Introduction
Serine protease inhibitors belong to the serpin superfamily, acting mainly as structurally conserved suicidal inhibitors (Yamasaki et al., Reference Yamasaki, Sendall, Harris, Lewis and Huntington2010). Over 70 serpin structures have been determined; these have been characterized as 40–50 kDa plasma proteins with domain typically consisting of 3 β-sheets and 8–9 α-helices. Serpins undergo a drastic conformational change and form a stable complex with the protease (Abraham et al., Reference Abraham, Pinto, Ghosh, Vanlandingham, Budd, Higgs, Kafatos, Jacobs-Lorena and Michel2005) to inhibit protease function (Law et al., Reference Law, Zhang, McGowan, Buckle, Silverman, Wong, Rosado, Langendorf, Pike, Bird and Whisstock2006). Although most serpins function to inhibit serine proteases, some inhibit other types of protein enzymes like cysteine protein enzymes (Gettins, Reference Gettins2002). Moreover, serpins that are more prevalent in invertebrates appear to be generated through alternative exon splicing (Zou et al., Reference Zou, Picheng, Weng, Mita and Jiang2009).
Serine protease inhibitors have now been identified in many kingdoms of life ranging from microorganisms to larger animals and have been shown to play a critical role in blood coagulation, complement activation, melanotic encapsulation and Spätzle processing (Irving et al., Reference Irving, Pike, Lesk and Whisstock2000). In insects, resembling serpins were first isolated from the hemolymph of silkworm larvae (Sasaki & Kobayashi, Reference Sasaki and Kobayashi1984) and were shown to participate in the immune response mediated by prophenoloxidase (proPO) activation. Mediated by serine protease, proPO is triggered by a cascade of proteolytic cleavage sites to generate phenoloxidase (PO), which oxidizes phenolic compounds to produce melanin (Bilang Liu et al., Reference Bilang Liu, Han, Jia, Xiang and He2012). Therefore, when microorganisms invade, proPO localizes melanization to the surface of invading microorganisms (Tong et al., Reference Tong, Jiang and Kanost2005).
In Manduca sexta, serpins inhibit hemolymph proteases (HPs) involved in the defense response against pathogen infection and tissue damage (Kanost, Reference Kanost1999; Jiang, Reference Jiang2008). Arabidopsis thaliana serpins inhibit metacaspase-9 cysteine protein enzyme (Irving et al., Reference Irving, Pike, Lesk and Whisstock2000). In Bombyx mori, serpin-15 (Bmserpin-15) was shown to inhibit prophenoloxidase activation in B. mori hemolymph, while injection of Bmserpin-15 significantly reduced transcript levels of antimicrobial peptides in the fat body (Liu et al., Reference Liu, Wang, Yang, Qian, Wei, Dai, Li, Zhu and Liu2015). In Ostrinia furnacalis, the levels of serpin-1 transcripts in the integument were higher and shown to be inhibited by challenges with Staphylococcus aureus and Escherichia coli (Zhang et al., Reference Zhang, Wu, Tang, Zhang, Xu, Zhao, Wang and Feng2016). Furthermore, Spn77B was shown to induce systemic expression of the antifungal peptide Drosomycin via the Toll pathway (Tang et al., Reference Tang, Kambris, Lemaitre and Hashimoto2008). These results demonstrate that serpins are an important factor involved in insect innate immunity.
Antheraea pernyi is an important silk-producing insect that lives in the wild. Although the organism exhibits significant advantages in disease resistance, disease outbreaks continue to cause severe economic loss each year. Hence, there is an urgent need to improve disease resistance in A. pernyi against pathogens. Fat bodies and hemolymph are considered important immune organs in insects and are involved in the immune response (Sun et al., Reference Sun, Wang, Qian, Dai, Li, Zhang, Zhu and Liu2015). Therefore, in this study, we conducted characterization, tissue distribution and expression patterns of serpin-1 in A. pernyi following various microorganism challenges. Our results indicate that Ap-serpin-1 might be involved in the innate immune responses of A. pernyi.
Materials and methods
Experimental animals
The Sericultural Research Institute of Henan, China, provided A. pernyi specimens for our experiments. We fed fresh leaves to the 1st through 4th instar larvae, indoors, three times a day, at 25°C, with humidity ranging from 65 to 75% and a photoperiod of about 14 h:10 h (light: dark). For our experiments, we sampled tissues from the midgut, silk gland, fat body, malpighian tubules, integument and hemolymph (Zhang et al., Reference Zhang, Dai, Wang, Qian, Wei, Li, Zhu and Liu2015).
Total RNA extraction and cDNA synthesis
We isolated total RNA from the fat body of A. pernyi 5th instar larvae using Trizol Reagent (Takara, Dalian, China), and followed the manufacturer's protocol. We synthesized the first-strand cDNA using the PrimeScript™ One Step real-time polymerase chain reaction (RT-PCR) Kit Ver.2 (Takara, Dalian, China), following the manufacturer's instructions. We checked the quality of cDNA by electrophoresis on a 1% agarose gel and spectrophotometry and stored the cDNA at −80°C until usage.
Cloning and sequence analysis of Ap-serpin-1
We obtained A. Pernyi serpin-1 cDNA fragment (Genbank succession number KY322717) from the cDNA library, which we constructed in our laboratory. We designed primers serpin-1F1 and serpin-1R1 (table 1) to amplify the Ap-serpin-1 cDNA and performed PCR using the following conditions: 10 min initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min 20 s, before a final elongation step at 72°C for 10 min. We analyzed the PCR product by 1% agarose gel electrophoresis, before verifying the sequence at Huada Company. We used DNAstar software (version 5.02) to predict the open reading frame (ORF). We determined the signal peptide using SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP-4.1/). We calculated the molecular weight of the Ap-serpin-1 protein using ExPASy (http://web.expasy.org/compute_pi/) and performed multiple sequence alignments using ClustalX2 (http://www.ebi.ac.uk/tools/clustalw2/index.html). We then constructed a phylogenetic tree by MEGA 5.1 using the neighbor-joining algorithm method (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011) and performed statistical analysis using 1000 bootstrap replicates.
Table 1. Sequences of primers used in this study.
Note: Restriction sites are underlined.
Expression and purification of recombinant of Ap-serpin-1 protein
We designed primers Ap-serpin-1-F1 and Ap-serpin-1-R1 (table 1) to amplify the Ap-serpin-1 cDNA sequence at the ORF and performed PCR as follows: 5 min initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min 20 s, and final elongation step at 72°C for 10 min. We purified the PCR product using a DNA Gel Extraction Kit (Axygen, Hangzhou, China) before ligating with PMD-19 T vector to obtain plasmid PMD-19T-Ap-serpine-1. After digestion with EcoR I and Sal I (Takara), we ligated the Ap-serpine-1 fragment into pET-28a (+) vector for protein expression in E. coli (Transetta DE3) cells (TransGen Biotech, Beijing, China). We used isopropyl-β-D-thiogalactopyranoside (IPTG) and induced protein expression for 5 h at 37°C. We harvested E. coli cells by centrifugation at 8000 × g for 10 min at 4°C, which we then completely lysed by sonication. Following further centrifugation at 12,000 × g for 15 min at 4°C, we purified the recombinant protein using a Ni-NTA Agarose column (QIAGEN, China) under native conditions, according to the manufacturer's protocol. We analyzed purified recombinant protein using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using an anti-FLAG antibody. We determined the protein concentration using the Bradford assay (Bradford, Reference Bradford1976).
Antibody preparation and titer determination
We prepared anti-Ap-serpin-1 polyclonal antibodies according to the method (Harlow & Lane, Reference Harlow and Lane1999) previously described. Briefly, we incubated 600 µg of purified protein with Freund's complete adjuvant and immunized male New Zealand rabbits. After 2 weeks, we used the purified protein diluted with Freund's incomplete adjuvant for the second immunization. We collected the antiserum after the third immunization boost and used western blotting to confirm the specificity of the antibodies. We determined the titer of the antibody using enzyme-linked immunosorbent assay (ELISA) (Buss et al., Reference Buss, Chan, Sluis, Domigan and Winterbourn1997). Briefly, we incubated Ap-serpin-1 with coated solution at 4°C overnight. After washing with PBST (blocking with 5% non-fat milk diluted with PBS containing 0.1% Tween-20), we incubated the recombinant protein with the primary antibody (diluted 1:1000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, respectively) at 37°C for 2 h. Then, we incubated the recombinant protein with goat anti-rabbit IgG (diluted 1:2000 with PBST; Beyotime, Shanghai, China) for 2 h at 37°C. Finally, we added the TMB substrate solution and used an ultraviolet spectrophotometer to measure optical density (OD) at 450 nm. The results of ELISA showed that the titer of Ap-serpin-1 antibody was 1:128000 (fig. 1).
Fig. 1. ELISA titer determination.
Expression of Ap-serpin-1 in various tissues and developmental stages
To determine the tissue-specific expression of Ap-serpin-1, we collected midgut, silk gland, fat body, malpighian tubules, integument and hemolymph from the third day of the 5th instar larvae. Moreover, we used the fat body (from 1st to 5th instar) from the larvae, pupae, moth and spawn to investigate the relative expression level of Ap-serpin-1 at different developmental stages. We extracted total RNA using the TRIzol reagent (Takara) and the first strand cDNA was synthesized using PrimeScript™ RT Master Mix (Takara). The primers used for PCR were designed using the online tool Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/) (table 1). We performed RT- PCR as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 57°C for 30 s, and a final extension at 72°C for 30 s. We applied a melting curve analysis (65–95°C) to confirm the unique and specific PCR products for each reaction. We determined the relative expression levels according to the 2−ΔΔCt method (Livak & Schmittgen, Reference Livak and Schmittgen2001). We normalized all quantitative RT-PCR (qRT-PCR) experiments using A.pernyi 18s rRNA gene as an internal control. We repeated each biological treatment three times and presented the data as the means ± standard error.
Western blotting
We selected tissues for total protein extraction using RIPA lysis buffer (Aidlab Biotech, Beijing, China) and determined protein concentration using the bicinchoninic acid (BCA) method (Walker, Reference Walker1994). We separated the protein extracts (20 µg each) using 12% SDS-PAGE and transferred the samples to a polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA) using the Mini Trans-Blot electrophoretic transfer system (Bio-Rad). We blocked membranes with 5% non-fat milk (diluted with PBS containing 0.1% Tween-20; PBST) for 3 h at room temperature, before incubating with the prepared anti-Ap-serpin-1 polyclonal antibody for 12 h at 4°C. Finally, we incubated the membranes with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma) for 1 h at room temperature. We detected the immunoblot signal using HRP-DAB Detection Kit (Tiangen, Beijing, China).
Expression analysis of Ap-serpin-1 induced by microorganisms
We divided the 5th larvae of A. pernyi into four groups; each group contained 18 larvae. We injected each larva with the heat-inactivated gram-negative bacterium (E. coli, 8 × 106 cfu), gram-positive bacterium (Micrococcus luteus 8 × 106 cfu), fungus (Beauveria bassiana 8 × 106 cfu) and virus (nuclear polyhedrosis virus, NPV; 8 × 106 PIBs), which we diluted in sterilized PBS. We used the larvae injected with sterilized PBS as the negative control. As the fat body and hemolymph are involved in the immune defense mechanism (Feng et al., Reference Feng, Huang, Song, Stanley, Lu, Zhang and Huang2011), we chose to analyze how Ap-serpin-1 expression was influenced by microorganism challenges in these tissues. We collected fat bodies and hemolymph from individual samples after 1.5, 3, 6, 12, 24 and 48 h. We collected the tissues of three larvae per sample and repeated the biological sampling three times. We determined the expression levels of Ap-serpin-1 by qRT-PCR as described above.
Result
Molecular characteristic of full-length Ap-serpin-1 cDNA
The complete cDNA sequence and deduced amino acid sequence analysis revealed that Ap-serpin-1 contains a putative conserved domain of the serpin superfamily. Full-length cDNA of Ap-serpin-1 consists of 837 bp that contains a 783 bp ORF, encoding a predicted 279 amino acid residue protein. The protein contains a signal peptide of 18 amino acid residues (MKITVCIFALAAMTLANG). The calculated molecular weight of the protein is 30.8 kDa with an isoelectric point (pI) of 5.24. The Ap-serpin-1 protein shared greatest similarities with the serpin from Antheraea mylitta (fig. 2).
Fig. 2. Phylogenetic analysis of Ap-serpin-1 with serpins from other species. The phylogenetic tree was constructed using the neighbor-joining algorithm and bootstrap values (1000 repetitions) of the branches are indicated. The name and GenBank accession numbers of the serpins are indicated.
Expression and purification of the recombinant Ap-serpin-1 protein
The Ap-serpin-1 protein was expressed in the pET-28a (+) vector in E. coli (Transetta DE3) cells. SDS-PAGE analysis showed that recombinant Ap-serpin-1 protein was successfully expressed and the expression was not influenced by different IPTG concentrations. Moreover, purified proteins appeared as a single band on the SDS-PAGE gel (fig. 3a). The apparent molecular weight was 32 kDa, which was little higher than the predicted molecular weight (30.8 kDa). The immunoblot assay using the anti-FLAG tag antibody confirmed the presence of recombinant Ap-serpin-1 protein (fig. 3b).
Fig. 3. Expression and purification of recombinant Ap-serpin-1. (a) Analysis of recombinant A. pernyi serpin-1 proteins on 12% SDS-PAGE gels. Bacterial proteins were collected after 4 h of induction with different isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations. M: protein molecular weight marker; Lane 1, PET-28a (+) control without recombinant proteins in E. coil (Transetta DE3) cells; Lane 2, non-induced cells; Lane 3, induction by 0.5 mM IPTG; Lane 4, induction by 1.0 mM IPTG; Lane 5, purification of recombinant protein. (b) Western blotting analysis of recombinant proteins using anti-His-tag antibodies. M: protein marker: Lane 1: non-induced cells: Lane 2: induction by 0.5 mM IPTG: Lane 3: induction by 1.0 mM IPTG.
Tissue distribution and developmental expression of Ap-serpin-1
We performed qRT-PCR to determine the expression levels of Ap-serpin-1 in different tissues and developmental stages. The analysis revealed that Ap-serpin-1 was expressed ubiquitously in all examined tissues, with higher expression levels found in fat bodies and least expression observed in hemolymph (figs 4a, 4b) Our qRT-PCR and western blotting results demonstrate that Ap-serpin-1 mRNA expression profile is consistent with the protein content profiles. Our qRT-PCR analysis of Ap-serpin-1 expression levels at different developmental stages of A. perenyi, indicated that Ap-serpin-1 expression was greatest at the pupal stage and lowest at the 4th larval stage. We recorded insignificant variations at other developmental stages (fig. 5).
Fig. 4. Tissue distribution of Ap-serpin-1 in the 5th instar larvae of A. pernyi. (a) Analysis of serpin-1 mRNA expression in tissues of larvae using quantitative real-time polymerase chain reaction (qRT-PCR). (b) Analysis of Ap-serpin-1 protein expression in tissues of larvae by western blotting. Ap-serpin-1 was detected using an anti-Ap-serpin-1 rabbit polyclonal antibody.
Fig. 5. Expression levels of Ap-serpin-1 during developmental stages. The sequence of period are 1st, 2nd, 3rd, 4th, 5th, pupae, moth and spawn.
Induced expression patterns of Ap-serpin-1 in the fat body and hemolymph
We explored the induced expression profiles of Ap-serpin-1 when infected with E. coli, M. luteus, NPV and B. bassiana. When the fat body from A. pernyi larvae were treated with B. bassiana, Ap-serpin-1 expression was significantly upregulated from 1.5 to 12 h, and peaked from 24 to 48 h compared with the control group (fig. 6a). When the larval fat bodies were treated with NPV and M. luteus, Ap-serpin-1 expression was significantly upregulated from 3to 48 h, respectively (figs 6b, 6c). Treatment with E.coli enhanced Ap-serpin-1 expression compared with the control group. Moreover, expression levels remained unchanged as a function of induction times (fig. 6d).
Fig. 6. Expression profiles of Ap-serpin-1 in the fat body of the 5th instar larvae after microorganism challenges. Ap-serpin-1 mRNA in the PBS-injected fat body was designated as the calibrator. (a–c and d) represent mRNA transcript level of Ap-serpin-1 in the fat body after challenges with B. bassiana, NPV, M. luteus and E. coli, respectively. Bars represent mean ± S.E. (n = 3). Bars labeled with different letters are significantly different (P < 0.05).
We also determined expression levels of Ap-serpin-1 in hemolymph following various biotic stresses using qRT-PCR. After B. bassiana treatment, Ap-serpin-1 expression was significantly upregulated at 24 to 48 h compared with the control group (fig. 7a). When the larvae were treated with E. coli, Ap-serpin-1 expression was upregulated, which gradually decreased from 1.5 to 48 h (fig. 7b). Upon treatment with M. luteus, Ap-serpin-1 transcription was only subtly upregulated at 1.5 to 12 h, and became strongly upregulated at 24 to 48 h (fig. 7c). Likewise, at 12 h, NPV treatment did not significantly upregulate Ap-serpin-1 expression, while, in other time period, we observed a significant increase (fig. 7d).
Fig. 7. Expression profiles of Ap-serpin-1 mRNA in the hemolymph of the 5th instar larvae of A. pernyi. Ap-serpin-1 mRNA in the PBS-injected hemolymph was designated as the calibrator. A, B, C and D represent mRNA transcript level of Ap-serpin-1 in hemolymph after challenge with B. bassiana, E. coli, M. luteus and nuclear polyhedrosis virus (NPV), respectively. Bars represent mean ± S.E. (n = 3). Bars labeled with different letters are significantly different (P < 0.05).
Discussion
Serpins are widely distributed in eukaryotes and considered to be involved in the regulation of several protease-mediated biological processes such as the immune response, metamorphosis and embryogenesis (Jiang & Kanost, Reference Jiang and Kanost1997; De Gregorio et al., Reference De Gregorio, Han, Lee, Baek, Osaki, Kawabata, Lee, Iwanaga, Lemaitre and Brey2002; Zhao et al., Reference Zhao, Dong, Duan, Wang, Wang, Li, Xiang and Xia2012). In the present study, we identified a serpin-1 gene from A. pernyi that encodes a protein with a molecular weight of 30.8 kDa. Phylogenetic analysis revealed that the Ap-serpin-1 was highly homologous to A. mylitta serpin-1, which contains the conserved signature of the serpin superfamily.
Our qRT-PCR analysis showed that Ap-serpin-1 was expressed in all examined tissues, with the highest expression levels in the fat body. As far as the developmental stages were concerned, higher expression levels were detected in the pupae. Zheng et al., documented higher expression level of serpin-1 in the fat body of Choristoneura fumiferana, which was highly expressed during the intermolt phase compared with molting phase (Zheng et al., Reference Zheng, He, Beliveau, Nisole, Stewart, Zheng, Doucet, Cusson and Feng2009). M. sexta serpin-1 was also reported higher larval fat body, which disappeared abruptly at the molting and wandering stages (Kanost et al., Reference Kanost, Prasad, Huang and Willott1995). Similarly, serpins-1b/1c are expressed predominantly in the fat body and subsequently exported to the hemolymph in Mamestracon figurate (Chamankhah et al., Reference Chamankhah, Braun, Visal-Shah, O'Grady, Baldwin, Shi, Hemmingsen, Alting-Mees and Hegedus2003). It is reported that after bacterial challenges, serpin-1J expression in M. sexta decreases the production of antimicrobial proteins like cecropin, attacin and hemolin via the Toll pathway response (An et al., Reference An, Ragan and Kanost2011). According to our results, Ap-serpin-1 expression in the fat body and hemolymph was significantly upregulated following microorganism challenges, and the expression patterns vary depending on the type of pathogens (E. coli, B. bassiana, M. luteus and NPV), which may be related to the difference in the signaling pathways involved in immune processes of A. pernyi.
Altogether, our results suggest that Ap-serpin-1 is involved in the immune response against pathogens. Although we investigated the expression level and patterns of Ap-serpin-1 in A. pernyi, the mechanisms involved in the interaction between Ap-serpin-1 and its effectors and its signaling pathways remain unclear. Further studies are needed to evaluate these molecular and signaling mechanisms in order to provide a clearer understanding of the interactions between Ap-serpin-1 and its effectors.
Acknowledgements
This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System [grant number CARS-22-SYZ10], the National Natural Science Foundation of China [grant numbers 31301715, 31472147], the Anhui Provincial Natural Science Foundation of China [grant number 1308085QC60], the Sericulture Biotechnology Innovation Team 410 [grant number 2013xkdt-05], and the PhD Programs in Biochemistry and Molecular Biology [grant number xk2013042].
