Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases

Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immune-responsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 microg/ml and increases to 30-75 microg/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.


INTRODUCTION
Synthesis of melanin by insects and other arthropods in response to infection results from a proteolytic cascade triggered by microbial cell wall components (1)(2)(3). In this innate immune response, microbes are recognized as foreign when soluble pattern recognition proteins bind to bacterial peptidoglycan or lipopolysaccharide or fungal β-1,3 glucan, initiating activation of a series of proteinases. Phenoloxidase (PO), the final enzyme in this cascade, exists in hemolymph as an inactive zymoge n, prophenoloxidase (proPO), which is activated by a specific proteinase.
Active PO catalyzes the hydroxylation of monophenols to o-diphenols and oxidation of odiphenols to quinones that can polymerize to form melanin. Cytotoxic molecules produced in this process, including quinones and reactive oxygen intermediates, may kill the invading microorganisms that are trapped by melanin (4).
Our understanding of the proPO activation cascade is still incomplete, and the number of proteolytic steps in the pathway is unknown. However, serine proteinases that directly activate proPO, known as proPO activating enzyme (PPAE) or proPO-activating proteinase (PAP), have been isolated and cloned from several arthropod species, including the silkworm, Bombyx mori (5,6), the tobacco hornworm, Manduca sexta (7-9), a beetle, Holotrichia diomphalia (10,11), and a crayfish, Pacifastacus leniusculus (12,13). These enzymes are composed of a carboxylterminal serine proteinase domain and at least one clip domain at their amino-terminus. A specific proteolytic cleavage between the domains activates the proteinase zymogens, but the two resulting chains remain covalently linked by a disulfide bond (14). The activated proteinase cleaves proPO at a conserved Arg-Phe bond at approximately residue 50 and leads to its activation (6)(7)(8)(9)(10)(11)(12)(13).
Three PAPs from M. sexta have been identified and cloned as cDNAs. PAP-1 contains by guest on July 10, 2020 http://www.jbc.org/ Downloaded from encodes the reactive center region, serpin-1 variants with different reactive site sequences have different inhibitory selectivity (21). The serpin-1 gene is constitutively expressed in fat body at a high level and, to lesser degree, in the granular hemocytes (22). The serpin-1 proteins are secreted into the plasma. M. sexta serpin-2 is expressed in cytoplasm of granular hemocytes in response to bacterial infection, but its function is not yet understood (19). There is also evidence for the existence of additional serpins in M. sexta, which are possibly encoded by different genes and may regulate the cuticular proPO activation (23).
Here we report the molecular cloning and analysis of an immune-responsive serpin (serpin-3) from M. sexta. Serpin-3, whose expression is stimulated by microbial infection, was shown to block the PPO activation cascade and to inhibit M. sexta prophenoloxidase activating proteinases.

EXPERIMENTAL PROCEDURES
Insects -M. sexta eggs were originally obtained from Carolina Biological Supply.
Larvae were reared as described by Dunn and Drake (24).
Hemolymph was collected into microcentrifuge tubes at 0, 1, 2, 6, 12, 24, 32, and 48 h after injection by cutting the dorsal horn. A few crystals of 1-phenyl-2-thiourea were added to each tube to prevent hemolymph melanization. Hemocytes were removed by centrifugation at 12,000×g for 10 min at 4°C. For total RNA preparation, hemocytes and fat body were collected by guest on July 10, 2020 http://www.jbc.org/ Downloaded from 7 Serpin-3 was expressed in a one l culture of E. coli strain XL1blue carrying the recombinant plasmid. Serpin-3 was expressed in an insoluble form and therefore was purified under denaturing conditions by nickel-affinity chromatography (32). An aliquot of the purified recombinant serpin-3 (1.5 mg) was further resolved by preparative SDS-PAGE and used to produce a polyclonal rabbit antiserum.
Production of soluble serpin-3 in a baculovirus expression system -The BamHI-XhoI cDNA fragment, containing the complete open reading frame for serpin-3, was inserted into pFastBac1 (Invitrogen Life Technologies). A recombinant baculovirus was generated from the resulting plasmid and amplified according to the manufacturer's instructions. For producing soluble serpin-3, Spodoptera frugiperda Sf9 cells (2×10 6 cells/ml) in 250 ml Sf-900 II serumfree medium were infected with the virus at a multiplicity of infection of 4 and incubated at 27°C with shaking at 140 rpm for 72 h. The conditioned medium was clarified by centrifugation at 500×g for 10 min, and the supernatant was placed in dialysis tubing and concentrated to 28 ml by covering the tubing with polyethylene glycol 8000, then dialyzed twice against 1.0 liter buffer A (20 mM Tris-HCl, 10 mM NaCl, pH 7.5) overnight at 4°C. The protein solution was applied to a 12-ml Q-Sepharose column and washed with 60 ml buffer A. The bound proteins were eluted with linear gradients of 10-220 mM NaCl (150 ml) and then 220-500 mM NaCl (50 ml) in buffer A. Fractions (2 ml each) were analyzed by 10% SDS-PAGE (33) and immunoblotting us ing serpin-3 antiserum as the first antibody. Concentration of the purified protein was determined by SDS-PAGE analysis along with a series of diluted ovalbumin standards. Intensity of the protein bands was digitized using Kodak Digital Science 1D gel analysis software. The amino terminal sequence of serpin-3 was determined by automated Edman degradation as described before (7).

Regulation of proPO activation in hemolymph by serpin-3 -A preparation of hemolymph
proteins that contains the components of the proPO activation cascade (proPO activation fraction) was produced as described previously (21). Ten µg of M. luteus in 1 µl sterile saline was added to 100 µl of PPO activating fraction (diluted 1:5 in water). At different incubation times (0-60 min), 10 µl aliquots of the reaction mixture were added to 700 µl phenoloxidase substrate solution (2 mM 3-hydroxytyramine in 50 mM sodium phosphate, pH 6.8) to measure PO activity (37). To test the inhibition of proPO activation, serpin-3 at final concentrations of 0-61 µg/ml was added to 1µ5 diluted proPO activation fraction (10 µl), along with 1 µg M. luteus.
PO activity was determined after incubation for 10 min at room temperature.
For detecting the formation of serpin-PAP complex, purified serpin-3 was reacted with the purified PAP-1 or PAP-3 in buffer B at a molar ratio of 1:1 or 1:2 (serpin/proteinase). In by guest on July 10, 2020 http://www.jbc.org/ Downloaded from control samples, diisopropyl fluorophosphate (DFP) at a final concentration of 5 mM was mixed with PAPs prior to the addition of serpin-3. After incubation at room temperature for 10 min, the reaction mixtures were treated with SDS sample buffer at 95°C for 5 min and resolved by 10% SDS-PAGE. Proteins transferred onto a nitrocellulose membrane were subjected to immunoblot analysis using 1:3000 diluted antisera against PAP-1 (7), PAP-3 (9), or serpin-3 as the first antibody.
Detection of serpin-PAP complexes in hemolymph by affinity chromatography -A serpin-3 immunoaffinity column was prepared according to Harlow and Lane (38). Briefly, 2.4 ml protein A-Sepharose beads (Sigma) were incubated with 4.8 ml serpin-3 antiserum in 19.2 ml phosphate-buffered saline (PBS; 0.8% NaCl, 0.02% KCl, 0.144% NaH 2 PO 4 , 0.024% KH 2 PO 4 , pH 7.4) for 1 h at room temperature to allow binding of the antibodies to protein A. Following a washing step, covalent coupling of antibodies to protein A was carried out by adding 20 mM dimethylpimelimidate in 0.2 M sodium borate, pH 9.0. After incubation at room temperature for subjected to immunoblot analysis as described above. The M. sexta serpin-1 gene contains twelve alternate forms of exon 9, each of which encodes a different reactive center loop (18,20). The serpin variants generated by alternative splicing differ in their inhibitory selectivities (21). To test whether the serpin-3 gene might have

Purification of Recombinant Serpin-3 Protein from E. coli and insect cells-We
expressed recombinant serpin-3 with an amino-terminal hexahistidine tag in E. coli and purified it by nickel affinity chromatography under denaturing conditions (Fig. 3A). While the purified serpin-3 was used as an antigen for producing a polyclonal rabbit antiserum, it was inactive in by guest on July 10, 2020 http://www.jbc.org/ Downloaded from the inhibition assays even after renaturation (data not shown). In order to characterize and study the function of serpin-3, we expressed it in insect cells using a baculovirus system. Serpin-3 was secreted into the cell culture medium utilizing its own signal peptide. Concentrated cell culture supernatant was separated by anion exchange chromatography on a Q-Sepharose column, and serpin-3 eluted at ~150 mM NaCl at pH 7.5. The purified serpin-3 from the baculovirus expression system (Fig. 3B) was used for characterization and functional analysis in later experiments. It has an apparent molecular mass of 56.8 kDa as estimated by SDS-PAGE, which is larger than the value calculated from the deduced amino acid sequence (48.9 kDa). This difference may be a result of glycosylation. The first five amino acid residues of the purified serpin-3 were determined to be Asp-Asp-Val-Asp-Pro, confirming the prediction that the signal peptide ends at residue 20 ( Fig. 1). We examined the induction pattern of serpin-3 protein in hemolymph by immunoblotting (Fig. 5). The constitutive serpin-3 concentration was 5-12 µg/ml in naive larvae. In saline- inhibited the activation of proPO by 90% at 61 ng/µl and 50% at 17 ng/ µl (Fig. 6). This result indicates that serpin-3 inhibits at least one proteinase of the proPO cascade.

Inhibition of prophenoloxidase activating proteinases-We tested whether inhibition of
PAPs by serpin-3 may explain the observed decrease in proPO activation. Recombinant M. sexta PAP-1 or PAP-3 were incubated with recombinant serpin-3 at different enzyme/inhibitor molar ratio s. The residual amidase activity decreased linearly as serpin-3 concentration increased, and complete inhibition occurred at a serpin/enzyme ratio of 1.6 for PAP-1 and 1.9 for PAP-3 (Fig.   7). We determined the second-order association rate constants (k assoc ) for inhibition of PAP-1 and PAP-3 by serpin-3 as an indicator of inhibitory selectivity. Serpin-3 had a k assoc of 7.5×10 5 M -1 s -1 for PAP-1 and 6.9×10 5 M -1 s -1 for PAP-3. Thus, serpin-3 inhibits PAP-1 and PAP-3 at rates comparable to those observed for other serpins in mammals and arthropods acting on their physiological target proteinases (Table 1).
In the inhibition reaction between a serpin and a susceptible proteinase, an inhibitor/enzyme complex is formed that is stable in SDS. Such complexes were detected when we incubated serpin-3 with either PAP-1 or PAP-3 (Fig. 8). The interaction of serpin-3 and PAP-1 resulted in the formation of a ~90 kDa complex under reducing conditions, which was absent from the controls lanes with only PAP-1 or serpin-3. This complex was recognized by both serpin-3 antibody and PAP-1 antibody ( Fig. 8A and B), indicating that it was composed of these two proteins. The combined apparent molecular mass of serpin-3 (residues 1-388) (51.5 kDa) and the PAP-1 catalytic domain (34 kDa) is close to the size of the complex (~90 kDa).
When the molar ratio of PAP-1 to serpin-3 was increased from 1:1 to 2:1, more of the serpin-3 was converted to the complex form (Fig. 8B, lanes 2 and 3). A band corresponding to a lower M r protein (51.5 kDa) was also detected by serpin antibody. Since this band was not present in the control of serpin-3 alone, it probably represents the cleaved serpin without the 5.3 kDa carboxyl-terminus (residues 389-435). This is consistent with the observation that more PAP-1 led to more of the cleaved form of serpin-3. The complex formation was blocked when an irreversible serine proteinase inhibitor, DFP, was preincubated with PAP-1 before addition of serpin-3 to the reaction mixture (Fig. 8B, lane 4). This result indicates that active PAP-1 is Next, we tested whether serpin-3 can inhibit and form a stable complex with the PAPs under more physiological conditions. Hemolymph collected from naive M. sexta larvae was incubated for 5 min with M. luteus to stimulate proPO activation. Serpin-3, constitutively present at a basal level, may inhibit the activated PAPs and form serpin-proteinase complexes during this process. After 5 min, remaining active serine proteinases were then inactivated with 5 mM DFP. These samples were applied to an immunoaffinity column made with serpin-3 antibody to isolate any complex formed (as well as free serpin-3). The proteins that bound to the affinity column were analyzed by immunoblotting using either PAP or serpin-3 antibodies.
Immunoblot analysis using PAP-1 antibodies revealed a ~90 kDa band, which is present at a much lower intensity in the control, untreated sample (Fig. 9A). Similarly, PAP-3 antibodies detected a band at about the same size in M. luteus-treated hemolymph, which was hardly detected in the control (Fig. 9B). This result demonstrated that in plasma, the activated PAP-1 and PAP-3 resulting from the bacteria-triggered activation of the PPO cascade can be inhibited by serpin-3, with consequent formation of a stable serpin-3-PAP complex. The complex in naive hemolymph may be due to a trace amount of enzyme that was spontaneously activated during hemolymph collection. The same samples were also examined using serpin-3 antibodies, which revealed two bands at the size range of serpin-3-proteinase complexes (Fig. 9C). We have not yet determined from this experiment which band represents which proteinase complex.

DISCUSSION
In the present study, we characterized the newly discovered serpin-3 from M. sexta hemolymph. It is constitutively expressed at a low level in fat body and secreted into hemolymph. After injection of bacteria or yeast, the serpin-3 mRNA and protein levels increase dramatically. We found that serpin-3 efficiently inhibits PAP-1 and PAP-3 and forms covalent complexes with the purified enzymes and with endogenous PAP-1 and PAP-3 in plasma. The in vivo half-life of PAP-1 or PAP-3, calculated as 1/k assoc ×[I], is 6-15s in naive larval hemolymph and 0.9-2.8s in induced larva l hemolymph, suggesting that serpin-3 could rapidly inhibit PAP-1 and PAP-3 under physiological conditions. Taken together, these data provide strong evidence that PAP-1 and PAP-3 are natural targets of serpin-3 and that serpin-3 blocks the proPO activation cascade by inhibiting the PAPs specifically and rapidly. Preliminary experiments suggest that PAP-2 may also be regulated by serpin-3 (Jiang et al., unpublished results). Several insect inhibitors that block the proPO cascade have been reported (21, 39-41, 46, 47), but the natural proteinase targets of these inhibitors have not been assigned. To our knowledge, this is the first report to provide direct evidence that an insect serpin can block the proPO activation cascade by specifically and rapidly inhibiting PAPs from the same species.
It is not surprising that PAP-1 and PAP-3 share a regulator because the enzymes are similar and they cleave the same substrates. They have an overall amino acid sequence identity of 42%, and many key residues are conserved. PAP-2 and PAP-3 cleave the two subunits of by guest on July 10, 2020 http://www.jbc.org/ Downloaded from proPO at Arg 51 within the sequence Asn-Arg-Phe-Gly (7,8,37,48). Serpin-3 contains Asn-Lys-Phe-Gly at its predicted P2-P2' position, which is strikingly similar to the proPO cleavage site.
This sequence similarity may explain the structural basis for the highly specific and efficient binding between the PAPs and serpin-3 The cleavage site of proPOs from different insect species and a crayfish are highly conserved with an occasional substitution of (P2) Asn to Ser or Thr and (P2') Gly to Ser (39).
Serpins with a P2-P2' sequence similar to the proPO activation site, perhaps homologs of serpin-3, may be present in other insect species to regulate proPO activation. Indeed, an immuneresponsive H. cunea serpin, which is 60% identical to serpin-3, was shown to inhibit proPO activation (39). Its target protease was not identified. Drosophila serpin 27A, which is 40% identical to serpin-3 and which has the same P2-P2' residues, is also immune inducible and inhibits activation of proPO (40,41). M. sexta serpin-3, H. cunea serpin, and Drosophila serpin 27A all have an extended amino-terminal sequence that is not present in most other serpins.
Spn27A mutants have higher PO activity than wild type flies, they develop melanized tissues, and they react to injury with uncontrolled melanization (40,41). The natural target of Spn27A is unknown, but the serpin inhibits a prophenoloxidase activating enzyme (PPAE) purified from the beetle Holotrichia diomphalia, suggesting that Spn27A may inhibit a Drosophila PPAE (40). M. sexta serpin-1J is another serpin that blocks proPO activation (21) and forms complexes with PAP-1, -2, and -3 (Wang et al.,unpublished results;9). The reactive site of serpin-1J is DRCC, which is less similar to the proPO cleavage sequence than the serpin-3 P2-P2' sequence. Once kinetic data for serpin-1J inhibition of the PAPs are available, we can compare the inhibitory efficiency and specificity of serpin-1J and serpin-3.