0001
Received: 27 Aug., 2014    Accepted: 13 Oct., 2014    Published: 01 Jan., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Swetha kumari et al., 2015, Expression and characterization of recombinant Juvenile Hormone Epoxide Hydrolase of Spilarctia obliqua, a major pest in Agri-Sericulture, Molecular Entomology, Vol.6, No.1 1-11 (doi: 10.5376/me.2015.06.0001)

Abstract

Juvenile Hormone (JH) modulates a variety of developmental and physiological processes in Insects. Previous studies suggest that the two hydrolytic enzymes namely Juvenile Hormone Esterase (JHE) and Juvenile Hormone Epoxide Hydrolase (JHEH) play important roles in the regulation of JH titer. Compared with JHE, JHEH is more essential regulatory enzyme and could be applied to control insects, because of its irreversible reaction on JH Mechanism. A full length of cDNA (1323bp) encoding juvenile hormone epoxide hydrolase gene from Spilarctia obliqua (SoJHEH) is isolated and subcloned into baculovirus vector system using pFastBac and pFastBac-HTA vectors. The bacmid-transformed SoJHEH is expressed in Insect cell line system and the expression of recombinant SoJHEH is confirmed by Western blotting. The enzyme activity of recombinant SoJHEH showed a high activity with JH III and very low with other general epoxide substrates. The deduced SoJHEH protein sequence showed the conserved domains of catalytic triad and oxyanion hole indicating the well preserved catalytic mechanism of epoxide hydration which can be explored further for bio control strategy at molecular level using insect’s own enzyme which can address the current problem of insect resistance.

Keywords
Spilarctia obliqua; Juvenile Hormone; Juvenile hormone epoxide Hydrolase; Baculovirus expression system; Characterization

Lepidopteran species are the most important pests of major annual and perennial crops, forests, and stored products throughout the world. More than 25% of the species that appear on a list of the 300 most important species threatening the country are from the order of Lepidoptera. Out of 30 most serious threats to agriculture, 50% of the species are Lepidopterans. Unfortunately control of lepidopterans worldwide is mostly done through the use of synthetic insecticides. This dependence on insecticides has contributed to the development of Insect resistance in many of the most serious pests and serious threats to environment.

Relevant examples include Spilarctia obliqua (Bihary Hairy caterpillar) as one among them, where resistance has developed. The adult is a brown moth with a 40-50 mm wing span and a red abdomen. Eggs are laid in clusters of 50-100, on the lower side of leaves. The larvae are covered with long yellowish to black hairs and are up to 5 cm long. Pupation takes place in the soil under dry foliage and debris. Young larvae feed gregariously mostly on the under surface of the leaves and cause loss by way of defoliation. In severe cases only bare stems are left behind (Agropedia-IITK, 2009, 2012).
Heavy dependence and frequent indiscriminate use of pesticides has also resulted in pesticide residues in consumable parts and earth crust showing a significant negative impact on the environment. Of particular importance to the agriculture is the destruction of the crop pollinators and beneficial insects – parasites and predators that maintain secondary pests under control. Development of alternative strategies to the use of the insecticides alone is therefore a major emphasis of most local, national and international organizations concerned with pest control.
The insect juvenile hormones are methyl esters of farnesoic acid 10, 11-epoxide (JH III) and analogous compounds, which function as important regulatory factors in embryogenesis, larval and adult development, metamorphosis, reproduction, diapause, migration, polymorphism, and metabolism (Nijhout 1994; Roe and Venkatesh 1990; de Kort and Granger 1996; Hammock, 1985). The two primary metabolic pathways of JH in insects are ester hydrolysis by JH esterase and epoxide hydration by an epoxide hydrolase (EH). The development of specific inhibitors of JH esterase has been instrumental in demonstrating a biological role for this enzyme in insects (Hammock et al., 1984, 1985; Prestwich et al., 1994; Abdel-Aal and Hammock, 1985; Harris et al., 1999).
Studies on exploiting Insect pest’s own hormones and their metabolizing enzymes can be explored as a molecular biocontrol tool, which can be made detrimental to the host (Kamita et al., 2005). From this point of view, the Juvenile hormone metabolism has been targeted for the biological control strategy. Juvenile hormone (JH) regulates key events in the life cycle of insects. It plays crucial role in the regulation of a number of physiological processes in the insect development and reproductive maturation.
The present study is focussed on the role of JH Epoxide Hydrolase (JHEH) for the biological control of Spilarctia obliqua. Epoxide hydrolases (EHs) are a/b-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs) (Kamita et al., 2012).
1 Methodology
The complete coding region for juvenile hormone epoxide hydrolase that has been already cloned from our lab, and sequenced from a fat body cDNA library derived from Bihary Hairy Caterpillar, Spilarctia obliqua, with GenBank Accession no. KC148541.1 was considered (M. Hema et al., 2012). The full length Spilarctia obliqua JHEH (SoJHEH) DNA sequence consists of 5’ UTR with 27bp followed by single ORF corresponding to 1323bp and 3’ UTR with 85bp followed by a Poly A tail (A)25.

1.1 Insilico Analysis and homology modelling at Catalytic site of deduced r-SoJHEH protein:

The primary and secondary structural analysis of Spilarctia obliqua juvenile hormone epoxide hydrolase protein using expasy tools namely ‘Compute pI/Mw’ (Bjellqvist et al., 1993, 1994; Gasteiger et al., 2005) and ‘Chou-fasman prediction method’, 'PSIpred’ (Buchan et al., 2013; Jones DT 1999) respectively were depicted. The transmembrane topology and signal peptide prediction of SoJHEH were determined using ‘TMpred’ (K. Hofmann and W. Stoffel, 1993) and ‘Phobius’ (Lukas Kall et al., 2004), expasy tools.

Homology modeling of recombinant SoJHEH was carried out using the Modeller 9.13 software (A. Sali and T. L. Blundell, 1993) and molecular software package Hyperchem professional 7.5. The JHEH from Bombyx mori (PDB ID: 4QLA.A) showed highest homology among the proteins reported as to crystal structure in Protein Data Bank with Spilarctia obliqua JHEH. Ten models were generated, out of which the one with highest DOPE (Discrete optimized protein energy) score was considered as final modeled SoJHEH structure.

1.2 SoJHEH Protein Expression:
Spilarctia JHEH ORF (named as SoJHEH) was subcloned into Sal I and Xho I cut pFastBac-1 baculovirus transfer vector (Invitrogen) and the recombinant vector was named as ‘pFastSoJHEH’. Authenticity of the positive clones was checked by colony PCR.

The SoJHEH ORF was in turn subcloned into Nco I and Xho I cut of pFastBac-HTA baculovirus transfer vector (Invitrogen) to determine the protein expression. The transformed PFastBac-HTA was named as ‘pFastHTSoJHEH’ and has been confirmed by colony PCR and restriction digestion.
The pUC/M13 Forward and Reverse primers that hybridize to the sites flanking the mini-attTn7 site within the lacZα-complementation region of bacmid DNA was used for the Transposition to occur (Invitrogen). The recombinant pFastHTSoJHEH was transformed using DH10α cells (Invitrogen). 1 µl of r-plasmid DNA was added in 1 ml of DH10α cells and incubated for 30 min in Ice. The mixture was subjected to heat shock at 42oC for 90 sec and immediately chilled with ice for 10 min. To this, 900 µl of LB media was added and kept for shaking for 4 hrs at 37oC. Later, the media was centrifuged and the pellet was spread over on LB-Agar plate and incubated at 37oC for Blue-white screening. The transformants were again confirmed by Colony PCR. The obtained transformation positive colonies were cultured and the recombinant plasmid DNA was isolated using Qiagen Midi Prep.
The Frozen Sf9 cells were revived and grown in monolayer culture using Graces Insect Medium and were maintained at 25oC. Cells were maintained at densities between 1×105 and 1×107 cells/ml. The cell viability was determined by Trypan blue Staining. The transformed pFastHTSoJHEH was transfected with 8µl of Cellfectin (Invitrogen) and 5 µl of recombinant bacmid DNA following the manufacturer’s manual (Invitrogen) in Sf9 cells having 100% confluency with a count of > 1×106 cells/ml.
1.3 Determination of SoJHEH recombinant protein by Western blotting:
Post 72 hrs, the Sf9 cells were harvested at a low centrifugation of 500 gm for 5 min at 4oC. The obtained supernatant, P1 Viral stock was separated and stored at -80oC to conduct bioassays. Since the pFastBac-HTA vector was having 6X His-tag, the detection of recombinant SoJHEH protein was done by western blotting. The Cell pellet obtained was washed with PBS and lysed with Radioimmunoassay (RIPA) buffer having 1 M Dithiothreitol (DTT) and 0.3 M Phenyl methyl sulphonyl flouride (PMSF). After final centrifugation, final supernatant and pellet were processed for SDS-PAGE. 2X SDS was added to both and heated at 75oC for 10 min. 10% SDS-PAGE gel was casted and the samples having control, transfected fractions and positive-His protein were loaded and separated. The expected size bands (~51KDa) were seen in transfected cell pellet fraction.
The separated proteins were transferred on to PVDF Membrane using Wet Method. The membrane was washed with TBST and was blocked by using NAP blocker for an hour. Followed by, the membrane was treated with primary Rabbit Anti-His antibody (Abcam AB 9108)with a composition of 3 µl of primary antibody in 9 ml of TBST-NAP blocker mixture for 1hr followed by secondary antibody Goat Anti-Rabbit IgG–Horseradish Peroxidase (GE Lifesciences) in 1:4500 dilutions with successive TBST washes. The membrane was treated with 750 µl each of two western blot substrates and the fluoresced bands were developed onto the X-ray Film.
1.4 Plaque Assays:
Plaque assays were conducted to determine the titer of the obtained baculoviral stock. Grace’s plaquing medium was prepared. 20 ml of heat-inactivated FBS was mixed with 100 ml of incomplete Grace’s insect medium (GIM). 25 ml of the above complete GIM was combined with 12.5 ml of sterile, distilled water and 12.5 ml of the melted 4% low melting agarose gel in the empty 100 ml bottle, mixed gently and was kept at 40oC water bath till it was used. Sf9 cells were harvested and 1.5 ml of cell suspension was aliquoted into each well of two 6-well plates. The cells were allowed to settle at the bottom of the plate by incubating at room temperature for 1 hour. The cell monolayers were observed. An 8-log serial dilution (10–1 to 10–8) of the clarified baculoviral stock with incomplete Grace’s Insect Cell Culture Medium was prepared. 0.5 ml of the baculoviral stock was mixed in 4.5 ml of medium in 12 ml disposable tubes (10–1 to 10–8). The 6-well plates containing Sf9 cells and the tubes of diluted virus were taken into the sterile hood and were labelled.
The medium was removed from each well and was immediately replaced with 1 ml of the appropriate virus dilution. Cells with virus were incubated for 1 hour at room temperature. Following one hour incubation, cells and the bottle of plaquing medium from the 40oC water bath was moved to a sterile hood and the medium containing virus from the wells was replaced with 1.5 ml of plaquing medium. The agarose overlay was allowed to harden for 10 – 15 min at room temperature. The cells were then incubated at 27oC for 7–10 days and the obtained plaques were observed and counted.
1.5 Expression and partial purification of SoJHEH:
Cells of Trichoplusia ni (Hi-5) were infected in 500 ml volumes at a multiplicity of infection (MOI) of 0.2 – 0.5 Pfu/cell and cultured with ESF921 medium (Expression system) at 27oC for the recombinant baculovirus to express JHEH. After 5days, the cells were harvested at 65 hrs post infection and centrifuged at 230×g for 15 min at 5oC. The pellet was resuspended in 20 ml of homogenization buffer-Buffer A (100 mM Tris-HCl, pH 8.0 having 1 mM EDTA, 1 mM DTT and 1 mM PMSF). The cell suspension was homogenized using a Turax-25 homogenizer for four half-minute pulses alternating with 1-2 min of chilling on ice. The homogenate was subjected to ultra centrifugation at 1,63,000×g for 60 min at 5oC. The pellet from this centrifugation was suspended in homogenization buffer containing 0.5% (V:V) Triton X-100 (100 mg wet weight of cells per ml of homogenization buffer) and homogenized on ice as above with three long bursts. The homogenate from this was centrifuged again at 1,63,000×g for 60 min at 5oC. The supernatant from this centrifugation was flash frozen in liquid nitrogen and stored at -80oC.
1.6 Determination of Kinetic constants & Enzyme assays:
The specific activity of SoJHEH for JH III and other close Epoxide substrates namely cis-Stilbene oxide (c-SO), trans-Stilbene oxide (t-SO), trans-Diphenyl propene oxide (t-DPPO) was determined by partition assay as described by Wixtrom and Hammock 1985. The partition assay was performed with 100 µl of 100 mM sodium phosphate buffer, pH 8.0, containing enzyme (SoJHEH), 50 µm substrate, 1% V/V ethanol, and 0.1 mg/ml of bovine serum albumin (BSA) at 30oC so that no more than 15% of the substrate was hydrolysed during the incubation period. For JH III, the same conditions were followed except the concentration i.e., 5 µM substrate was used. All the assays were done in triplicate and repeated thrice for each concentration of substrate. The Michaelis-Menton constant Km and Vmax of SoJHEH for JH III were determined using specific activity values that were obtained with eight different concentrations of JH III (ranging from 0.5 µM to 75 µM) through non linear regression using sigma plot enzyme kinetics module 1.1 (Systat software). To prevent the hydrolysis of the ester group of JH III by contaminating esterases, 10min long pre-incubation at 30oC was done using a highly strong esterase inhibitor, 3-Octyl thio-1,1,1-trifluoro propane-2-one (OTFP, 10 µM). Background hydrolytic activity was calculated for each concentration of all substrates using control Hi-5 cell microsomes by subtracting activity obtained from SoJHEH Hi-5 cell microsomes.
2 Results and Discussion:
In Insect development, JHEH is one of the key enzymes controlling the JH levels. According to A. Seino et al 2010, when compared to JHE, which controls the JH levels in the haemolymph, JHEH plays more important role for two reasons. First, being a non-secreted enzyme, it can decompose JH and adjust the JH concentrations approximately in each organ at each developmental stage. Second, JHEH yields diol, an irreversibly hydrolysed metabolite, creating a lasting impact on the Insect development.
As a part of this study, the following analysis have been made and discussed here under.
2.1 Insilico Analysis of JHEH from Spilarctia obliqua:
A full length JHEH encoded from cDNA of Spilarctia obliqua (KC148541) showed 1460 nts long and contained 1323 nts long open reading frame flanked by 5’ UTR sequence of 27 nts and 3’ UTR sequence of 85 nts with a poly A25 tail (Figure 1). The deduced protein of SoJHEH showed 440 amino acid residues and had a predicted mass of 49475.9 Daltons and PI of 6.32. The secondary structural analysis of SoJHEH (Figure 2, Table 1) showed that 168 amino acids involved in helices formation, 71 in extended strands, 17 in β-turns and 184 in random coils. ‘Phobius’ and ‘TMpred’ results showed that the first 1–20 amino acids constituted the signal peptide and 1 – 21 amino acids overlapping transmembrane region at N-terminus respectively (Figure 3).


Table 1 Primary and Secondary structural analyses of SoJHEH


Figure 1 Nucleotide and deduced amino acid sequence of Spilarctia obliqua JHEH (Highlighted text: Transmembrane region (Red, 1-2aa), Signal peptide (Green, 1-20aa), Oxyanion Hole: Y288-Y351, red &HGFP motif (143-146), Catalytic triad (D217-H408-E329, violet)


Figure 2 Secondary structural features of SoJHEH


Figure 3 Biochemical parameters of SoJHEH sequence

The epoxide hydration has been well conserved across various species and genera. In mammals, the epoxide hydrolysis mechanism is a two step process (Borhan B et al., 1995).
In first step, the two tyrosine residues polarize the epoxide group by hydrogen-bonding. Simultaneously, the nucleophilic carboxylic group of Aspartic acid, on the opposite side of the catalytic cavity makes a backside attack on the epoxide, which is usually at the least sterically hindered and most reactive carbon. The nucleophilic acid is oriented and stimulated by Histidine and a second carboxylic amino acid (Glutamic or Aspartic acid) resulting in the opening of the epoxide forming the hydroxyl–alkyl–enzyme intermediate (Figure 4). In second step, a water molecule is allowed to activate by the acid–histidine pair by the distant orientation of Histidine (Figure 4). This water molecule attacks the carbonyl of the ester, releasing the diol product and the original enzyme (Pinot F et al., 1995; Morisseau et al., 1998; Argiriadi MA et al., 2000; Yamada T et al., 2000; Schiott B and Bruice TC, 2002).


Figure 4 Catalytic Mechanism of EH (The amino acid residue numbers correspond to the Human sEH) (Source: Christophe Morisseau and Bruce D. Hammock. 2005. Epoxide Hydrolases: Mechanisms, Inhibitor Designs, and Biological roles. Annu. Rev. Pharmacol. Toxicol. 45:311-33)

Upon clustering and conserved Domain analysis of deduced SoJHEH protein (Figure 5), it has been clearly found that all the amino acids involved in the catalytic mechanism were conserved in r-SoJHEH. Among the protein structures reported in PDB, Bombyx mori JHEH (PDB ID: 4QLA.A) showed highest sequence homology (E-value: 9e-123) to r-SoJHEH. The proposed catalytic triad of SoJHEH was located at putative active site as shown in fig 6. They were predicted to be
Asp217-His408-Glu329 and the other amino acids forming the Oxyanion hole, Y288-Y351 and HGFP motif (143-146aa) were also found in SoJHEH indicating the conserved mechanism of epoxide hydrolysis of juvenile hormone with an intermediate formation of hydroxyl alkyl-enzyme intermediate.


Figure 5 Multiple Sequence alignment showing conserved catalytic activity amino acid residues

Like all epoxide hydrolases, SoJHEH is also characterized by a nucleophile-histidine-acid catalytic triad and had a two-step mechanism involving the formation of the covalent intermediate (Figure 6).


Figure 6 Schematic representation of Catalytic Mechanism of SoJHEH protein (Using Hyperchem)

2.2 Expression and Partial Purification of SoJHEH
The SoJHEH ORF was sub cloned into Nco I and Xho I cut pFastBac-HTA baculovirus transfer vector (Invitrogen). The transformed PFastBac-HTA was named as PFastHTSoJHEH (Figure 7A) and has been confirmed by colony PCR and restriction digestion (Figure 7B & 7C).


Figure 7 Subcloning of SoJHEH into pFastBac-HTA transfer vector

The recombinant SoJHEH pFastBac HT-A construct was purified and transformed into DH10
αE. coli for transposition into the Bacmid. The obtained white colonies were screened and the recombinant bacmid DNA was analysed by PCR using M13 Primers and the presence of the gene insert into the recombinant bacmid was verified (Figure 8A).


Figure 8 Bacmid Transformation and Transfection

The recombinant Bacmid DNA was transfected into Sf9 Cells at 1×106 Cells/ml using Cellfectin reagent. Post 24 hrs showed different sized cells with nuclei filling the entire cells. Late 48 hrs, cells stopped the growth and getting detached when compared with the control cells. Granular appearance indicating the signs of viral budding was observed. Post 72 hrs of the Infection, the virus was harvested from the cell culture medium and is known as P1 Viral Stock (Figure 8B).
2.3 Western Blotting:
The pellet fraction was then subjected to Cell lysis. The transfected cells with different time points were shown in the Figure 8B. The expressed protein was determined by Western blotting. The results of Western blotting for the determination of SoJHEH recombinant protein were as shown in Figure 9.


Figure 9 Determination of SoJHEH protein expression

2.4 Viral titer Calculation: 
Plaque assays were conducted and the obtained plaques were counted in each dilution and the viral titer was calculated using the formula given below and the titer obtained was in the range of 1 × 106 to 1 × 107 pfu/ml for P1 viral stock Figure 10.


Figure 10 Sigma plot graph showing Vmax and Km of SoJHEH activity

Titer (pfu/ml) = No. of plaques × Dilution factor × (ml of inoculum per well)-1
2.5 Partial purification of r- SoJHEH:
The microsomal preparations were analyzed on SDS PAGE using 4-12% Tris-glycine gels and have been stained by following the Biorad protocols. Further the gels were analysed on ‘ImageJ’ program to check with the efficiency of the protocol of microsomal preparation. Protein concentrations were determined using Bicinchoninic Acid (BCA, Pierce) protein assay reagents to generate a standard curve.
2.6 Hydrolytic Activity of SoJHEH with c-So, t-SO, t-DPPO and JH III:
SoJHEH hydrolysis have been tried with general EH substrates like c-SO (cis stilbene oxide), t-SO (trans stilbene oxide) and t-DPPO (trans diphenyl propene oxide). The results showed very negligible activity for general epoxides i.e., for c-SO and t-SO it showed less than 0.01 and t-DPPO it showed less than 0.05±0.04 nmol/mg/min. This indicates that the SoJHEH has no or nil activity in hydrolyzing general Epoxide groups (Table 2).


Table 2 Hydrolysis of general epoxide substrates and JH-III by SoJHEH

While the SoJHEH hydrolysed JH III with a Vmax of 30.2 ± 1.7 nmol min-1mg-1 of JH III diol formed. This is similar or faintly lower Vmax when compared to authentic and recombinant JHEHs from other Lepidopteran insects such as Manduca sexta i.e, MsJHEH (Touhara and Prestwich, 1993), Bombyx mori i.e, Bommo-JHEH (Zang et al., 2005), the coleopteran Tribolium castaneum i.e, TcJHEH-r3 (Tsubota et al., 2010) and the leaf hopper, Homalodisca vitripennis i.e, Hovi-mEH (S. G. Kamita et al., 2013).
The Km value of SoJHEH for JH III was 10.8±2.1 µM, the value that was nearly 50 fold higher than that of MsJHEH and 30 fold higher than that of Bommo-JHEH suggesting that JH III is a better substrate for this Spilarctia obliqua JHEH.
3. Conclusion:
JHEH has proven its impact in controlling the JH levels of the Insect pest. This is the first observation of JHEH activity in Spilarctia obliqua, which synchronized with the findings of other lepidopteran, coleopteran and dipteran insects. We have expressed and characterized the juvenile hormone epoxide hydrolase of Spilarctia obliqua (SoJHEH), which is an economically significant major lepidopteron pest on mulberry and many other food crops in India. On the basis of its high affinity for JH-III, it’s structurally conserved domains and trends in enzyme hydrolysis, it is understood that SoJHEH plays a crucial role in maintaining JH concentrations by converting it into JH-diols in an irreversible manner and is the biologically active JHEH of Spilarctia obliqua. This study explores the dual enzyme regulating system of JH which controls varied biological activities of the insect pest and can further throw insights for developing innovative biocontrol ways.
Acknowledgements
The authors acknowledge the Department of Biotechnology (DBT), Ministry of Science and Technology, India for its financial support to some part of this work. We are also thankful to late Dr. J. Nagaraju, Centre for DNA Fingerprinting and Diagnostics for accepting us to access his lab to carry out certain parameters of this work.
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