key: cord-0882253-shw799ts
authors: Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan
title: Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity
date: 2015-05-29
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2015.04.026
sha: abf7357ff5204fc96ea4a9d5a7447ce3456ff16e
doc_id: 882253
cord_uid: shw799ts

The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae.

Cry d-endotoxins produced by Gram-positive spore-forming Bacillus thuringiensis (Bt) are highly toxic toward a wide range of insect larvae [1] . Of particular interest, the Cry4Ba toxin from Bt subsp. israelensis is specifically active against mosquito larvae of the genus Aedes and Anopheles, vectors of various human diseases including dengue fever, chikungunya, yellow fevers and malaria [2, 3] . After ingestion by susceptible insect larvae, these Cry toxins which are produced as insoluble protoxins are solubilized in the larval midgut lumen (generally alkaline pH for dipteran and lepidopteran larvae) prior to their activation by gut proteases to yield 65-kDa active toxins [2, 4] . According to all the known Cry structures, the activated toxins consist of three distinct functional domains: an a-helical bundle (DI), a b-sheet prism (DII) and a b-sheet sandwich (DIII). DI and DII have been revealed as membrane-pore formation and receptor recognition, respectively [5] . Recently, we have demonstrated that charge-reversal mutations at Asp 454 (D454R and D454K) located within a receptor-binding loop of Cry4Ba-DII markedly increases the toxin activity against lesssusceptible Culex larvae, suggesting a role of the charged sidechain in determining target specificity [6] .

A number of activated Cry toxins are shown to bind to a variety of specific receptors located on the midgut epithelium cells [7] . This toxin-receptor interaction would promote toxin oligomerization and membrane-pore formation, resulting in midgut cell lysis and the eventual death of the target larvae [7] . Specific binding to their individual receptors is believed to be the critical determinant in Cry Abbreviations: AaeAPN, Aedes aegypti membrane-bound aminopeptidase N; AeaALP, Aedes aegypti membrane-bound alkaline phosphatase; CAT, chloramphenicol acetyltransferase; GPI, glycosylphosphatidylinositol. toxin specificity. In mosquito larvae, various membrane-bound proteins were identified as functional receptors for several Cry toxins, including Aedes and Anopheles cadherin-like proteins for Cry11Aa and Cry4Ba, respectively [8, 9] , Aedes alkaline phosphatases (ALP) for Cry11Aa and Cry4Ba [10e12], Anopheles ALP for Cry11Ba [13] , Aedes aminopeptidases N (APN) for Cry4Ba and Cry11Aa [14, 15] and Anopheles APN for Cry11Ba [16, 17] . Recently, the Cyt2Aa toxin from Bt subsp. darmstadiensis has also been reported as another binding protein for Cry4Ba to exert synergistic toxicity against Aedes aegypti larvae [18] . However, the structural basis for Cry4Ba toxin-receptor interaction is still unclear and need to be explored.

The APN family is a class of M1 zinc-metalloprotease/peptidase superfamily that cleaves neutral amino acids from the N-terminus of polypeptides [19] . Members of this family are classified as gluzincins that contain a consensus Zn 2þ -binding sequence [HEXXH-(X18)-E, where X stands for any amino acid] and a GAMEN sequence in the active site [19] . To date, crystal structures of the M1 superfamily are available for various species, e.g. Escherichia coli (PDB ID: 2HPT), Plasmodium falciparum (PDB ID: 3EBG), Thermoplasma acidophilum (PDB ID: 1Z5H) and human (PDB ID: 2YD0) [20] . They all show similar overall structures, with domain II being the most conserved and domain IV the most variable [21] . In addition to being studied for their typical role in digestion, APNs have also been extensively studied as tumor-related targets, receptors for coronavirus and bacterial toxins [21, 7] .

Previously, we have demonstrated in A. aegypti mosquito larvae that knockdown of three different GPI-anchored APN isoforms (AaeAPN2778, AaeAPN2783 and AaeAPN5808) via RNA interference resulted in the decrease of Cry4B toxicity [14] . Here, we provide further insights into the molecular structure and specific binding to the Cry4Ba toxin of two functionally expressed AaeAPN isoforms, AaeAPN2778, AaeAPN2783, on the Sf9 insect cell membrane.

Three pairs of AaeAPN-specific primers were designed (see Supplementary Table 1 ) based on three putative GPI-anchored APN sequences from A. aegypti genome database (www.vectorbase.org): AaeAPN2778, AaeAPN2783 and AaeAPN5808, where the numbering is referred to the last four digits (underlined) of transcript identification from VectorBase (AAEL012778-RA, AAEL012783-RA and AAEL005808-RA). Then, potential GPIanchoring sites were predicted by four different GPI-prediction programs: PredGPI (gpcr2.biocomp.unibo.it/gpipe/pred.htm), big-PI Predictor (mendel.imp.univie.ac.at/sat/gpi/gpi_server.html), FragAnchor (http://navet.ics.hawaii.edu/~fraganchor/NNHMM/ NNHMM.html) and GPI-SOM (http://gpi.unibe.ch/). Signal peptide and N-glycosylation sites were further predicted by SignalP and NetNGlyc1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/), respectively.

First-strand cDNA was synthesized from RNA transcripts of the 5th instar A. aegypti larval midgut using oligo-dT primer. Coding sequences of these three AaeAPNs were individually amplified via the cDNA product with Phusion DNA polymerase (Finnzymes). The expected size-amplicons were purified, ligated to the pENTR/SD-TOPO vector and transformed into One-Shot TOP10 competent cells, following the manufacturer's protocol (Invitrogen). Recombinant pENTR/AaeAPN plasmids were randomly selected and their sequences verified by DNA sequencing.

Each of recombinant pENTR/AaeAPN plasmids was selected for an in vitro Lamda Recombination (LR). Individual AaeAPN fragments were transferred from pENTR/AaeAPN plasmids to Baculodirect Linear DNA (Invitrogen) by site-specific recombination. Briefly,~200 ng of pENTR/AeAPN plasmids were mixed with Baculodirect C-term Linear DNA (~200 ng) and LR Clonase II for 18 h at 26 C. The recombinant AaeAPN expression vectors were subsequently transfected into the insect Sf9 (Spodoptera frugiperda 9) cells using Cellfectin, following manufacturer's protocol (Invitrogen).

Cell lysates collected from Sf9 cells expressing each recombinant AaeAPN were separated by SDS-PAGE. The protein bands corresponding to AaeAPN proteins were excised from the gel and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis (MaXis, Bruker).

Cry4Ba-R203Q mutant toxin (one tryptic cleavage site was eliminated by substitution at Arg 203 with Gln) which still retains high larvicidal activity [22] was used in this study. E. coli strain JM109 expressing the R203Q mutant was grown in LB medium containing 100 mg/mL ampicillin at 37 C until OD 600 reached 0.3e0.6. Toxin expression was induced with 0.1 mM isopropyl-b-D thiogalactopyranoside for 4 h and harvested by centrifugation at 6000Â g. The cell pellet was re-suspended in 100 mM KH 2 PO 4 (pH 5.0) containing 0.1% Triton X-100 and 0.5% NaCl, and further disrupted by sonication (VCX750-Sonics Vibra Cell™) with the following parameters: 5 cycles of amplitude 60%, 10-s ON, 30-s OFF with a total time ON ¼ 1 min/cycle. Protein inclusions were collected by centrifugation at 6000Â g, washed and subsequently re-suspended in distilled water. The concentration of the partially purified inclusion was determined using the Bradford-based protein microassay. Cry4Ba toxin inclusions were solubilized in carbonate buffer (50 mM Na 2 CO 3 /NaHCO 3 , pH 9.0) at a final concentration of 1 mg/mL for 1 h at 37 C. The solubilized toxin was activated by trypsin (tolylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin; Sigma) at a ratio of 1:10 enzyme/toxin (w/ w). The 65-kDa activated toxin was further purified by sizeexclusion chromatography (Superose 12 10/300, GE Healthcare) and eluted with the carbonate buffer (pH 9.0) at a flow rate of 0.4 mL/min. The purified Cry4Ba protein fraction was then concentrated with a 10-kDa-MWCO Millipore membrane (Merck KGaA) and analyzed by SDS-PAGE.

100 mg/mL of Cry4Ba toxin (mixed with 1 mL Sf-900 medium (Invitrogen) was applied to the 5-day post-infected Sf9 cells expressing recombinant AaeAPNs, AaeALP (Aedes membranebound alkaline phosphatase) and CAT control (chloramphenicol acetyltransferase) at~8 Â 10 5 cells/mL in a 6-well culture plate and further incubated at 26 C for 2 h. The cell morphology was observed under light microscope. To determine viability of cells, PrestoBlue™ cell viability assay (Life Technologies, USA) was performed. Briefly, 100 mL PrestoBlue™ reagent was added directly to cells in 900 mL culture medium, incubated for 10 min at 37 C and then spectrofluorometrically quantitated (excitation at 530 nm; emission at 590 nm). The significance of the data between samples was analyzed using Student's t-test.

Interactions between the Cry4Ba toxin and AaeAPNs were investigated by double immunofluorescence staining. After 5-day post-infection, Sf9 cells expressing AaeAPNs (AaeAPN2778 or AaeAPN2783), CAT or AaeALP were incubated with 100 mg/mL of the activated Cry4Ba toxin at 26 C for 2 h. Cells were washed twice with PBS and collected by centrifugation at 3000Â g. The cell pellets were fixed with 4% paraformaldehyde in PBS buffer and then blocked with 3% BSA followed by 1-h incubation with primary antibodies, either mouse monoclonal antibody-2F(1H2) specific to the Cry4Ba domain III at 1:100 dilution or rabbit polyclonal antibodies against human APN (anti-CD13, ab9389, Abcam) at 1:50 dilution. The cells were rinsed twice with PBS and collected by centrifugation, followed by another 1-h incubation with secondary antibodies, either FITC-conjugated rabbit anti-mouse IgG (Jackson Immuno Research) or Alexa Fluor ® 647-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) at 1:100 dilution. The unbound conjugate was removed by washing with PBS. The stained cells were collected by centrifugation and deposited on the glass slide and mounted with a coverslip. The immuno-stained cells were then examined on a confocal laser scanning microscope (Olympus FV1000).

Plausible homology models of AaeAPN2778 and AaeAPN2783 were constructed using SWISS-MODEL, a protein structure homology-modeling server (http://swissmodel.expasy.org/). For template protein identification, the homologous structure was identified by searching the Protein Data Bank (PDB) using BLAST algorithm (http://www.rcsb.org/). ClustalW program was employed to identify conserved and variable regions between these two AaeAPNs and the template proteins. After manual adjustment, alignment files were subjected to SWISS-MODEL server in alignment mode. Stereochemical quality and Ramachandran plot analyses of both AaeAPN models were performed using QMEAN [23] and RAMPAGE [24] servers.

Membrane-bound APNs have been identified as Cry toxin receptors in various species of insect larvae such as Bombyx mori, Heliothis virescens, Ostrinia nubilalis and Manduca sexta [25e28]. In Anopheles and Aedes mosquito larvae, several APNs were also identified as putative receptors for the Cry4Ba, Cry11Aa and Cry11Ba toxins [14e16]. Here, three different full-length genes encoding AaeAPN2778, AaeAPN2783 and AaeAPN5808 were cloned from A. aegypti larval midgut RNA transcripts with specific primers using RT-PCR strategy. The PCR products obtained were of the expected sizes, i.e. 3003-bp for AaeAPN2778, 2868-bp for AaeAPN2783 and 2848-bp for AaeAPN5808 (Fig. 1, inset) . The translated cDNA sequences of AaeAPN2778, AaeAPN2783 and AaeAPN5808 revealed open reading frames of 1000, 955 and 947 amino acid residues (see Supplementary Fig. 1 ) with a predicted molecular mass of 112, 107 and 108 kDa, respectively. The APN conserved sequences, HEXXH_E and GAMEN sequences [19] were identified in all three AaeAPN clones, indicating that these three AaeAPNs belonged to the member of M1 zinc-metalloprotease/ peptidase superfamily. Moreover, these three AaeAPNs also contained a putative N-terminal signal peptide together with their predicted GPI-anchored sites on the C-terminus (Fig. 1) that strongly suggested these AaeAPNs as membrane-bound proteins attached to the cell surface via a GPI-anchor.

To generate the recombinant AaeAPN proteins, baculovirus expression was performed. The infected Sf9 cells were harvested after 5 days for protein expression analysis. Both AaeAPN2778 and AaeAPN2783 were detected as dominant protein bands with sizes corresponding to the predicted mass of 112 and 107 kDa, respectively (Fig. 2C) . For the controls, 30-kDa and 58-kDa protein bands were observed as expected for CAT and AaeALP-expression, respectively (Fig. 2C) . However, no protein of the expected size was found for AaeAPN5808. Thus, the specific toxin-receptor interaction study (see below) employed only the 112-kDa AaeAPN2778 and the 107-kDa AaeAPN2783.

Further analysis by LC-MS confirmed that the 112-and 107-kDa protein bands indeed represent the expressed products of AaeAPN2778 and AaeAPN2783, respectively; multiple peptide sequences were found to correspond to both AaeAPN2778 ( 165 RLSNSEDGFYISSYVNKD 182 , 524 RRTYETGDIIISQERF 539 , 791 RSMII-SALGCSENKQ 805 ) and AaeAPN2783 ( 92 RQITVTHVKL 101 , 280 KVINA-LEDYLQVKY 293 , 743 KDIDPNLKG 751 , 831 RVGLETAMKFLDANAATIDR LYNGGSFGGRA 861 ). It is noteworthy that the size of the expressed protein bands correspond to the predicted 112-kDa AaeAPN2778 and 107-kDa AaeAPN2783, suggesting that the recombinant APN proteins were not glycosylated, at least, in the Sf9 cells. This finding was similar to another Cry4Ba receptor, i.e. AaeALP, which is found not to be glycosylated in Sf9 cells [12] and is functionally expressed in E. coli [29] . Also, another dipteran APN from Anopheles mosquitolarvae (AgAPN2) expressed in E. coli was shown not to have glycosylation for Cry11Ba binding [17] unlike some identified lepidopteran APNs that need the sugar moiety for binding to their toxin counterparts [7, 28] .

In vitro cytotoxicity assays were carried out to test whether the two recombinant AaeAPN isoforms can serve as a functional receptor for the Cry4Ba toxin. It was found that when Cry4Ba (100 mg/ mL) was added, Sf9 cells expressing AaeAPN2778, AaeAPN2783 or AaeALP appeared to undergo cell lysis as visualized under light microscope ( Fig. 2A) . To quantitate this observation, cell viability assay was performed. The result showed an increase in percent mortality of Sf9 cells expressing AaeAPN2778 (35.0 ± 4.5%) and AaeAPN2783 (27.0 ± 2.2%) when compared to the control Sf9 cells expressing CAT (10.0 ± 0.8% cell mortality) (Fig. 2B ). Cell mortality mediated by these two AaeAPNs was similar to AaeALP (31.7 ± 2.8% cell mortality) which has been previously identified as a Cry4Ba receptor [12] , suggesting that both AaeAPN2778 and AaeAPN2783, like AaeALP, share a common role as a toxin receptor mediating the Cry4Ba cytotoxicity. Moreover, this result was in agreement with our previous study that AaeAPN2778-or AaeAPN2783-knockdown larvae became less susceptible to the Cry4Ba toxin [14] , strongly suggesting that these two APN isoforms serve as functional receptors for the Cry4Ba toxin in A. aegypti mosquito-larvae.

Further studies via double-labeling immunofluorescence experiments showed that Cry4Ba-immunoreactivity was seen in AaeAPNs-and AaeALP-expressing Sf9 cells but not in CATexpressing cells (Fig. 3) . Both the Cry4Ba toxin and AaeAPNs were detected on the cell membrane surface with extensive colocalization ( Fig. 3I and L) , indicating that Cry4Ba was able to bind only to Sf9 cells-expressing membrane-bound AaeAPNs or AaeALP. These results also suggested that both AaeAPN isoforms were expressed in Sf9 cells as membrane-bound proteins. Taken altogether with the cytotoxicity results suggest that specific binding of Cry4Ba to its single counterpart (AaeAPN2778 or AaeAPN2783) is sufficient for mediating the toxin activity against individual receptor-expressing Sf9 cells without the need of pre-interaction with another receptor. Nevertheless, it has been shown both in vivo and in vitro for lepidopteran-specific Cry1A toxins that pre-binding of the toxin to cadherin-like receptors is required for toxin oligomerization prior to interacting with second membrane-bound receptors (e.g. APNs or ALPs) for insertion and pore formation [7, 30] . Multiple sequence alignments of the deduced amino acid sequences of AaeAPN2778, AaeAPN2783 and AaeAPN5808 against APNs from An. gambiae mosquito-larvaeÀAgAPN [17] and humanÀHuERAP1 [20] . HuERAP1 is used as the reference sequence denoting secondary structure elements. The conserved GAMEN and gluzincin sequences involved in Zn 2þ -binding or substrate-binding are boxed in yellow and green, respectively. The predicted N-terminal signal peptide is underlined; the putative GPI-anchor site is highlighted in red. Inset is RT-PCR analysis of total RNAs from A. agypti larval midguts, showing the cDNAs of all three full-length transcripts as their sizes indicated on each lane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) AaeAPN2778-domain IV (shaded green/grey) and AaeAPN2783-domain IV (shaded magenta/white) structures revealing negatively charged patches on the surface exposed loops. All structures were generated by using UCSF Chimera program. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Currently, there is no three-dimensional (3D) structure of insect APNs, homology-based models of AaeAPNs were therefore built based on the highest sequence similarity. Human endoplasmic reticulum aminopeptidase 1 (PDB 2YD0) [20] was therefore selected as a template for homology modeling as it showed the highest similarity with 50% and 46% to AaeAPN2778 and AaeAPN2783, respectively. To validate the models, the stereochemical quality of AaeAPN2778 and AaeAPN2783 showed the overall QMEAN score of 0.609 and 0.591, respectively, suggesting good quality models as estimated model reliability is between 0 and 1, with higher values for more reliable structures [23] . In addition, Ramachandran plot analysis revealed that 97.3% and 97.6% of AaeAPN2778 and AaeAPN2783 amino acid residues, respectively, are present in the most favored and additional allowed positions [24] , suggesting that both 3D-modeled structures are mostly in the sterically favorable main-chain conformations.

When compared to the X-ray structure of other members of the M1 superfamily including E. coli APN (ePepN, PDB ID: 2HPT), P. falciparum APN (PfAM1, PDB ID: 3EBG) and human endoplasmic reticulum APN (HuERAP1, PDB ID: 2YD0) [20] , the two AaeAPNs show overall structures similar to these known APN structures consisting of four structural domains: a b-sandwich (domain I), a thermolysin-like ab fold (the most conserved domain II), a small bsandwich (domain III) and an a-helical bundle (domain IV) (Fig. 4A ). This suggests that AaeAPNs displayed high structural conservation among the M1 aminopeptidase family. It should be noted that in the most variable domain IV there is no predicted N-/ O-glycosylation site in the Thr-rich C-terminal stalk regions (see Supplementary Fig. 1 ) which are believed to be a part of Cry1Abinding regions for the lepidopteran APNs [7] , suggesting different toxin-binding sites between lepidopteran-and mosquito-APNs. Interestingly, when compared the variable domain IV of these two AaeAPNs among insect APNs that have been reported as Cry toxin receptors including Lymantria dispar APN [31] , M. sexta APN [32] , H. virescens APN [26] and An. gambiae APN [17] , the conserved negatively charged residues were found only on the two AaeAPNs: Glu 734 , Asp 735 , Asp 775 and Glu 802 for AaeAPN2778; and Glu 724 , Asp 725 , Asp 765 and Glu 790 for AaeAPN2783 (see Fig. 4B , as indicated by *). These conserved-negatively charged patches placed them on the surface-exposed loops of both AaeAPN structures (Fig. 4C) , possibly being involved in binding to the Cry4Ba toxin via ionic interactions and hence determining target specificity against Aedes larvae. Further studies, to better understand more critical insights into Cry4Ba toxin-receptor interactions, charged reversed mutagenesis on these putative interaction sites would be of great interest. Likewise, point mutations to positively charged sidechains on receptor-binding loop regions of the Cry4Ba toxin itself might perhaps improve the toxin activity and specificity against Aedes larvae, consistent with previous findings that Cry4Bacharged-reversal mutants (D454R and D454K) exhibit increased binding affinity and larval toxicity to Culex larvae [6] .

There are no conflicts of interest to declare.

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In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae

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Families of zinc metalloproteases

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1

Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis

QMEAN: a comprehensive scoring function for model quality assessment

Structure validation by Ca geometry: phi,psi and Cb deviation

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

Identification, isolation, and cloning of a Bacillus thuringiensis CryIAc toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens

Study of the aminopeptidase N gene family in the lepidopterans Ostrinia nubilalis (Hubner) and Bombyx mori (L.): sequences, mapping and expression

Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin

Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin

Toxicity and mode of action of insecticidal Cry1A proteins from Bacillus thuringiensis in an insect cell line

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin

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This work was supported in part by grants from the Thailand Research Fund (IRG-57-8-0009 and BRG-53-8-0007). A PhD scholarship from the Development and Promotion of Science and Technology Talents Project (to A.A.) is gratefully acknowledged.

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.04.026.

Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.04.026.