Cloning, expression, and characterization of prophenoloxidase from Antheraea pernyi.

A r t i c l e

CLONING, EXPRESSION, AND CHARACTERIZATION OF PROPHENOLOXIDASE FROM Antheraea pernyi Wang Xia Lu∗ School of Medical Devices, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, P. R. China and Benxi Institute of Medicines, Shenyang Pharmaceutical University, Benxi, Liaoning Province, P. R. China

Du Yue∗ School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, P. R. China

Zhang Jing Hai School of Medical Devices, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, P. R. China and Benxi Institute of Medicines, Shenyang Pharmaceutical University, Benxi, Liaoning Province, P. R. China

Wen Daihua, Zhao Ming Yi, and Wu Chun Fu School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, P. R. China

Zhang Rong School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, P. R. China and Benxi Institute of Medicines, Shenyang Pharmaceutical University, Benxi, Liaoning Province, P. R. China

∗ Wang Xia Lu and Du Yue contributed equally to this work. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30972770 and 31100647; Grant sponsor: Liaoning key laboratory of Pattern Recognition, Shenyang key laboratory of Medical Devices; Grant number: F13-292-1-00; Grant sponsor: Research Foundation of Shenyang Pharmaceutical University for Young Teachers; Grant sponsor: Young Teacher Career Development in Shenyang Pharmaceutical University. Correspondence to: Zhang Rong, School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning Province 110016, P. R. China. E-mail: [email protected]

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 88, No. 1, 45–63 (2015) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21219

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Archives of Insect Biochemistry and Physiology, January 2015

Prophenoloxidase (PPO) is an essential enzyme in insect innate immunity because of its role in humoral defense. In this study, we have cloned a full-length cDNA of Antheraea pernyi prophenoloxidase (ApPPO) with an open-reading frame encoding 683 amino acids, and the deduced amino acid sequence of ApPPO exhibited a high similarity with those of lepidoptera. The expression of ApPPO was inducible so that the mRNA level was significantly upregulated in the microbial challenged tissues, including fat body, hemocytes, and midgut. To better investigate the enzymatic and immunological properties of ApPPO, recombinant ApPPO (rApPPO) was produced in Escherichia coli. Several functional verification experiments were performed after studying the enzymatic properties. It was found that rApPPO could be stimulated by the microbial challenged larvae hemolymph and then killed bacteria in the radial diffusion assay. Furthermore, rApPPO also induced the transcription of C 2014 Wiley cecropins after injected into the larvae 24 h later. Periodicals, Inc. Keywords: innate immunity; PPO-activating system; prophenoloxidase (PPO); Antheraea pernyi

INTRODUCTION Hosts defend themselves against pathogen through various strategies including behavioral modification, physical barriers, and finally their immune system (Pierick and Tom, 2009). In contrast to mammals, invertebrates lack antibodies, lymphocytes, or other features of the vertebrate adaptive immunity and therefore must depend on the innate immune system completely (Aspán et al., 1995; Taro et al., 2012). Insects are the most abundant and diversified group of organisms that have acclimatized to the majority of ecological niches. They possess a robust immune system permitting them to occupy diverse habitats (Rajagopal et al., 2005) through humoral and cellular defense responses. In general, the insect cellular immunity involves circulating hemocytes (which perform phagocytosis of small bacteria) and encapsulation (around large parasites), while the humoral factors comprise plasma proteins and peptides, including recognition proteins, clotting proteins, antimicrobial peptides, and even the PPO-activating (where PPO is prophenoloxidase) system proteins (Destoumieux et al., 1997; Söderhäll and Thörnqvist, 1997; Strand, 2008) The PPO-activating system, which is also called the PPO-activation cascade, plays a vital role in invertebrates in the defense processes against pathogens, parasites, and cuticular sclerotization (Moon et al., 2002). The system involves several serine proteinases and is an efficient non-self-recognition cascade that can be triggered by minute amounts of lipopolysaccharides, peptidoglycan from bacteria, or β-1,3-glucans from fungi (Ai et al., 2009). After recognition and the activation of serine proteinases, endogenous phenols can be oxidized by the key enzyme, activated phenoloxidase (PO) into quinones, and then subsequent auto-catalyze into melanin (Jesy et al., 2013). As an important part of the immune defense, melanization is mainly involved in the process of sclerotization, pigmentation, and wound healing of the cuticle as well as in defense reaction (nodule

Archives of Insect Biochemistry and Physiology

Cloning, Expression and Characterization of Prophenoloxidase

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formation, phagocytosis, or encapsulation; An et al., 2013; Wang et al., 2014). Nowadays, studies on insect PPO activation cascade have been conducted primarily in Aedes aegypti, Drosophila melanogaster, Bombyx mori, Manduca sexta, and Tenebrio molitor (Ashida et al., 1990; Ashida and Yoshida, 1990; Asada et al., 1993; Hall et al., 1995; Yasuhara et al., 1995; Chosa et al., 1997; Asada, 1998; Asada and Sezaki, 1999; Lee et al., 1999; Satoh et al., 1999; Lee et al., 2000Asada et al., 2003; Gupta et al., 2005; Junsuo et al., 2005). It has generally been accepted that upon recognition of aberrant surfaces or foreign invaders, PPO in the blood plasma is activated through a limited proteolysis at its Nterminus by the clip-domain of serine proteinases that are called PPO-activating factors (PPAFs) or PPO-activating proteins (PAPs; Kanost et al., 2004). However, the overall process leading to PPO activation is so complicated and it is still not clear how many factors are directly or indirectly involved in the PPO-activating mechanism (Ashida and Brey, 1998). PO (monophenol, L-Dopa: oxidoreductase; SC 1.14.18.1.) is a multifunctional copper-containing oxidase that catalyzes the hydroxylation of tyrosine to 3,4dihydroxyphenylalanine (dopa) or the oxidation of ortho-diphenolic substances to their relative quinones that then polymerize to form melanin (Tsao et al., 2009). In insects, POs mainly exist in inactive form (PPO) that are highly conserved and belong to the family of type 3 copper-containing enzymes. In several insects it has been reported that PPOs are mainly produced by hemocytes in circulation and hematopoietic organs, while it has also been observed that PPOs are produced by cells in the silkworm hindgut where they melanize feces for microbial flora elimination (Lu et al., 2014b). So far, besides cleaved by PPAFs in vivo, PPO can also be activated by chemicals in vitro, such as sodium dodecyl sulfate (SDS), cetylpyridinium chloride (CPC), ethanol, 2-propanol, etc. With the development of biomolecular technology, research in immune proteins from the gene perspective has become a hotspot. The first PPO gene was cloned from the freshwater crayfish Pacifastacus leniusculus (Aspán et al., 1995). Till date, nine PPO genes in Anopheles gambiae, 10 in A. aegypti, three in D. melanogaster, two in B. mori and M. sexta, and only one PPO gene in the honeybee Apis mellifera (Lu et al., 2014a) have been reported according to the literature. All of these insect PPOs contain two putative tyrosinase copper-binding motifs with six histidine residues, and are predicted by a regulated exocytosis process due to the lack of signal sequences. Recently, we have identified and characterized two pattern-recognition proteins, 1,3-β-D-glucan recognition protein (Ap-βGRP) and C-type lectin (Ap-CTL) from Antheraea pernyi (Ma et al., 2013; Wang et al., 2013). These results provide evidence for the existence of PPO-activating system in A. pernyi during pathogen invasion. In order to further elaborate the molecular mechanism of the PPO-activating system, we reported the cDNA cloning, primary structure analysis, and mRNA expression of PPO from A. pernyi (ApPPO) for the first time in this study. Then, a series of verified experiments were carried out using the recombinant protein expressed in Escherichia coli (rApPPO) to study its enzymatic properties and immune behaviors, including the optimal enzymatic conditions, substrate specificity, PO activity stimulated by the microbial challenged larvae hemolymph, and the antibacterial ability. It is also indicated that additional injection of rApPPO in vivo could raise the transcription level of cecropins, which connects the function of PO with antibacterial peptides production for the first time.


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MATERIALS AND METHODS Insects and Chemicals Antheraea pernyi larvae were purchased from Shenyang Agricultural University and reared in the laboratory on a natural diet of quercus mongolica leaves at 27°C. On day 8, the fifth instar larvae were chilled on ice for 5 min and the native hemolymph was collected as described by Zhang and colleagues (Zhang et al., 2003). L-dopa, dopamine, 4-hychoxyproline ethylester, pyrogallic acid, 4-methyl catechol, and catechol were also purchased (Sigma Aldrich, St. Louis, MO). cDNA Cloning of ApPPO Total RNA was extracted from fat body of the 24-h postchallenged A. pernyi larvae with Trazol reagent (Invitrogen, Carlsbad, CA). Then, the RNA sample was subjected to synthesize the first-strand cDNA using TM II Reverse Transcriptase according to the manufacturer’s introductions (Invitrogen). C-F1/R1 (5 -AATCTNCAYCATTTGCAYTTGC-3 /5 -CATRTGRTGNGGCAANCCRC ANCC-3 ), primers for the cDNA fragment amplification of ApPPO were designed based on the highly conserved amino acid sequences of other known insect PPOs. The purified PCR product was cloned into the vector, pMD-18T (TAKARA, Dalian, CN), and then sequenced using T7 primers (GENSCRIPT, Nanjing, CN). RACE To obtain the 5 and 3 ends of ApPPO, rapid amplification of cDNA ends (RACEs) was performed based on the manufacture of 5 RACE system and 3 RACE system for RACEs (Invitrogen), respectively. Both 5 RACE and 3 RACE products were purified, cloned into pMD-18T vectors, and sequenced. The full-length cDNA sequence of ApPPO has been assembled by the Seqman program of DNASTAR. Sequence Analysis of ApPPO The cDNA sequence of ApPPO was analyzed with several bioinformatics analytic tools. Sequence analysis tools of the ExPASy Molecular Biology Server of Swiss Institute of Bioinformatics, SignalP, and computer pI/MW Server, were used to analyze the deduced ApPPO protein sequence. The probable N- and O-glycosylation sites were predicted by NetNGlyc 1.0 and NetOGlyc 2.0 prediction servers, respectively. Comparison of ApPPO cDNA sequence against the nonredundant public sequence in the Genbank database was carried out using BLASTX. The alignment of multiple amino acid sequences was created by ClustalW program and a phylogenetic tree was generated from ClustalW guide tree data using the DNAMAN program. Production of Recombinant ApPPO (rApPPO) A gene fragment encoding all the amino acid residues of ApPPO (1–683aa) was first cloned into the expression vector pET-28a(+) with the restriction enzyme sites, NdeI and EcoRI (TAKARA), and then transformed into E. coli (strain DE3). Following sequencing, the positive colony was selected and cultured in LB medium containing 50 μg/ml Archives of Insect Biochemistry and Physiology


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kanamycin. Till the logarithmic growth phase of bacteria reached 1.0 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 0.5 mmol/l CuSO4 (final concentration) was added to induce the expression of rApPPO with a six-histidine tag at the amino terminus. Bacteria were then harvested by centrifugation, sonication, and SDS-Polyacrylamide gel electrophoresis (PAGE) analysis. Recombinant ApPPO was mainly expressed in the form of inclusion body. After dissolving in 8 mol/l urea solution, the soluble denatured protein was purified by affinity chromatography using Ni2+ -NTA agarose (Sigma Aldrich) and evaluated by SDS-PAGE. For renaturation, the purified denatured sample was first dialyzed against 50 mmol/l sodium phosphate buffer with 0.1% Triton X-100 and 1.0 mol/l urea, pH 8.0, to remove most urea. The whole removal process was conducted gradually (8–1 mol/l, urea gradient) for at least 24 h at 4°C. And the sample was then dialyzed against the buffer without urea to refold at 4°C for 8 h. Bradford assay was performed to evaluate the concentration of rApPPO and rApPPO was used as an antigen to produce rabbit antiserum for biological analysis. Activation of rApPPO Recombinant ApPPO could be stimulated by incubating with a trace amount of hemolymph from A. pernyi larvae, which not only contained the PPO-activating enzymes, but also had a low basal PO activity (Ashida and Brey, 1998). Thus, in this section, rApPPO was first activated by the PAPs involved in minute amount of hemolymph before examining the PO activity. Fifty microliter rApPPO (40 μg/ml) in 50 mmol/l Phosphate-Buffered Sodium (PBS), pH 8.0, was preincubated with 5 μl cell-free plasma at 30°C for 10 min, 445 μl substrate solution (MH, 1 mmol/L 4-methylcatechol and 2 mmol/L 4-hychoxyproline ethylester in 50 mmol/l PBS, pH 8.0) was then added and the mixture was incubated at 30°C. The PO activity was monitored continuously at the absorbance of 520 nm using a microplate reader. One unit of PO activity was defined as 0.01 absorbance increase at A520 nm /min. The cleavage of rApPPO peptide in the PO activity assay was detected by immunoblot using anti-rApPPO antiserum as mentioned above. Samples without rApPPO or hemolymph (replaced by appropriate buffer) were examined as negative control. The Optimal Enzymatic pH and Temperature of rApPO The optimum pH was determined by measuring the enzyme activity as described above in the range of 4.0–10.0 at 30°C. In total, 20 mmol/l citric acid, PBS, and Tris-HCl were used to prepare various pH solutions. The same reaction system at pH 7.8 was carried out to examine the optimal enzyme temperature of rApPO at the range of 15–60°C. As negative control, the same amount of protein solvent was used instead of rApPPO. Substrate Specificity In total, 10 mmol/l L-dopa, MH, pyrogallic acid, 4-methyl catechol, dopamine and catechol were dissolved in 20 mmol/l Tris-HCl buffer, pH 7.0, respectively. Each sample mentioned above was used to determine whether the substrate of rApPO by monitoring the PO activity induced by the substrate at its own maximum absorption as described above. The PO activity induced by A. pernyi hemolymph at the same concentration was used as positive control. Archives of Insect Biochemistry and Physiology

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Kinetic Studies Michaelis constant (Km) of rApPO for substrate (pyrogallic acid and dopamine) was examined as follows: A. pernyi hemolymph (5 μl, 20 μg proteins/ml) was preincubated with rApPPO (50 μl, 400 μg/ml) at 30°C for 10 min, then, substrate in 20 mmol/l Tris-HCI buffer (pH7.0) was added to the reaction mixture to provide an increasing concentration gradients in a final volume of 1 ml. Kinetic parameters were calculated from a plot of 1/V vs. 1/[S] using Lineweaver and Burk’s method. mRNA Expression of ApPPO The mRNA expression of ApPPO was determined by semiquantitative RT-PCR: 5 μg total RNA was isolated from tissues of 24-h postchallenged larvae (immune challenged by a mixture of formaldehyde-killed E. coli, S. aureus, C. albican, and S. Cerevisiae, 2 × 106 cells/ml, respectively), including hemocytes, fat body, midgut, and integument. First-strand cDNA synthesis was then carried out from total RNA by reverse transcription, followed by subsequent amplified using pfu polymerase (TAKARA). A pair of rps eight primers (5 -GAAGTTGGTAATGTAATGCCCGTG-3 /5 CAGGATTGTGACCGATAACTGTGG-3 ) were used as an internal reference control for normalization and the native larvae without challenged were used as negative control. Moreover, to examine the effect of microbial challenge at different time points, the transcription levels of ApPPO in fat body after immune challenged (0, 12, 24, 48, and 72 h) were also determined as mentioned above. PO Activity Assay and Radial Diffusion Assay of rApPO PO activity assay and radial diffusion assay were both performed to examine the immune function of rApPO in A. pernyi hemolymph. Different amount (0, 1, 5, or 50 μg) of cell-free plasma preincubated rApPPO was mixed with 5 μl laminarin (soluble β-1,3-glucan, 200 μg/ml), PO activity was then monitored as mentioned in section “Activation of rApPPO.” Simultaneously, radial diffusion assay was carried out using the pre-incubated samples as follows: 5 μl samples were loaded into the wells (3 mm in diameter) in the underlay, in which washed mid-logarithmic phase bacteria (E. coli and S. Aureus, respectively) were trapped. The underlay agar consisted of sterile LB medium containing 1.2% (w/v) agarose (Sigma Aldrich). After 3 h for diffusion of proteins into underlay medium, a 10 ml nutrient-rich overlay medium containing 6% TSB and 1% agarose was added. The plates were then incubated overnight at 37°C and the diameters of clearing zones were measured with scale lupe and normalized in units (1 mm = 10 units). As the comparison group, the samples pre-incubated with the same amount of BSA instead of rApPPO were both examined in the PO activity and radial diffusion assays. The Transcription Levels of Cecropins Induced by rApPPO In Vivo Samples of rApPPO in insect saline (20 μl sample containing 20 μg protein) were injected into the fifth instar larvae with 20-gauge needles by puncturing the cuticle in the intersegmental region between two abdominal segments. Twenty-four hours after injection, the total RNA of larvae fat body were extracted and the mRNA expression levels of cecropin B and D in A. pernyi were measured by semi-quantitative RT-PCR. Larvae without injection served as negative controls and the ribosomal protein S8 gene (rps8) was used as positive internal control. Archives of Insect Biochemistry and Physiology


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Statistical Analysis All the experiments were repeated at least three times and similar results were obtained. Each point represents mean ± SEM (n = 3). The results were subjected to analysis of t-test and P < 0.05 was considered statistically significant. RESULTS cDNA Cloning and Phylogenetic Analysis of ApPPO A novel PPO cDNA sequence from the lepidoptera A. pernyi was obtained from the larval fat body (Fig. 1). By using degenerate primers designed from the highly consensus amino acid sequences of lepidoptera, a fragment of 1536 bp was first amplified by means of RT-PCR, and then 5’- and 3’-RACE were carried out to obtain the 5’- and 3’-ends cDNA fragments of ApPPO using pairs of specific primers derived from the obtained cDNA fragment mentioned above. Finally, it is constructed that the full-length sequence of ApPPO cDNA consists of 2322 bp with an open reading frame of 2049 bp, which encoding 683 amino acids residues (from nucleotide 89 to nucleotide 2137). There was no potential signal peptide, N-linked glycosylation site, or O-linked glycosylation site in ApPPO amino acid sequence. The molecular mass of the deduced ApPPO protein was evaluated to be 782,877 Da and the calculated isoelectric point was 6.46. It was predicted that the proteolytic cleavage site was between R51 and F52 (arrow) due to its close proximity with the cleavage sites of other insect PPOs. Moreover, four conserved motifs, copper-binding site A, copper-binding site B, thiol ester region-like motif, and a conserved motif at the C-terminal end were identified as underlined in Figure 1 (Region I–IV). A BLAST search of Genbank sequence data base was performed using the deduced amino acid sequence of ApPPO. It was indicated that ApPPO was a novel member of the Hemocyanin superfamily and the putative protein contained three domains: Hemocyanin N domain (all-alpha domain) from positions 23 to 135, Hemocyanin M domain (copper-containing domain, active domain) from positions 136 to 393, and Hemocyanin C domain (Ig-like domain) from positions 399 to 652. (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) ApPPO was also found to be homologous to other insect members of the Hemocyanin superfamily from the alignment result of ClustalW multiple sequence alignment program (Fig. 2A). Moreover, ApPPO contained all of the six conservative cysteine residues found in insect PPOs (*), which were considered to be ligands for copper ion. A phylogenetic tree was constructed using the DNAMAN program and the phylogenetic structure indicated three major clades of these 10 insect PPOs. Just as predicted, ApPPO belonged to the clade that contained M. sexta PPO, but not to the clade containing B. mori and B. mandarina PPOs. This result was consistent with the phylogenetic relationship of most immune proteins we have obtained from A. pernyi in other studies (Fig. 2B). Production of Recombinant ApPPO (rApPPO) Purification of natural ApPPO from A. pernyi larvae hemolymph is challenging since it is unstable and losing activity rapidly during preparation (data not shown). However, it requires such sufficient purified protein to analyze its characterization and enzymatic properties that we are committed to produce recombinant ApPPO (rApPPO) in a prokaryotic expression system. In this study, rApPPO was expressed in E. coli with a six-histidine Archives of Insect Biochemistry and Physiology

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Figure 1. Nucleotide and deduced amino acid sequences of the Antheraea pernyi PPO (ApPPO). The number of nucleotide is shown on the left of the nucleotide sequence. The start codon ATG is shown in bold and the termination codon (TGA) is marked with an asterisk (*). Possible polyadenylation signal (AATAAA) and poly (A)+ tail are doubly underlined. Arrow shows a possible cleavage site for proteolytic activation of the enzyme. Conserved motifs are underlined. Note in region I: copper-binding site A, region II: copper-binding site B, region III: thiol ester region-like motif and in region IV: a conserved motif at the C-terminal end.

at the amino-terminus. After ultrasonication, the insoluble inclusion body was solubilized in 8 mol/l urea and the denatured protein was purified through Ni2+ -NTA affinity chromatography immediately. Renaturation was then carried out to recover the biological activity of rApPPO through dialysis to remove urea gradiently. Finally, as was analyzed by 10% SDS-PAGE and Western blot shown in Figure 2C, the purified protein migrated as a single band at approximately 80 kDa (Fig. 2C, lane 1). However, in Western blot assay, the natural ApPPO had migrated a little faster than rApPPO using anti-rApPPO antibody (Fig. 2C, lanes 2 and 3). This migration change was thought to be affect by the extra histidine tag. Archives of Insect Biochemistry and Physiology


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Figure 2. Multiple sequence alignment and phylogenetic analysis of ApPPO with other insect PPOs together with SDS-PAGE and Western blot analysis of ApPPO. (A) Multiple sequence alignment. ApPPO: Antheraea pernyi PPO; BmxPPO1: Bombyx mori PO subunit-1 precursor (NP_001037335.1); BmaPPO: Bombyx mandarina PPO (ACC69184.1); SfPPO1: Spodoptera frugiperda PPO subunit-1 (ABB92834.1); MysPPO1: Mythimna separata PPO-1 (BAM76811.1); HvPPO1: Heliothis virescens PPO-1 (ABH10016.2); MsPPO1: Manduca sexta PO subunit1 (O44249.3); PiPPO: Plodia interpunctella PPO (AAU29555.1); GmPPO: Galleria mellonella PPO (AAK64363.1); CfPPO1: Choristoneura fumiferana PPO-1 (ABW16859.1); Shading indicates degree of overall conservation of each site: black, identical; gray, conservative; *: conserved histidine residues of copper-binding A and B; the thiol ester-like motif (GCGWPQHM) is shown double-underlined. B: Phylogenetic analysis. The phylogenetic tree was constructed by the neighbor-joining method. A value of 0.05 indicated the similarity between the species; C: SDS-PAGE and Western blot analysis of ApPPO. Lane M, molecular weight markers; lane 1, SDS-PAGE and Commassie Blue staining of rApPPO purified by Ni2+ affinity chromatography under denaturing conditions (0.4μg). Lane 2 and 3, Western blot analysis of rApPPO (0.4μg) and the natural ApPPO in larvae hemolymph (10μg) using antibody against rApPPO, respectively. The arrow indicates the 80kDa rApPPO.


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Activation of rApPPO In previous research, it has been ascertained that there is a complete PO-activating system components in the hemlymph of A. pernyi that can be stimulated by microbes. Therefore, PO activity assay and Immunoblot analysis were both carried out to investigate the activation of rApPPO stimulated by A. pernyi hemolymph. However, it is reported in many insects that PPOs express at an extremely low level under the normal physiological conditions continuously for protecting themselves in situation that pathogen invaded. To minimize the effect of natural ApPPO on the PO activity assay, the reaction conditions, including the concentration ratio of rAppPO and cell-free plasma, reaction buffer, and incubation time, were normalized. Finally, the optimum reaction conditions were determined in which not only rAppPO could be activated as much as possible, but also the impact of natural ApPPO was minimized. The PO activity was examined at the maximum absorption wavelength of MH, λmax 520 nm. As shown in Figure 4A, the PO activity increased significantly in the sample in which rApPPO was treated with hemolymph (P < 0.05), comparing to controls. In order to further study the activation mechanism of rApPPO by larvae hemolymph, immunoblot was used to analyze the cleavage of rApPPO. In Figure 4B, four bands were recognized by anti-rApPPO antibody (band 1, 2, 3, 4). Lanes 1 and 2 were negative controls (bands 1 and 2), whereas in lanes 3 and 5, rApPPO in both heat-treated only and larvae hemolymph-incubated group remain inactive. Comparatively, when native A. pernyi larvae hemolymph was treated with heating, the natural ApPPO was degraded (lane 4, band 3). This phenomenon indicated that (1) it is not heating that stimulated the activation of rApPPO. (2) The activator(s) for ApPPO (e.g., PPAFs) remained in the inactive form, but heating treatment set up the PPO-activating cascade so that the activator(s) was activated to cleave ApPPO. However, in the group of mixing rApPPO with larvae hemolymph followed by heating, besides band 3, another smaller band (lane 6, band 4) was detected. In combination with the PO activity assay, we speculated that bands 3 and 4 may naturally activate ApPO and rApPO, respectively. This hypothesis should be proved further in future. Optimal Temperature and pH To examine the influence of temperature and pH on PO activity, the enzyme activity of rApPO was determined at different temperatures and pH values. It was found that the optimum pH of PO was approximate 7.8 (Fig. 4A), higher than that of B. mori and B. mandarina (pH 7.0), but lower than that of Heliothis virescens (pH 9.0; Lockey and Ourth, 1992) and D. Melanogaster (pH 8.0; Asada and Sezaki, 1999). rApPO had an optimum temperature of 40°C (Fig. 4B) coinciding with that of B. mori, higher than that of D. melanogaster (30°C; Asada and Sezaki, 1999), but lower than that of H. virescens (45°C; Lockey and Ourth, 1992). However, the differences of optimum pH and temperature might correlate with different species, buffers, substrates, or tissue-specific differences. In addition, it was found that the pH value had an enormous impact on the activity of PO. Substrate Specificity of rApPO and Enzyme kinetics The substrate specificity of rApPO is shown in Figure 4C, MH and pyrogallic acid were proved to be superior substrates than others. Moreover, dopamine was proved to be a better substrate than dopa for the enzyme, while catechol and 4-methyl catechol were poor substrates for rApPO. Nevertheless, 4-methyl catechol and catechol were reported Archives of Insect Biochemistry and Physiology


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superior substrates for PO from both M. sexta and B. mandarina, (Hall et al., 1995). Even though the substrate specificity of rApPO was different from that of POs in M. sexta and B. mandarina, dopa, which is often used as a substrate for routine assay of insect PO, and was not an appropriate substrate for them. In the light of these results, routine use of dopa as a substrate for the insect enzyme is not recommended. The kinetic properties of rApPO were studied using different concentrations of pyrogallic acid and dopamine as specific substrates. Using the Lineweavere-Burk model, the Km value of rApPO for pyrogallic acid and dopamine was calculated as 1.81 mmol/l and 3.03 mmol/l, respectively (Fig. 4D). The mRNA Expression of ApPPO in Response to Microbial Challenge Semi-quantitative RT-PCR was performed to measure the relative expression levels of ApPPO transcript in different tissues in response to microbial challenged (Fig. 3C). Firststrand cDNAs were reverse transcripted from the total RNA extracted from a variety of tissues, and a 720-bp fragment was then amplified using a pair of specific primers designed from the cDNA sequence of ApPPO. As was shown, ApPPO was mainly expressed in fat body on the physiological condition (control), while in the immune-challenged group (induced), ApPPO were expressed in fat body, hemocytes, and midgut, though fat body was still the highest expression tissue. However, ApPPO was not expressed in integument no matter in the physiological or immune-challenged condition. Next, ApPPO expression in fat body at different time points after inoculation was measured (Fig. 3D). No significant changes in the expression of ApPPO occurred with time following saline injection (data not shown). But the expression of ApPPO could not be significantly upregulated by microbial challenge until 12 h after injection, and this increase sustained for hours. The transcript level of ApPPO was found to be highest at 48 h, but began to decrease ever since 52 h after injection (not shown), the transcript level at 72 h dropped to about the same as that of 12 h postinjection. As a loading control, rps8 was used and remained unchanged throughout the experiments. Enzymatic Contribution of ApPPO in Innate Immunity To identify the immune behavior of ApPPO, several experiments were performed on the enzymatic contribution of ApPPO in A. pernyi innate immunity system, including the PO activity (Fig. 4E) and antibacterial ability (Fig. 4F) of rApPPO induced by the immunechallenged larvae hemolymph in vitro, and the transcription of cecropins in response to rApPPO induction in vivo (Fig. 3F). PO activity assay was first carried out to test whether rAppPO obtained in this study could be activated by the PO-activating system in the larvae hemlymph when soluble PAMP, including LPS, mannan, laminarin, DAP-PGN, LTA, and Lys-PGN, was added. In order to prove that the PO activity was indeed produced by rApPO instead of other proteins, the PO activity of BSA induced by hemolymph was also examined. Take laminarin for example, the PO activity results were shown in details (Fig. 4E). It was exactly detected that the native hemolymph produced PO activity after immune challenged (Sample: 0 μg), but the level was too low to compare to the samples added with rApPO. Moreover, scarcely any difference of PO activity was detected between the native hemolymph and the samples added with BSA, even though the content of BSA increased from 1 to 50 μg. However, when rAppPO incubated with stimulated larvae hemolymph, it produced much higher PO activity, and the level rose sharply with the concentration of rAppPO Archives of Insect Biochemistry and Physiology

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Figure 3. Analysis of the stimulation of rApPPO and mRNA expression. (A) PO activation assay of rApPPO induced by Antheraea pernyi larvae hemolymph. The results of PO activity assay is shown as the mean ± SEM (n = 3) and the analysis of variance and Newman–Keuls test is P < 0.05; (B) Western Blot analysis of the activation of rApPPO. Lane 1, rApPPO (0.4 μg); Lane 2, native A. pernyi larvae hemolymph (10 μg); Lane 3, the mixture of rApPPO and native A. pernyi larvae hemolymph without heating; Lane 4, native A. pernyi larvae hemolymph treated with heating; Lane 5, the mixture of rApPPO and native A. pernyi larvae hemolymph treated with heating. (C) Analysis of the mRNA expression of ApPPO in different tissues. Semi-quantitative RT-PCR was performed to detect the mRNA transcript of ApPPO in different tissues in response to challenges. Five micrograms of total RNA was obtained from fat body (Fb), hemocytes (Hc), midgut (Mg), and integument (Ig) of challenged larvae, respectively. (D) The transcription level of ApPPO in fat body immune challenged at different time points (0, 12, 24, 48, and 72 h). The group of tissues from naive larvae was extracted as negative controls. F: The transcription of cecropins in response to rApPPO induction in vivo. Samples of rApPPO in insect saline were injected into the fifth instar larvae for 24 h. Semi-quantitative RT-PCR was performed to test the mRNA expression levels of cecropin B and D in A. pernyi fat body from the samples of rApPPO injected or not. Larvae without injection served as negative controls. Antheraea pernyi ribosomal protein S8 (rps8) was used as an internal reference control. The average relative expressions were representative of three independent repeats ± 1 SD.

increasing from 5 to 50 μg. The PO activity of rAppPO was approximate 2.4 times (1 μg), 3.1 times (5 μg), and 7.4 times (50 μg) of that produced by the BSA group at the same content, respectively. Similar results were also detected when the larvae hemolymph was immune challenged by other soluble PAMPs (data not shown). Since the experiment result above provide a hint that rApPPO could be activated by immune-challenged hemolymph, we put forward a hypothesis that bacteria could be killed by the melanins formed from quinones that are catalyzed by activated rApPO. To confirm this hypothesis, radial diffusion assay was performed. After cultured overnight at 37°C, the antibacterial activity against E. coli and S. aureus was examined by measuring the diameter of clearzones. Detailed result against E. coli is shown in Figure 4F: the antibacterial activity in samples of BSA at different contents hardly changed, which was as high as that of native hemolymph without immune challenged (0 μg). The samples of 1 μg rApPPO led to Archives of Insect Biochemistry and Physiology


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Figure 4. Characteristic of rApPO. (A) Optimal enzyme pH of rApPO; (B) optimal enzyme temperature of rApPO; (C) substrate specificity of rApPO; Substrate used were a: 4-methyl catechol, λmax 528 nm; b: MH, λmax 520 nm; c: L-dopa, λmax 490 nm; d: dopamine, λmax 475 nm; e: catechol, λmax 410 nm; f: pyrogallic acid, λmax 334 nm. (D) Enzyme kinetics analysis of rApPO; a: The kinetic properties of rApPO on pyrogallic acid; b: The kinetic properties of rApPO on dopamine; (E) PO activity assay of rApPPO induced by the larval hemolymph of Antheraea pernyi. 5 μl laminarin and different amounts of rApPPO (0, 1, 5, and 50 μg) were mixed with the larval cell-free hemolymph, after the substrate was added, the PO activity was monitored as Fig. 3A. The samples pre-incubated with the same amount of BSA instead of rApPPO were examined as negative control. (F) Radial diffusion assay. The antibacteria ability of rApPO activated by the immune challenged hemolymph was observed by measuring the diameters of clearing zones with scale lupe and expressed in units (1 mm = 10 units). As the comparison group, the samples pre-incubated with the same amount of BSA instead of rApPPO was also examined. Each point represents mean ± SD (n = 3).


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approximate twice antibacterial ability of that produced by the BSA group at the same content. The antibacterial ability was about 2.6 times and 8.2 times of that produced by the BSA group at the contents of 5 μg and 50 μg, respectively. Result against S. aureus was similar to that of E. coli, so the data are not shown in this article. At the same time, a phenomenon was observed that the solid culture medium color of clear zones was much black and sticky than that of bacteria growth area, which further provided evidence that melanins formed to inhibit the growth of bacteria. Anyway, from another point of view, whether another kind of antibacterial substance was induced by activated rApPO besides melanins? With this purpose, semi-quantitative RT-PCR was performed to evaluate the mRNA levels of cecropin B and D in A. pernyi in response to rApPPO induction (Fig. 3D). Though both cecropin B and D were expressed at a normal physiological needed concentration in a fat body, a slight increase of transcription rate in larvae 24 h post injection was observed. In the control group, injection of BSA with the same amount of rApPPO did not stimulate this transcription increase (data not shown).

DISCUSSION PPO-activating system is an essential innate immune response against microbial infections in invertebrates (Lei et al., 2010). Studies of PPO, the vital enzyme involved in this system, provide indispensable material basis for elucidating the molecular mechanism of immune defense response (Gupta et al., 2005). PPO cDNA has been isolated and well analyzed from lepidopteron, such as Galleria mellonella (Li et al., 2002), B. mori (Kawabata et al., 1995), and Hyphantria cunea (Park et al., 1997). In this study, we identified and characterized a cDNA clone of PPO from the lepidoptera A. pernyi, which is one of the most important economical insects in China. Consistent with other PPOs, ApPPO contains two copper-binding motifs (region I and II), a thiol ester-like motif (region III) and a conserved nonfunctional region (region IV; Fig. 1). The six histidine residues for the copper-binding motif A was found in positions 209, 213, and 239, and those for the copper-binding motif B was present at positions 370, 377, and 406 (Fig. 2A). Moreover, sequence around the putative activation site, 50 NRFG53 , was conserved in other eight lepidoptera PPOs sequences, except for CfPPO1 (data not shown). For Holotrichia diomphalia (H. diomphalia) and M. sexta PPOs, there are another putative cleavage sites for serine proteases, which are located between R162 and A163 for H. diomphalia PPO1 and M. Sexta PPO1, Q163 and A164 for H. diomphalia PPO2, and M. sexta PPO, respectively (Jiang et al., 1997; Kim et al., 2002). But this motif was not observed in BmxPPOs. (Kawabata et al., 1995). In ApPPO, besides the common cleavage site, Arg50 -Phe51 , there was another potential cleavage site as M. sexta PPO1, R162 -A163 . But whether it is necessary for ApPPO activation needs further analysis in the future. As most lepidopteron, none of the PPOs has a signal peptide for secretion, but the glycosylation modification varied in different species. Most lepidopteran PPOs such as M. sexta, Plutella xylostella (Du et al., 2010), and A. pernyi in this article are nonglycosylated, which is consistent with their lack of binding capability to concanavalin A. Though there were five possible N-linked glycosylation sites, B. mori PPOs do not harbor any detectable carbohydrate either (Kawabata et al., 1995; Yasuhara et al., 1995). However, this result was different from that obtained from G. mellonella PPO, which binds to concanavalin A and lentil lectin (Kopácek et al., 1995). Sequence analysis via the ClustalW multiple sequence alignment program showed highly sequence similarity between ApPPO and PPOs of B. mori (74.74%), B. mandarina (74.45%), S. frugiperda (74.60%), M. separata(72.99%), H. virescens (73.72%), M. sexta Archives of Insect Biochemistry and Physiology


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(72.3%), P. interpunctella (71.74%), G. mellonella (70.66%), and C. fumiferana (70.07%), whereas the amino acid sequence of ApPPO is only 46% identical with M. sexta PPO2 (Hall et al., 1995) and 47.42% with B. mori PPO2, respectively (Kawabata et al., 1995). All the results above suggested that ApPPO is likely to be a member of the PPO1 family, rather than PPO2. Furthermore, the phylogenetic analysis revealed that ApPPO was distinctly far away from the Tortricidae family PPOs (C. fumiferana), Pyralidae family (G. mellonella), Zygaenidae family (P. interpunctella), and Noctuidae family (H. virescens and M. separata; Fig. 2B). In insects, circulating hemocytes has been viewed as the unique source of PPO (Ashida and Brey, 1998). But in B. mori and other lepidopteron, oenocytoids were also reported to produce PPO (Strand, 2008; Lu et al., 2014a). Nowadays, the distribution of PPOs in different tissues has been investigated in a variety of organisms, but results were not seemed to be concordant. In M. sexta, analysis of the hemocytes by both immunofluorescence labeling and hybridization in situ indicated that M. sexta PPOs were synthesized exclusively in oenocytoids, rather than in granular cells or plasmatocytes (Jiang et al., 1997). Similarly, it was shown in recent study that epidermal cells in the hindgut of B. mori also produce PPO (Shao et al., 2012); whereas in A. pernyi, ApPPO was an inducible immune protein that constitutively expressed in fat body of native larvae and the content could be upregulated during pathogen invasion. Yet, according to the result of semiquantitative RT-PCR, ApPPO was mainly expressed in hemocytes and fat body in response to immune challenges. Besides, inferior mRNA level was also observed in midgut. However, no band was detected from either control or induced integument. The result was roughly consistent with the conclusion that fat body was the tissue source of most insect plasma proteins (Iwama and Ashida, 1986). After immune challenged by formaldehyde-killed bacteria and fungi, cuticular melanization was observed from A. pernyi larvae at 24 h postinjection. The larvae began melanizing until death when the quantities of injected microorganism increased progressively. Comparison was carried out to observe the constitution difference of hemolymph between challenged and native larvae by SDS-PAGE under nonreducing condition. Interestingly, an additional band of high molecular weight (over 100 kDa) was observed in the challenged sample, which was speculated to be PO-containing complex as reported in other insect species. To further study the biochemical and structural property of ApPO, vast amount of purified zymogen was required. By now, many insect PPOs have been purified through ammonium sulfate fractionation and chromatography. For example, two isoforms of PPO were isolated from the pupae of D. melanogaster (Fujimoto et al., 1993) through three-step chromatography following ammonium sulfate fractionation. By using the similar purification methods, PPO isoforms were then obtained from the cuticles of B. mori (Asano and Ashida, 2000), the larval hemolymph of Sarcophaga bullata, A. aegypti, and Ostrinia furnacalis (Chase et al., 2000; Feng and Fu, 2004; Li et al., 2005; Feng et al., 2008), respectively. Most purified insect PPOs were identified as heterologous dimerization, but the enzyme purified from Holotrichia diomphalia appears to be homogeneity, which forms a dimer with only one kind of 79 kDa protein (Kwon et al., 1997). Nonetheless, the approaches of natural purification are so complicated that it does not meet the requirement for comprehensive characterization. At the same time, it was conceivably difficult to obtained natural purified ApPO due to its instability and viscidity. However, according to references, most insect PPOs were found to be nonglycosylated, which provided the feasibility to express recombinant PO in E. coli of its zymogen form (PPO). Recombinant PPO of Spodoptera litura had been expressed successfully in E. coli at 37°C, and three large quantities of recombinant D. melanogaster PPOs expressed in E. coli were obtained using Archives of Insect Biochemistry and Physiology

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modified conditions at 16°C for activation analysis (Li et al., 2012). Then the mechanism of PPO1 activation induced by α-chymotrypsin was well studied using recombinant D. melanogaster PPO1 obtained from E. coli as mentioned above (Lu et al., 2014a). Based on these contributions, in this study we expressed rApPPO in E. coli with Cu2+ added in the culture medium during induction at 37°C, and the biological activity of rApPPO was recovered by appropriate renaturation methods. PO activity assay was performed using both renatured rApPPO and native PPO in the larval hemolymph. Though the enzyme activity of rApPPO was lower than that of native PPO, yet scarcely any E coli-derived component was involved. In the immune behavior identification experiments, PO activity assay was carried out to test whether rApPPO could be activated by the PO-activating enzymes from hemolymph. The PO activity results were shown in details in the case of Laminarin pre-stimulated hemolymph (Fig. 4E). It was exactly detected that rApPPO was cleaved to the active form, rApPO. Though similar results were also detected in groups of other soluble PAMPs at the same content (data not shown), the activation levels differ from each other. On the whole, the hemolymph stimulated by Gram-positive bacteria (LTA and Lys-PGN) exhibited higher ability of rApPPO activation than that of fungi (laminarin and mannan), while the group of Gram-negative bacteria (LPS and DAP-PGN) showed the lowest activity. Since the signal pathway of PPO-activating cascade in A. pernyi has not been clear, the reason for this different activity is yet to be explained. However, we put forward two possibilities: (1) The PPO-activating system response is discriminatory to the same amount of PAMPs. Once the same amount of different PAMPs was recognized specifically by relative patternrecognition proteins, different signaling pathways were finally delivered to the PPOactivating cascade, leading to varying degrees of PO activity. It was summarized that the system is more sensitive to the Gram-positive bacteria and fungi than to the Gram-negative bacteria. (2) The PPO-activating system is complicated in that many PPAFs and SPHs are involved. After PAMPs were recognized by pattern-recognition proteins, different PPAFs were activated and subsequently stimulate the activation of rApPPO. As was reported, the PPO-activating system and Toll signaling pathway was crossed in some species (An et al., 2009), which provides a possibility that the change of members in PPO-activating system could inflect Toll signaling pathway directly or indirectly. In this study, as products of Toll pathway, both cecropin B and D were transcriptional induced by rApPPO injection (Fig. 3F). To further certify the relevance of PO with Toll signaling pathway, the transcription level of Spätzle in response to rApPPO injection will be studied next.

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Molecular cloning and expression analysis of a hemolin-like molecule from Antheraea pernyi.

Morphological and molecular characterization of Nosema pernyi, a microsporidian parasite in Antheraea pernyi.

Identification and Characterization of a Novel Microvitellogenin from the Chinese Oak Silkworm Antheraea pernyi.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

Structural and functional characterization of the actin-1 gene promoter from the Antheraea pernyi (Lepidoptera: Saturniidae).

Nucleation of hydroxyapatite on Antheraea pernyi (A. pernyi) silk fibroin film.

Understanding the variability of properties in Antheraea pernyi silk fibres.

Mineralization and biocompatibility of Antheraea pernyi (A. pernyi) silk sericin film for potential bone tissue engineering.

DNA sequence organization in the lepidopteran Antheraea pernyi.

Genome Sequence of Enterococcus pernyi, a Pathogenic Bacterium for the Chinese Oak Silkworm, Antheraea pernyi.

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): purification and characterization.

Molecular characterization and phylogenetic analysis of the eukaryotic translation initiation factor 4A gene in Antheraea pernyi (Lepdoptera: Saturniidae).

Interspecific hybrids of Antheraea roylei and A. pernyi - a cytogenetic reassessment.

Genetic characterization of a novel Iflavirus associated with vomiting disease in the Chinese oak silkmoth Antheraea pernyi.

The complete mitochondrial genome of Antheraea pernyi strain 731 (Lepidoptera: Saturniidae).

Zn superoxide dismutase in the Chinese oak silkworm (Antheraea pernyi).

Powdered Wound Dressing Materials Made from wild Silkworm Antheraea pernyi Silk Fibroin on Full-skin Thickness Burn Wounds on Rats.

Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation.

N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

Spermine-modified Antheraea pernyi silk fibroin as a gene delivery carrier.

DNA complexes for targeted gene delivery in HEK 293 and HCT 116 cells.

Inhibitors of eicosanoid biosynthesis influencing the transcripts level of sHSP21.4 gene induced by pathogen infections, in Antheraea pernyi.

A short-type peptidoglycan recognition protein from the silkworm: expression, characterization and involvement in the prophenoloxidase activation pathway.