Serine protease SP105 activates prophenoloxidase in Asian corn borer melanization, and is regulated by serpin-3

Melanization reaction, resulting from the activation of prophenoloxidase, is a vital immune response in insects for encapsulating and killing the invasive organisms. Prophenoloxidase needs to be proteolytically activated by its upstream prophenoloxidase-activating protease (PAP) in melanization. Identification and characterization of PAPs facilitates the understanding of the molecular mechanisms involved in insect immunity. We here cloned a full-length cDNA for a serine protease, named as SP105, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of SP105 encodes 424-amino acid residue protein with a 19-residue signal peptide. Sequence comparison indicates that SP105 is most similar to Manduca sexta PAP3, a defined prophenoloxidase-activating protease. qRT-PCR analysis showed that SP105 mRNA levels increased significantly after a bacterial injection. Recombinant SP105 directly cleaved and activated Asian corn borer prophenoloxidase and therefore acted as the prophenoloxidase-activating protease. Additionally, SP105 formed SDS-stable complexes with a serine protease inhibitor, serpin-3, and its activity in activating prophenoloxidase was efficiently inhibited by serpin-3. Our work thus illustrated a prophenoloxidase-activating protease and revealed its regulation by serpin-3. The results would allow further advances in the understanding of the melanization in Asian corn borer and other insects.


Results
Molecular cloning and sequence analysis of Asian corn borer SP8 and SP105. We have identified 13 potential clip domain serine proteases from Asian corn borer larvae 43 . Among them, serine protease 8 (SP8) was predicted to mediate the menalization response in Asian corn borer 44 . As a first step to characterize SP8's function, we designed primers on the basis of the known transcriptome 43 and tried to clone SP8 in full length nucleotide sequence. Interestingly, we also identified another serine protease gene, nominated as SP105, in addition to the expected SP8. The cDNA sequences of SP8 and SP105 were successfully submitted to NCBI, with GenBank accession number as KT751522 and KT751521, respectively. They shared 82.4% identity in nucleotide acid sequences. The conceptual protein deduced from nucleotide sequence of SP8 and SP105 consists of 424 amino acid residues, including a predicted 19-residue secretion signal peptide. There are one and two putative N-linked glycosylation sites, and eleven and eight potentially O-linked glycosylation sites in deduced SP8 and SP105, respectively (Fig. 1A). The calculated molecular mass and isoelectric point of the mature protein are 43.1 kDa and 6.38 for SP8, 43.6 kDa and 6.35 for SP105, respectively.
Asian corn borer SP8 and SP105 are each composed of two amino-terminal clip domains connected by a linker region to a carboxyl-terminal S1 family serine protease domain containing a catalytic triad consisting of His, Asp, and Ser residues (Fig. 1). They are most similar in amino acid sequence to M. sexta PAP3, a clip domain serine protease directly activating prophenoloxidase in melanization process 13 (Fig. S1). The predicted proteolytic activation sites (↓ ) are located at ADNK 163 ↓ ITGG 167 in both SP8 and SP105 (Fig. 1B). The important determinants of the enzyme specificity are predicted to be Asp 368 , Gly 395 , and Gly 406 in SP8 and SP105 (Fig. 1A), suggesting they are trypsin-like protease cleaving its substrate after arginine or lysine residue 45 .
As mentioned above, we successfully obtained two full-length cDNA sequences during amplifying Asian corn borer SP8. However, three clones in nine sequenced SP8 samples had a mutant residue from Cys 38 to Arg 38 , which is absolutely conserved in all defined clip domains. Additionally, recombinant SP8 with Arg 38 failed to be activated with unknown reasons (Fig. S2). Therefore, we only focused on SP105 in the studies that followed.
Gene expression profiles of Asian corn borer SP105. We analyzed the mRNA levels of Asian corn borer SP105 in the various development stages, different tissues, or different pathogen inducements using qRT-PCR methods. SP105 transcripts in fifth instar larvae were significantly more than that in other developmental stages. Although SP105 expression level remained consistent in three, fourth instar larvae and pupae, it was still significantly higher than in the egg, first and second instar larval stage ( Fig. 2A). In different tissues, SP105 was expressed at significantly higher levels in hemocytes than in head, gut, and fat bodies. SP105 transcripts increased up to 14 folds in hemocytes (Fig. 2B). Moreover, qRT-PCR assay showed that SP105 mRNA levels increased significantly in the larva challenged by Escherichia coli, Micrococcus luteus or Beauveria bassiana conidia (Fig. 2C).
Purification and activation of recombinant proSP105 Xa . In order to investigate the potential function of SP105 in Asian corn borer, we produced active SP105 in vitro. Considering SP105 is expressed as a zymogen (proSP105) and its endogenous activating enzyme is currently unknown, we produced a recombinant form of proSP105 (proSP105 Xa ) in Sf9 cells. In proSP105 Xa , the predicted activation site was manually mutated from ADNK 163 to IEGR 163 to permit its activation by commercially available bovine Factor Xa. Recombinant proSP105 Xa was secreted into medium using its own secretion signal peptide. SDS-PAGE analysis indicated that purified proSP105 Xa had an apparent mass of ~50 kDa, approximately 7 kDa larger than that predicated based on its cDNA sequence (Fig. 3A). The increased mass is likely due to glycosylation, as the protein sequence contain ten putative glycosylation sites (Fig. 1A).
Incubation of purified proSP105 Xa with Factor Xa resulted in decreased intensity of the 50-kDa zymogen band and the appearance of a 38-kDa band corresponding to the catalytic domain of proSP105 Xa (Fig. 3B), as expected for activation cleavage. After conditioned medium containing recombinant wild type proSP105 with 6× His tag was incubated with Asian corn borer plasma, the new band representing the catalytic domain of proSP105 appeared at the same position (38 kDa) (Fig. S3). It suggests that the cleavage of proSP105 Xa by Factor Xa could simulate the activation of proSP105 in vivo. Additionally, the activation of proSP105 Xa by Factor Xa was confirmed by testing whether activated SP105 Xa could hydrolyze the colorimetric substrate IEARpNA. As shown in Fig. 3C, proSP105 Xa lacked IEARase activity, but after the zymogen was activated by Factor Xa, IEARase activity increased significantly above that of Factor Xa alone, which could also hydrolyze the substrate. These results indicated that Factor Xa cleaved and activated proSP105 Xa . SP105 Xa activated native and recombinant Asian corn borer PPO. When purified proSP105 Xa zymogen was added into Asian corn borer plasma, the 38-kDa band corresponding to the catalytic domain was detected (Fig. S4A), although the putative activation sites in proSP105 Xa had been mutated from original ADNK 163 to IEGR 163 . The addition of recombinant proSP1 Xa , another Asian corn borer serine protease with IEGR 114 as activation sites 44 , also led to the appearance of 33-kDa product corresponding to the catalytic domain of proSP1 Xa (Fig. S4B). It seems that unknown protease in Asian corn borer plasma possessed partial activity of Factor Xa to cleave its downstream protease after IEGR residues, and therefore could weakly activate proSP105 Xa .
After Asian corn borer plasma was incubated with Factor Xa alone, the antibodies against Asian corn borer PPO2 identified an 80-kDa band corresponding to PPO2 zymogen. The incubation of Asian corn borer plasma with proSP105 Xa zymogen resulted in the appearance of a new band with low intensity at 72-kDa corresponding to Asian corn borer PO2, and a slight increase in PO activity (Fig. 4A,B). After plasma was incubated with Factor Xa-activated proSP105 Xa , the 72-kDa band corresponding to Asian corn borer PO2 was observed at higher intensity, the reaction mixture turned black, and PO activity increased significantly (Fig. 4A,B). It indicates that active SP105 Xa causes cleavage and activation of Asian corn borer PPO2. The position of the peptide bond cleaved during activation is indicated by red arrow. In proSP105 Xa , the putative activation site was changed from ADNK to IEGR. Three glycine and six histidine residues were added to the carboxyl terminus of SP105 and shown in bold.
To explore whether PPO is a substrate of SP105, we incubated SP105 Xa and recombinant Asian corn borer PPO2. As in Asian corn borer plasma, western blot analysis of purified PPO2 detected a band with apparent molecular weight of 80 kDa. No change was observed after incubation with proSP105 Xa zymogen. The addition of Factor Xa alone into plasma led to the weak appearance of 72-kDa band corresponding to Asian corn borer PO2, but no significant change in PO activity, suggesting the weak cleavage of recombinant PPO2 by Factor Xa. However, when active SP105 Xa was incubated with purified PPO2, the band of 72 kDa was observed with strong intensity and PO activity of the mixture increased significantly (Fig. 4C,D).

SP105 Xa was directly inhibited by Asian corn borer serpin-3.
In a previous study, we identified a serine protease inhibitor, serpin-3, which regulates melanization cascade in Asian corn borer 46 . To investigate whether serpin-3 functioned via inhibiting SP105, we firstly checked whether serpin-3 could form an SDS-stable complex with SP105 Xa because the formation of such complex is a characteristic feature of serpin-target protease reaction 30 . We mixed SP105 Xa with recombinant serpin-3, and detected the appearance of a high molecular weight complex by western blot, using antibodies against His or serpin-3. Anti-His serum recognized both proSP105 Xa zymogen and serpin-3 as ~53-kDa band, which migrated to the same apparent position in SDS-PAGE (Fig. 5A, left panel). The incubation of recombinant serpin-3 with Factor Xa alone resulted in the appearance of two bands at ~48 and 45 kDa, possibly due to the nonspecific degradation of serpin-3 by Factor Xa. However, when serpin-3 was mixed with Factor Xa-activated SP105 Xa , the intensity of the 38-kDa band corresponding to the catalytic domain of proSP105 Xa decreased, and a new immunoreactive band at ~92-kDa position (the expected size of a serpin-3/SP105 complex) was observed, which was also recognized by antibody against serpin-3 ( Fig. 5A, right panel).
To confirm that this complex formation indeed leads to the inhibition of SP105, we tested the IEARase activity of SP105 Xa in the presence of serpin-3. SP105 Xa 's activity decreased linearly as serpin-3 concentration increased (Fig. 5B). The stoichiometry of inhibition was 2.4, indicating that under the experimental conditions serpin-3 almost exclusively acted as an inhibitor rather than a substrate of SP105.
Since SP105 could cleave Asian corn borer PPO2, inhibition of SP105 by serpin-3 theoretically would suppress its cleavage of the substrate PPO2. To test this hypothesis, we incubated Asian corn borer PPO2 with Factor Xa-activated SP105 Xa in the absence or presence of serpin-3. When SP105 was pre-treated with serpin-3, more PPO2 was detected as 80-kDa band corresponding to PPO2 zymogen, and the 72-kDa band representing active PO2 decreased in intensity (Fig. 5C). The PO activity of the reaction mixture containing serpin-3 decreased significantly (Fig. 5C, right panel). Taken together, SP105 was inhibited by serpin-3.

Discussion
Melanization of invading microbes is one important strategy that protects insects against infection 1-3 . In melanization cascade, prophenoloxidase (PPO)-activating protease (PAP) functions as the terminal protease to convert PPO into active phenoloxidase (PO) 12,13,15,20,21 . Although PAPs have been investigated for many years, understanding of PAPs in most insects is still incomplete. Here, we identified a serine protease gene, SP105, from Asian corn borer during amplifying expected SP8. Recombinant SP105 protein directly cleaved and activated Asian corn borer PPO2 in vivo and in vitro, which suggests that SP105 acts as a PAP in Asian corn borer melanization. In addition, we clarified that SP105's activity was regulated by a serine protease inhibitor, serpin-3.
So far, veritable PAPs have only been determined in some model insects such as A. gambiae 20 , B. mori 23 , D. melanogaster 9,24 , H. diomphalia 25 , M. sexta 12,26 , and T. molitor 16,19 (Fig. S1). In our previous work, we revealed that Asian corn borer SP13 directly cleaved D. melanogaster PPO1, and therefore potentially functioned as a PAP in Asian corn borer 44 . In this study, we resolved the previous problem and successfully obtained soluble Asian corn borer endogenous PPO, which meant we could perform experiments to check whether SP105 enable to cleave and activate its conspecific PPO. The results indicated that SP105 indeed activated native and recombinant Asian corn borer PPO2 (Fig. 4), and works as a PAP in PPO activation pathway. Therefore, we have identified two PAPs in Asian corn borer larvae so far. Similar situation also existed in M. sexta, in which three PAPs (PAP-1/-2/-3) have been characterized 12,26 . However, only one PAP was reported in other insects except for these two insect species. Based on the findings from Asian corn borer and M. sexta, we postulate that there should be more PAPs to be identified than currently illustrated in most insects. It ought to be common that multiple PAPs cooperatively activate PPOs in the melanization reactions, considering the key roles of PO in the melanization and the importance of the melanization against the microbial infection in insects.
In addition, it is notable that Asian corn borer SP105 also cleaved D. melanogaster PPO (Fig. S5). Consistently, Asian corn borer SP13 activated native and recombinant D. melanogaster PPO 44 , and A. gambiae CLIBP9 activated M. sexta PPO 20 . It suggests that the cleavage of PPO by PAP is not completely restricted in the conspecific insects. The possible reason is that PPOs from heterogeneous insects have the identical or highly conserved putative activation cleavage sites 33,46 , and its upstream activating protease recognizes and cleaves the same cleavage site without regard for the resource of PPO substrate. Therefore, in some small-size insects with limited amount of hemolymph for PPO purification or in the insects lacking sequence information for recombinant PPO production, it might be a reasonable strategy to make use of available heterologous PPO to clarify the putative function of a serine protease in PPO activation. Furthermore, some reports indicated that the proteases secreted from the invading microorganisms, such as fungal virulence factor Pr1, could proteolytically activate Persephone (Psh) protease in D. melanogaster Toll pathway 47,48 . It will be interesting to explore whether the secreted protease(s) from invading microbes also could directly activate host PPO since PAP cleaving PPO is not necessarily specific.
Although SP13 and SP105 redundantly function as PAP in Asian corn borer melanization response, there is still some remarkable differences between them. Firstly, SP13 and SP105 are typical clip-domain serine proteases containing type-2 clip domain with two helices between Cys-3 and Cys-4 27,28,43 (Fig. 1). Clip domains are proposed to be sites for interactions of proteases with their activators, cofactors, and substrates 3,27 . SP13, most similar in amino acid sequences to M. sexta PAP1, contains a single type-2 clip domain, whereas SP105 which has the highest similarity to M. sexta PAP3 contains two type-2 clip domains (Fig. S1). Secondly, SP13 zymogen was activated by SP1 in PPO activation cascade in Asian corn borer 44 . However, SP1 failed to cleave proSP105 in our reduplicative experiments (Fig. S6), suggesting that SP1 acts upstream of proSP13, but not of proSP105, in Asian corn borer melanization. Similar results have been also reported in M. sexta PPO activation response, in which proPAP1 and proPAP3 are involved in two branched pathways and is activated by two separate serine protease, HP6 and HP21, respectively 13,14 . Therefore, we conclude that SP105 and SP13 belong to different type of PAPs and propose a hypothesis for the PPO activation in Asian corn borer. More than one cascade contributes to the PPO activation in Asian corn borer upon the challenge of foreign microbes. In one branch, the microbial infection leads to the activation of unknown serine protease(s) which then activates proSP1. Active SP1 then processes proSP13, and active SP13 in turn activates PPO. In another branch, the invading of pathogens or parasites results in the sequential activation of a serine protease other than SP1, then proSP105 is proteolytically activated. Active SP105 further converts PPO to PO. Other cofactors such as serine protease homologs (SPHs) also participate in this process. SPHs are similar in amino acid sequence to S1 family serine proteases but lack proteolytic activity Factor Xaactivated SP105 Xa cleaved Asian corn borer PPO2 in plasma (A) and as purified protein (C), causing a significant increase in PO activity (B,D). In (A,C), the sizes and positions of the molecular weight markers are indicated on the right. His or Asian corn borer PPO2 antiserum was used as primary antibodies in immunoblot analysis. Circles, proSP105 Xa ; asterisks, catalytic domain of proSP105 Xa ; hollow diamonds, PPO2 zymogen; solid diamonds, cleaved and activated PO2. In (B,D), the bars represent mean ± S.D. (n = 6). Statistically significant differences are represented by different letters (one-way ANOVA followed by Newman-Keuls test, P < 0.05).
due to the mutation of the catalytic residues 49 . SPHs have been reported to be essential for the prophenoloxidase activation in M. sexta 50 and H. diomphalia 51 . We infer that there also exist similar SPH(s) in Asian corn borer to facilitate SP105 in activating PPO.
On the other hand, the activity of SP105 in cleaving PPO was blocked by serpin-3 (Fig. 5). In our previous report, serpin-3 also prevented SP13 from cleaving PPO 46 . It is not surprising that a single serpin regulates multiple target proteases. Actually, M. sexta serpin-3 inhibits all three PAPs in PPO activation cascade 38,40 . Serpin-1J in M. sexta not only regulates PAP-1 to -3 in melanization, but also restricts HP8 in Toll pathway 26,40,52 . Target specificity of inhibitory serpins is controlled by the sequence and tertiary structure of their reactive center loop, which allows the binding and cleavage by target proteases 32,33 . Serpin is thus kind of substrate of its target protease to some extent. We inferred that the serine proteases with the common downstream substrate would be potentially inhibited by the same serpin in insect immune response. More evidence will be presented to support this hypothesis as more serpin -target protease regulatory units are characterized in other insects. It also provides a useful (C) Serpin-3 inhibited the cleavage of recombinant PPO2 by SP105. Factor Xa-activated SP105 Xa (0.2 μ g) was incubated with a 1-fold molar excess of serpin-3, then incubated with recombinant Asian corn borer PPO2 (0.3 μ g). The mixtures were subjected to 7.5% SDS-PAGE and immunoblotting using His (left panel) or PPO2 (middle panel) antiserum. Circle, proSP105 Xa ; asterisk, catalytic domain of proSP105 Xa ; triangle, serpin-3; hollow diamond, PPO2 zymogen; solid diamond, activated PO2. PO activity of the mixtures (mean ± S.D., n = 4) was monitored using dopamine as substrate (right panel). Asterisk means that is significantly different from the control (unpaired t test, two-tailed p < 0.05).
Scientific RepoRts | 7:45256 | DOI: 10.1038/srep45256 clue to seek more cognate proteases for a specific serpin, or to identify the putative serpin for a serine protease which has identical substrate preference to the known protease in a defined serpin-protease unit.

Molecular cloning and sequence analysis of Asian corn borer SP8 and SP105. In our previous
study, a clip domain serine protease gene, SP8, was predicted to mediate the melanization response in Asian corn borer 44 . Based on its cDNA sequence from assembled Asian corn borer transcriptome 43 , primers (Table S1) were designed to amplify the full-length cDNA encompassing the entire reading frame using cDNA from the whole body of fifth instar larvae collected 20 h after injection with 3 μ l of M. luteus (3 μ g/μ l). The products were cloned into pMD19-T vector, and the nucleotide sequences were confirmed by DNA sequencing. In addition to expected SP8, another clip domain serine protease gene with 82.4% identity to SP8 in nucleotide sequences was obtained during this process. It was designated as SP105.
We carried out a series of sequence analysis for identified SP8 and SP105. The deduced amino acid sequences were obtained by using the Translate tool provided by the Swiss Institute Bioinformatics. Analysis of deduced amino acid sequences, including prediction of signal peptide, molecular weight, isoelectric point, and glycosylation sites, was executed in the EXPASY (Expert Protein Analysis System) proteomics server (http://www. expasy.org). Multiple sequence alignment was performed by using the CLUSTALW program, along with the same region in other invertebrate serine proteases with defined functions. The information about the sequences (with GenBank accession number) used for the alignment were found in the figure legend for Fig. S1. Phylogenetic trees were constructed by the neighbor-joining method using MEGA Version 5 software 53 . For neighbor-joining method, gaps were treated as characters, and statistical analysis was performed using the bootstrap method with 1000 replicates.

Quantitative reverse transcriptase (qRT)-PCR analysis of the expression profiles of SP105.
To investigate the transcriptional changes of SP105 during the various stages of Asian corn borer, total RNA samples were individually prepared (n = 5) from three different stages including egg, larvae, and pupa using TRNzol Reagent (TIANGEN). One μ g of RNA equally from 5 individual RNA samples in each stage was treated with DNase I and converted into first-strand cDNA using FastQuant RT Kit (TIANGEN). The cDNA products independently from 3 biological replicates were diluted 10-fold for use as template in qRT-PCR experiments. Specific primers were designed and listed in Table S1. Asian corn borer ribosomal protein L8 (rpL8) was used as an internal standard to mormalize the expression levels. The qRT-PCR was performed on a Applied Biosystems 7500 Real-Time PCR System (Life Technologies) using the SYBR Premix EX Taq TM Kit (TAKARA), according to the manufacturer's instructions. The thermal cycling conditions for qRT-PCR were 95 °C for 30 s followed by 40 cycles of 95 °C for 30 s and 60 °C for 34 s, then 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s, and 60 °C for 15 s. The relative expression of genes was calculated using 2 −ΔΔCt method 54 .
To determine the expression patterns of SP105 in different tissues of Asian corn borer, total RNA samples were isolated separately from combined heads, midguts, fat bodies, and hemocytes from 20 day 0 fifth instar larvae. The synthesis of first-strand cDNA and qRT-PCR analysis was performed as described above.
To check the expression profiles of SP105 under different inducement conditions, day 1 fifth instar larvae from the same batch were injected into the hemocoel with 3 μ l of sterile water containing formaline-killed E. coli DH5α (2 × 10 5 cells/μ l), dried M. luteus (3 μ g/μ l), B. bassiana suspension (2 × 10 5 conidia/μ l, B. bassiana conidia suspension was prepared as described previously 55 ), or sterile water as a control. After 20 h (10 h for B. bassiana treatment), each five larvae from challenged and control group were collected, and total RNA samples were individually prepared. The following qRT-PCR analysis was conducted as described above.

Preparation of recombinant Asian corn borer proSP105 and prophenoloxidase-2 (PPO2).
To produce recombinant proSP105, a cDNA fragment encoding the entire proSP105 coding region including the signal peptide was amplified by PCR using primers listed in Table S1 and cDNA from M. luteus-injected larvae. The forward primer included a BamH I site, and the reverse primer contained three codons for glycine and six codons for histidine residues followed by a stop codon and a NotI site. The PCR product was cloned into pMD19-T vector and then digested with BamH I and NotI (TaKaRa). The digested product was recovered by agarose gel electrophoresis, and then inserted into the corresponding restriction sites in pFastBac1 vector (Invitrogen). The resulting proSP105 plasmid, after sequence confirmation, was used as template to produce mutant proSP105 (proSP105 Xa ) plasmid according to Chiu's method 56 . In proSP105 Xa , the predicted activation site ADNK 163 was replaced with IEGR 163 to allow the cleavage and activation by commercially available bovine Factor Xa 14 . After sequence verification, the resulting plasmids were used to generate a recombinant baculovirus using Cellfectin ® II Reagent (Invitrogen). For the production of proSP105 Xa , Sf9 cells (2 × 10 6 cells/ml) in 500 ml of Insect-Xpress protein-free medium (Lonza) were infected with the recombinant baculovirus at multiplicity of infection (MOI) of 3, then incubated at 28 °C with shaking at 150 rpm. The culture was harvested at 48 h post infection, and cells were removed by centrifugation at 5000 × g for 15 min at 4 °C. The cell-free medium was used to further purify recombinant proteins following the method described previously 44 .
For expressing recombinant Asian corn borer prophenoloxidase-2 (OfPPO2), the coding region of mature OfPPO2 was amplified by PCR using specific primers listed in Table S1, in which NcoI and XhoI sites were added into the 5′ -end of forward and reverse primers, respectively. The PCR products were digested and subcloned into the same restriction sites in the expression vector pET28a (Novagen). After sequence verification, the plasmids were used to transform E. coli strain BL21 (DE3). The OfPPO2 was then expressed and purified following the methods described previously 46 . Five milligram of purified OfPPO2 was used as antigen for the production of a rabbit polyclonal antiserum (Beijing CoWin Bioscience Co. Ltd.).
Activation of recombinant proSP105 Xa by Factor Xa. To test whether proSP105 Xa could be activate by Factor Xa, 0.25 μ g of purified recombinant proSP105 Xa was incubated with 0.2 μ g of bovine Factor Xa (New England Biolabs) in the reaction buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM CaCl 2 , pH 8.0) at 37 °C for 4 h. The mixtures were separated by 10% SDS-PAGE followed by immunoblot analysis with anti-His as primary antibodies.
The activation of proSP105 Xa was confirmed by measuring the amidase activity of activated SP105 Xa with 200 μ l of 50 μ M acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (IEARpNA) in 0.1 M Tris-HCl (pH 8.0), 0.1 M NaCl and 5 mM CaCl 2 as colorimetric substrate. The amidase activity was measured by monitoring changes in absorbance at 405 nm in a microplate reader (Bio-Tek Instrument, Inc.). One unit of amidase activity was defined as Δ A 405 / min = 0.001.

Assays of Asian corn borer PPO activation by SP105 Xa .
To measure the ability of recombinant SP105 in activating Asian corn borer PPO in vivo and in vitro, 0.2 μ g of proSP105 Xa or Factor Xa-activated SP105 Xa were incubated at 37 °C with 0.2 μ g of purified OfPPO2 or 0.5 μ l of plasma collected from day 1 fifth instar Asian corn borer larvae. In control reactions, active SP105 Xa was replaced with Factor Xa alone. One hour later, reaction mixtures were subjected to 7.5% SDS-PAGE and immunoblot analysis. To determine PO activity, 0.7 μ g of recombinant proSP105 Xa or SP105 Xa were mixed with 0.5 μ l of plasma. Additionally, 1.6 μ g of recombinant proSP105 Xa or SP105 Xa were incubated with 20 μ g of purified OfPPO2. After incubation at either room temperature for 10 min (plasma) or 37 °C for 30 min (recombinant OfPPO2), PO activity in the reaction mixtures was measured using dopamine as substrate 13 . One unit of PO activity was defined as the amount of enzyme producing an increase in absorbance (Δ A 490 ) of 0.001 per min.

Detection of SDS-stable serpin-3/SP105 complexes by immunoblot analysis.
Purified recombinant proSP105 Xa (0.2 μ g) was activated by Factor Xa as described above, and mixed with purified serpin-3 46 at molar ratio of 1:1 (proSP105:serpin-3). In control samples, proSP105 Xa or Factor Xa was omitted from the mixture. After incubation at room temperature for 30 min, the reaction mixtures were subjected to 10% SDS-PAGE and immunoblot analysis with mouse anti-His (1:2,000) or rabbit anti-serpin3 (1:500) as primary antibodies.

Analysis of SP105 inhibition by serpin-3 with IEARpNA as substrates.
To directly measure the inhibitory potential of serpin-3 on SP105 function, we incubated 0.3 μ g of SP105 Xa with recombinant serpin-3 at different molar ratios. In control reactions, the same amount of Factor Xa used to activate proSP105 Xa was substituted for active SP105 Xa . After incubation at room temperature for 30 min, the residual amidase activity was measured as described above. Amidase activity of SP105 Xa was defined as the activity of SP105 Xa minus the activity of Factor Xa alone. Analysis of SP105 inhibition by serpin-3 using OfPPO2 as substrates. Activated SP105 (0.2 μ g) was mixed with serpin-3 at a molar ratio of 5:1 (serpin-3:SP105). After incubation at room temperature for 30 min, 0.3 μ g of OfPPO2 was added to the reaction mixtures, and incubated at 37 °C for 1 h. The cleavage of OfPPO2 in the mixtures was visualized by immunoblot analysis using antiserum against OfPPO2 (1:500 diluted) or His tag (1:2,000 diluted). The residual PO activity in the mixtures was measured using dopamine as substrate as described above.