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  • Infective juveniles of the Steinernema carpocapsae Breton strain were produced in larvae from the Galleria mellonella moth, harvested in a White trap and stored in tap water at 10°C for 1–2 months before use. Larvae from G. mellonella, Pseudalaetia unipuncta and Spodoptera littoralis were supplied by the insectarium in our Department. RNA Extraction and Full-length cDNA Cloning: The full-length sc-srp-6 cDNA was cloned using a previously isolated 866-bp expressed sequence tag (EST) from the parasitic stage of S. carpocapsae (GenBank accession number GR977748.1). Total RNA was isolated as previously described [23], and first-strand cDNA was synthesised using Superscript III reverse transcriptase (Invitrogen) and an oligo (dT) primer. The full-length cDNA was produced by rapid amplification of cDNA ends (RACE) using the SMART RACE cDNA Amplification Kit (Clontech-Takara) with the SRP-5′ primer (5′-TAG AAG CGA CCG TCT TGG GTG-3′) to isolate the 5′ end and an oligo-d(T)20 and SRP-3′ primer (5′-TTC CCG AAG AGT GAG ACC-3′) to isolate the 3′ end. The reaction mixture contained 50 ng of cDNA, 2.5 µl 10×PCR buffer, 10 mM dNTPs, 0.4 µM of each primer, 1.6 mM MgCl2 and 0.2 µl Taq DNA polymerase in a final volume of 25 µl. The amplification comprised an initial denaturation step at 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 3 min. Amplified cDNA fragments were TA-cloned into the pCR4-TOPO vector using the TOPO TA Cloning kit (Invitrogen) and send to sequence by a costumer service (STABvida, Portugal). In silico Analysis of sc-srp-6: The sc-srp-6 full-length cDNA was used in a BLAST search query (http://ncbi.nlm.nih.gov/blast), and sequence alignments were created with the ClustalW program (http://www.ebi.ac.uk/clustalw). Protein motifs were predicted using SMART (http://smart.emblheidelberg.de), and SignalP 3.0 was used to identify the signal peptide (http://www.cbs.dtu.dk/services/SignalP). For phylogenetic reconstruction, sequences were aligned with Muscle (www.ebi.ac.uk/Tools/msa/muscle/). A phylogenetic tree was reconstructed with maximum likelihood using the PhyML program (www.atgc-montpellier.fr/phyml/), and robustness was assessed by the bootstrap method (100 pseudoreplicates). Human serpin was used as an out-group to root the phylogeny. A 3D structure prediction was obtained using the I-TASSER online platform (http://zhanglab.ccmb.med.umich.edu/I-TASSER). Sc-srp-6 mRNA levels were profiled in different nematode stages by quantitative RT-PCR. Nematodes were produced in G. mellonella larvae exposed to IJ in Petri dishes padded with wet filter paper and collected from parasitised insects after dissection. L3 nematodes were isolated from the gut and hemocoel, L4 nematodes were isolated from the hemocoel and adults and L1/L2 stages were isolated from the body tissues. Approximately 20 specimens were collected from each stage from three separate exposures and homogenised in liquid nitrogen to produce cDNA as previously described. Controls without reverse transcriptase were included for each sample, and data were normalised against 18S rRNA. Sc-srp-6 was amplified with SRP-6F (5′-GGG GAA GAC GAG TCAG GAG A-3′) and SRP-6R (5′-CTC GGC TTG AGG GTT GCT GA-3′) primers. The 18S rDNA was amplified with the 18SF (5′-GCT AAT CGG AAA CGA AAG TC-3′) and 18SR (5′-CAT CCA CCG AAT CAA GAA AG-3′) primers using an ABI 7900 HT Real Time PCR thermocycler (Applied Biosystems). Amplifications were performed in triplicate with each reaction consisting of an initial heating step at 95°C for 10 min, followed by 60 cycles of 95°C for 15 s and 60°C for 60 s. RT-PCR data were analysed using the comparative CT method according to the manufacturer′s recommendations. The data are expressed as the mean ± standard error from six independent experiments. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni multiple comparison tests (Statistical Package for the Social Sciences software, SPSS version 13.0). Differences were considered significant at P<0.05. The full-length sc-srp-6 coding sequence was amplified using the forward primer SRP-6heF, which includes a BamHI restriction site (5′-GGA TCC ATG TTG CCG ACT CCT AAG ACC AAT-3′) and the reverse primer SRP-6heR, which includes an XhoI restriction site (5′-CTC GAG ATA GAA GTC CCA CGA AGA G-3′). The 1.3-kb product was purified from a 1.5% agarose gel using the QIAEX II Gel Extraction Kit (Qiagen) and digested with BamHI and XhoI. The fragment was then inserted into the pET23a expression vector in frame with an N-terminal T7 tag (Novagen). The vector was transformed by thermal shock into the E. coli C41(DE3) and Rosetta™ II(DE3) strains. The identity and integrity of the inserts were confirmed by sequencing. A single colony from each transformed E. coli strain was incubated in 5 ml Luria broth (LB) containing 100 µg/ml ampicillin at 37°C with shaking at 250 rpm overnight. These cultures were used to inoculate 20 ml of LB plus antibiotics as described above and were incubated at 37°C until an OD600 of 0.6 was reached. Sc-SRP-6 expression was induced in both strains under several different conditions including 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 3 hrs, 1 mM IPTG at 37°C for 3 hrs, 0.5 mM IPTG at 20°C overnight and 1 mM IPTG at 20°C overnight. Cells from the different cultures were harvested by centrifugation at 4,000×g for 15 min. The pellet was then suspended in 2 ml 0.05 M Tris-HCl (pH 7.4) containing 0.5 M NaCl and 1 mg/ml lysozyme and frozen at −20°C overnight. After thawing, the lysate was supplemented with 1 ml of 1 M MgCl2 and 600 µl of 1 mg/ml DNAase and then incubated for 30 min at room temperature with orbital agitation. Cell debris was removed by centrifugation at 12,000×g for 20 min, and the supernatant was collected and analysed to detect the recombinant protein. Solubilisation, Refolding and Purification of Recombinant Sc-SRP-6: E. coli Rosetta (DE3) cells were cultured in 2 L LB, and recombinant protein expression was induced with 0.5 mM IPTG followed by 3 hrs of incubation at 37°C. Cells were recovered and lysed as described above. The lysate was washed for 4 hrs in 1 L 0.05 M Tris-HCl (pH 7.5) containing 0.5 M NaCl (TN), followed by 4 hrs in TN containing 1% Triton X-100, and the pellet with inclusion bodies was recovered by centrifugation at 6,500×g for 20 min at 4°C. The inclusion bodies were solubilised overnight in 40 ml of 8 M urea containing 0.7% β-mercaptoethanol under orbital agitation. The solution was adjusted to 200 ml with the same buffer and dialysed in a cellulose membrane tube against 20 mM Tris-HCl (pH 8.0) for 48 h at 4°C followed by two more buffer changes. Solubilised proteins were concentrated using a 5000 molecular-weight cutoff membrane (Centricon, Millipore), diluted 1∶1 in 20 mM Tris (pH 8.0), 5 mM imidazole, 150 mM NaCl, 5 mM DTT and purified by nickel-chelate affinity chromatography (Ni-Sepharose, GE Healthcare) in an AKTA FPLC system (GE Healthcare). The Sc-SRP-6 fraction was concentrated and dialysed using a Centricon column and was subsequently refolded for 3 hrs in 300 mM L-arginine, 50 mM Tris (pH 8.0), 50 mM NaCl and 5 mM DTT. The refolded Sc-SRP-6 was then applied to a MonoQ column (GE Healthcare) that was equilibrated with 20 mM Tris-HCl (pH 8) and eluted in a linear gradient of 1 M NaCl to eliminate minor contaminants. The protein concentration was determined using the BCA Kit (Thermo Scientific), and purity was assessed by 10% SDS-PAGE. Recombinant Sc-SRP-6 was analysed by 10% SDS-PAGE with a Mini-PROTEAN II gel system (Bio-Rad), and proteins were stained with Coomassie brilliant blue dye. For western blot, proteins separated by SDS-PAGE were electroblotted onto a PVDF membrane using a mini Trans-Blot Cell (Bio-Rad). The membrane was blocked with 0.01 M Tris-HCl (pH 7.5) containing 0.1 M NaCl, 5% (w/v) BSA and 0.05% (v/v) Tween-20 for 30 min at room temperature (RT) and then incubated with peroxidase-conjugated mouse anti-His6-tag antibody diluted (1∶5,000) in blocking solution for 2 hrs at RT. Membranes were washed three times for 10 min with 0.01 M Tris-HCl (pH 7.5), 0.1 M NaCl. Antibodies were detected by incubation with tetramethylbenzidine peroxidase substrate (Sigma) according to the manufacturer′s recommendations. MALDI-TOF/TOF (Matrix-assisted Laser Desorption/Ionization-Time-of-Flight) Tandem Mass Spectrometry: Proteins were digested in-gel with trypsin, and the peptides were purified and concentrated using an R2 pore microcolumn. The peptides were applied to a MALDI plate with 0.5 µl α-cyano-4-hydroxycinnamic acid matrix (5 mg/µl in 50% acetonitrile, 5% formic acid) and prepared for MALDI as previously described [28]. The m/z spectra were acquired in a 4,700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems) in the MS and MS/MS modes. Searches were performed with a minimum mass accuracy of 50 ppm for the parent ions, an error of 0.3 Da for the fragments, one missed cleavage in the peptide masses and carbamidomethylation of Cys and oxidation of Met as fixed and variable amino acid modifications, respectively. The confidence threshold for protein identification was set to 95% (p<0.05). To obtain the highest confidence score for the identification of proteins involved in serpin complexes, the masses originating from Sc-SRP-6 were excluded from the peptide mass list used in the database search. Protein identification was performed using the MASCOT program (www.matrixscience.com) to search the UniProtKB database (downloaded on 10/06/2012). Circular Dichroism of Recombinant Sc-SRP-6: To assess the correct folding of recombinant Sc-SRP-6, the far-UV circular dichroism (CD) spectrum of the protein was acquired. Purified protein was run on size-exclusion Sephadex 200 chromatography columns to remove putative aggregates and eluted in 50 mM phosphate buffer (PB) (pH 8.0). CD experiments were performed on a DSM-20 CD spectrophotopolarimeter (OLIS) controlled by GlobalWorks software using protein concentrations ranging from 0.15 to 0.35 mg/ml. Far-UV CD spectra were recorded between 190 and 260 nm using a 1-mm path length cuvette. CD spectra were run with a step-resolution of 1 nm, an integration time of 5 sec, and slit width of 0.6 nm, at 37°C. The spectra were averaged over two scans and corrected by subtraction of the buffer signal. Data are expressed as the mean residue molar ellipticity [Θ]MRW, which is defined as [Θ]MRW = Θobs (0.1MRW)/(lc), where Θobs is the observed ellipticity in millidegrees, MRW is the mean residue weight, c is the concentration in milligrams per millilitre and l is the cuvette path length in centimetres. The secondary structure contents were calculated with the CONTINL, CDSSTR and Selcon3 algorithms [29]–[31] against the CLSTR reference basis set, which contains soluble and denatured proteins with known secondary structure. The inhibitory activity of purified Sc-SRP-6 was tested against trypsin, α-chymotrypsin and elastase from bovine pancreas, subtilisin from Bacillus licheniformis and thrombin from bovine plasma. Twenty microlitres of Sc-SRP-6 (3.5 µg/µl) was incubated with 10 µl of each enzyme (1 µg/µl) for 10 min at 37°C in microtiter plates with the total volume adjusted to 45 µl with 0.1 M Tris-HCl (pH 8) containing 0.1 M NaCl and 1 mM CaCl2. The hydrolytic activity of the remaining enzyme was quantified in a final concentration of 1 mM of the BApNA, Suc-AAPFpNA, Suc-AAPLpNA, Z-GGLpNA and BPVA-pNA (Sigma-Aldrich) chromogenic substrates for trypsin, α-chymotrypsin, elastase, subtilisin and thrombin (Sigma-Aldrich), respectively. Sc-SRP-6 was replaced with buffer in the control reactions. The formation of p-nitroaniline was monitored in an ELISA microplate reader at 405 nm, and the percentage of enzyme inhibition caused by Sc-SRP-6 was calculated as follows: % inhibition = ((total enzyme activity units – residual enzyme activity units)/total enzyme activity units) * 100. The activity of trypsin or chymotrypsin was determined with specific substrates (1 mM) in the presence of different concentration of Sc-SRP-6 (0, 8, 22 and 30 µM) under the conditions described above. The fractional activity (velocity of enzyme with Sc-SRP-6/velocity of enzyme without Sc-SRP-6) was plotted against the ratio of serpin/enzyme concentrations, and the x-axis intercept as a value for the stoichiometry of inhibition (SI) was determined by linear regression analysis. The inhibition constant (Ki) for Sc-SRP-6 against trypsin and chymotrypsin was estimated using a Dixon plot. A single regression line for each substrate concentration (0.05, 1 and 2 mM) was obtained, and the Ki was calculated from the intersection of the three lines. The inhibitory mechanism was determined using Lineweaver-Burk plots. Regression lines for trypsin and chymotrypsin were produced using the inverse of the initial rate for each Sc-SRP-6 concentration plotted against the inverse of the substrate concentration. To determine the optimal pH, Sc-SRP-6 was mixed with each enzyme, and the pH adjusted to 5, 6 and 7 with 0.1 M PB, 0.1 M NaCl and to 8 and 9 with 0.1 M Tris-HCl, 0.1 M NaCl. After incubation for 10 min at 37°C, the inhibition of enzyme activity was determined under the conditions described above. To identify serpin/trypsin and serpin/α-chymotrypsin complexes, reactions between serpin and enzyme concentrations equal to and above the SI were analysed by SDS-PAGE, under non-reducing conditions. To search for serpin and plasma protein SDS-stable complexes, Sc-SRP-6 was incubated with LPS-activated plasma (prepared as described in the phenoloxidase activation section) at 1∶20 and 1∶10 concentration ratios in 50 mM Tris-HCl (pH 8.0) for 30 min at 37°C. The reaction mixture was boiled at 90°C in non-reducing loading buffer for 10 min and then resolved on a 10% SDS-PAGE gel. In controls with phenylmethylsulfonyl fluoride (PMSF) the samples were preincubated with 5 mM PMSF for 10 min at RT. Sc-SRP-6 and complexes were detected in-gel using the InVision™ His-tag Stain signal (Novex) and excised after staining with Coomassie brilliant blue. Isolation of Insect Mid-gut Proteases and Inhibition Assays: Digestive enzymes were extracted from chilled and excised mid-guts from 20 fourth-instar G. mellonella, P. unipuncta and S. littoralis larvae. After rinsing in ice-cold 20 mM Tris-HCl (pH 8), the mid-guts were homogenised on ice in 1 ml of buffer using a Polytron homogeniser at 12,000 rpm. Debris was removed by centrifugation at 15,000×g for 20 min at 4°C, and the supernatant was filtered through a 0.2 µm nitrocellulose membrane (Millipore). The filtrate was fractionated in a Sephacryl S-200 column (GE) in 20 mM Tris-HCl (pH 8), and the fractions were screened for trypsin and chymotrypsin activities with 2 mM BApNA and Suc-AAPFpNA, respectively. Active fractions for both enzymes were pooled. In the inhibition assays, insect enzymes were pre-incubated with 30 µM Sc-SRP-6 in 20 mM Tris-HCl (pH 8) for 30 min at 30°C. The activity was determined using the referred specific substrates for each protease. Assays were performed in triplicate. P. unipuncta neonates were fed an artificial diet supplemented with 200 µM Sc-SRP-6 at 0.2% (w/v), which was replaced under the same conditions every 2 days. In the controls, Sc-SRP-6 was replaced with Ringer solution. To follow the digestion of ingested materials, albumin-bromphenol blue was prepared as described [32] and added to the artificial diet. Twenty larvae were individually dispersed in 24-well multi-well plates with perforated lids and maintained at 25°C with a 16/8 hrs (L/D) cycle. The assay was performed in triplicate. Larval weight and mortality were recorded every 2 days and compared using one-way ANOVA. Differences were considered significant at P<0.05. PO activation in G. mellonella plasma was quantified as previously described [21]. The hemolymph was collected by puncturing the ventral pro-leg of fourth-instar larvae and draining it into an anticoagulant solution (Ringer containing 20 mM EDTA instead of CaCl2) at a 5∶1 final ratio (v/v). The solution was centrifuged at 1,500×g for 3 min at 4°C to remove the hemocytes, and 40 µl of the plasma solution was added to 10 µl of Sc-SRP-6 (3 µM/µl) and incubated for 5 min on ice. As a control, Sc-SRP-6 was replaced with the anticoagulant. The samples were then activated with 5.5 µl (10 µg/ml) of lipopolysaccharide (LPS E. coli O55:B5, Sigma) dissolved in 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl and incubated for 15 min at 25°C. The plasma produced was centrifuged at 5,000×g for 5 min at room temperature, and the supernatant was recovered and supplemented with 20 µl of 15 mM L-DOPA (Sigma), dissolved in 20 mM Tris-HCl (pH 7.5) and incubated for 15 min at 37°C. The reaction was monitored at 490 nm using an ELISA microplate reader (Bio-Rad). PO activation was measured as the relative change in OD at reaction time points 0 and 15 min. Experiments were performed in triplicate. A drop clot assay was performed by adding 30 µM Sc-SRP-6 to a plasma drop on a slide that was then activated with LPS (10 µg/ml). The slide was placed in a humidified chamber for 30 min. To measure the incorporation of melanin in clots after incubation with and without Sc-SRP-6, the supernatant and adherent materials were carefully separated by aspiration with a fine tip followed by visualisation and photography by light microscopy. To determine whether clot fibres formed, plasma drops prepared and incubated as above were fixed with 2% glutaraldehyde in 10 mM phosphate buffer (PB) (pH 7) at 4°C for 15 min. The glutaraldehyde was removed from the slides by carefully washing with 10 mM PB, and it was replaced with 70% ethanol. The floating clot strands were carefully transferred to a new slide and observed by phase-contrast microscopy. In the same assay, a portion of the clot was transferred to PB plus 8% NaCl, stained with 10 mg/ml peanut agglutinin conjugated with FITC (PNA-FITC, Sigma) and observed under a fluorescence microscope (Zeiss) at 480 nm. An encapsulation assay was performed with Sephadex G200 beads. Beads were added to the LPS-activated plasma, incubated for 30 min in a humidified chamber and fixed in 2% glutaraldehyde. Preparations were processed for scanning electron microscopy as previously described [33]. Analysis of Reaction from Injury: Fourth-instar larvae were lightly anesthetised by chilling and then pierced with an ultrafine needle in the ventral midline between segments A3 and A4 under a stereomicroscope. Fifteen microlitres of Sc-SRP-6 (0.3 µM) was applied to the wounds, and the larvae were maintained in Petri dishes for 30 min at RT. The larvae were then dissected to expose the incision site, and the wounds analysed and photographed under a stereomicroscope.
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