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Prophenoloxidase-activating Proteinase-2 from Hemolymph ofManduca sexta

2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês

10.1074/jbc.m205743200

1083-351X

Haobo Jiang, Yang Wang, Xiao‐Qiang Yu, Michael R. Kanost,

Insect symbiosis and bacterial influences

Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invading pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new serine proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating proteinase-2 (PAP-2), differs from another PAP, previously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg51but yielded a product that has little phenoloxidase activity. However, in the presence of two serine proteinase homologs, active phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The serine proteinase homologs associate with a bacteria-binding lectin in M. sextahemolymph, indicating that they may be important for ensuring that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or hemocytes, but it became abundant in these tissues after the insects were injected with bacteria. Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invading pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new serine proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating proteinase-2 (PAP-2), differs from another PAP, previously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg51but yielded a product that has little phenoloxidase activity. However, in the presence of two serine proteinase homologs, active phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The serine proteinase homologs associate with a bacteria-binding lectin in M. sextahemolymph, indicating that they may be important for ensuring that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or hemocytes, but it became abundant in these tissues after the insects were injected with bacteria. phenoloxidase prophenoloxidase-activating proteinase precursor of proprophenoloxidase-activating proteinase prophenoloxidase-activating enzyme prophenoloxidase-activating factor serine proteinase homolog prophenoloxidase diisopropyl fluorophosphate acetyl-Ile-Glu-Ala-Arg-p-nitroanilide 1-phenyl-2-thiourea matrix-assisted laser desorption ionization concanavalin A high performance liquid chromatography IEARpNa-hydrolyzing enzyme Phenoloxidase (PO)1 is implicated in several defense mechanisms in insects, including cuticle sclerotization and melanotic encapsulation (1Ashida M. Yamazaki H. Molting and Metamorphosis. 1st Ed. Japan Scientific Society Press, Tokyo1990: 239-265Google Scholar, 2Sugumaran M. Söderhäll K. Iwanaga S. Vasta G. New Directions in Invertebrate Immunology. 1st Ed. SOS Publications, Fair Haven, NJ1996: 355-374Google Scholar, 3Ashida M. Brey P.T. Molecular Mechanisms of Immune Responses in Insects. 1st Ed. Chapman & Hall, London1998: 135-172Google Scholar). Quinones produced by phenoloxidase may also participate in wound healing and killing of sequestered parasites and pathogens (4Nappi A.J. Vass E. Adv. Exp. Med. Biol. 2001; 484: 329-348Crossref PubMed Scopus (78) Google Scholar). To minimize detrimental effects of the reactive intermediates to host tissues and cells, arthropod phenoloxidases known so far are all produced as inactive proenzymes and require specific proteinases for proteolytic activation. Activation of proPO in insects is probably mediated by a serine proteinase cascade, analogous to the coagulation pathway and complement system in human plasma (1Ashida M. Yamazaki H. Molting and Metamorphosis. 1st Ed. Japan Scientific Society Press, Tokyo1990: 239-265Google Scholar, 2Sugumaran M. Söderhäll K. Iwanaga S. Vasta G. New Directions in Invertebrate Immunology. 1st Ed. SOS Publications, Fair Haven, NJ1996: 355-374Google Scholar, 5Sugumaran M. Kanost M.R. Parasites and Pathogens of Insects. 1st Ed. Academic Press, Inc., New York1993: 317-342Crossref Scopus (80) Google Scholar, 6Kanost M.R. Jiang H. Wang Y., Yu, X., Ma, C. Zhu Y. Adv. Exp. Med. Biol. 2001; 484: 319-328Crossref PubMed Scopus (54) Google Scholar). Components of this proteinase system in insects may be already present in circulating hemolymph or released from hemocytes or fat body when pathogens or parasites are encountered. Recognition of invading microorganisms or aberrant host tissues may trigger the autoactivation of the first proteinase in the pathway, leading to the sequential activation of other components in the system through limited proteolysis. Prophenoloxidase-activating proteinase (PAP) (also known as prophenoloxidase-activating enzyme (PPAE)) is the terminal enzyme that directly converts proPO to PO. The reactions catalyzed by phenoloxidase can then result in melanization of foreign organisms trapped in capsules or hemocyte nodules (7Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1116) Google Scholar). Presumably, protein-protein interactions ensure that the defense response occurs near the site of infection. Inhibitors of serine proteinases and phenoloxidases may further reduce unwanted damage caused by the active enzymes (8Kanost M.R. Dev. Comp. Immunol. 1999; 23: 291-301Crossref PubMed Scopus (371) Google Scholar). The molecular mechanisms of proPO activation have been investigated in several insect species. We have isolated a PAP from a cuticular extract of the tobacco hornworm, Manduca sexta (9Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar). This serine proteinase, now renamed PAP-1, hydrolyzes a synthetic peptidyl-p-nitroanilide substrate but requires another protein factor for generating active PO. A cDNA clone for PAP-1 was obtained from an M. sexta hemocyte library. Sequence comparison indicated that the protein belongs to a family of arthropod serine proteinases containing a clip domain (10Jiang H. Kanost M.R. Insect Biochem. Mol. Biol. 2000; 30: 95-105Crossref PubMed Scopus (326) Google Scholar, 11Gorman M.J. Paskewitz S.M. Insect Biochem. Mol. Biol. 2001; 31: 257-262Crossref PubMed Scopus (149) Google Scholar). In this paper, we report the purification and characterization of a second serine proteinase from M. sexta hemolymph that activates proPO. To distinguish it from PAP-1 isolated previously from cuticles (9Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar), we designate this new proteinase PAP-2. Molecular cloning of PAP-2 indicates that it is most similar to the silkworm PPAE and has two clip domains at its amino terminus (12Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The PAP-2 zymogen is present at a higher level in hemolymph of M. sexta larvae that were challenged with killed bacteria. PAP-2 requires serine proteinase homologs from M. sexta plasma (13Yu, X-Q., Jiang, H., Wang, Y., and Kanost, M. R. (2003)Insect Biochem. Mol. Biol., in pressGoogle Scholar) as cofactors for proPO activation. Since the serine proteinase homologs associate with immulectin-2, a C-type lectin isolated from Manducahemolymph (13Yu, X-Q., Jiang, H., Wang, Y., and Kanost, M. R. (2003)Insect Biochem. Mol. Biol., in pressGoogle Scholar, 14Yu X-Q. Kanost M.R. J. Biol. Chem. 2000; 275: 37373-37381Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 15Yu X-Q. Kanost M.R. Adv. Exp. Med. Biol. 2001; 484: 191-194Crossref PubMed Scopus (27) Google Scholar), they may serve as anchoring/auxiliary factors for PAP-2 so that proPO activation only occurs as a local defense response against nonself. M. sexta eggs were originally purchased from Carolina Biological Supply, and larvae were reared on an artificial diet (16Dunn P. Drake D. J. Invertebr. Pathol. 1983; 41: 77-85Crossref Scopus (185) Google Scholar). Pharate pupae with metathoracic brown bars were chilled and dissected for hemolymph collection. When all tissues were cautiously removed from the integument with a spatula, hemolymph was pooled and collected carefully with a 1-ml pipetter, avoiding contaminating tissue fragments. This method yields significantly greater amounts of hemolymph than cutting a proleg of pharate pupae, because their hemolymph volume is quite low. Individual hemolymph samples (0.8–1 ml/insect) were immediately mixed with 100% saturated ammonium sulfate (pH 7.0) to prevent the rapid melanization of hemolymph, which occurs at this developmental stage. The ammonium sulfate was adjusted to 50% saturation, and the suspension was stored at −70 °C. Hemolymph collected this way contains many active proteinases and is stable for at least 2 years. For the bacterial induction experiment, fifth instar M. sexta larvae (day 2) were injected with Micrococcus luteus (10 μl/larva, 10 μg/μl; Sigma), formalin-killed Escherichia coli XL1-blue (108 cells/larva, 10 μl), or H2O (10 μl) as a negative control. proPO was purified from M. sexta larval hemolymph as described previously (17Jiang H. Wang Y., Ma, C. Kanost M.R. Insect Biochem. Mol. Biol. 1997; 27: 835-850Crossref PubMed Scopus (156) Google Scholar). Column fractions (10 μl) were mixed with 0.5 μg of proPO and 20 mm Tris-HCl, 5 mmCaCl2, pH 7.5, to a final volume of 20 μl. The reaction mixtures were incubated in microplate wells on ice for 60 min. PO activity was measured by adding 200 μl of 2 mm dopamine in 50 mm sodium phosphate, pH 6.5, to each sample well. Absorbance at 470 nm was then monitored continuously using a microplate reader (Molecular Devices). One unit of PAP activity is defined as the amount of enzyme yielding PO that produces an increase of 0.001 absorbance units/min. Amidase activity was assayed by using acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (A0180; Sigma) as a chromogenic substrate. Samples of column fractions (10–20 μl) were mixed with 200 μl of 50 μm substrate in 0.1m Tris-HCl, 0.1 m NaCl, 5 mmCaCl2, pH 7.8. One unit of activity is defined as ΔA 405/min = 0.001. Since a detailed, optimized purification scheme is presented below, we briefly describe here how PAP-2 was first isolated from M. sexta hemolymph. Unless otherwise specified, conditions for separation remained the same in the first and second PAP-2 purification. The plasma (42.5 ml) from bar stage prepupae was first fractionated with 15–40% saturation of ammonium sulfate and then dialyzed before separation on a hydroxylapatite column. Fractions were assayed for PO and proPO-activating activities. The fractions that activated proPO were pooled, concentrated in a Centriplus-30 (Millipore Corp.), and applied to a Sephacryl S100-HR column. Since proPO-activating activity was low in this and the following steps, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide hydrolysis (Sigma) was used to monitor an amidase activity (IEARase) that activates proPO only in the presence of other protein factors. Combined fractions of IEARase were passed through concanavalin A (ConA)-Sepharose 4B (5 ml; AmershamBiosciences) and Jacalin-agarose columns (5 ml; Vector Laboratories) to remove minor proteins that are difficult to separate in the final step. The unbound fraction from the lectin affinity columns was concentrated in a Centriplus-30 concentrator, and buffer exchange was carried out in the same centrifugal filter device. The sample was separated by HPLC on an MA7Q anion exchange column (Bio-Rad). Fractions containing the IEARase activity were individually concentrated in a Microcon-30 concentrator (Millipore Corp.). The partially purified IEARase (PAP-2) was treated with SDS-sample buffer containing 2-mercaptoethanol and separated by electrophoresis on a 12% polyacrylamide gel (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The protein was then transferred to a polyvinylidene difluoride membrane and stained with Amido Black (Sigma). The 35-kDa polypeptide, which corresponded to a [3H]DFP-labeled band detected as described by Jianget al. (9Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar), was subjected to automated Edman degradation. To determine the amino-terminal sequence of its 25-kDa light chain, purified PAP-2 (2 μg) was mixed with 1 μg of pyroglutamate aminopeptidase (Roche Molecular Biochemicals) in 100 μl of 100 mm sodium phosphate (pH 8.0) containing 10 mmEDTA, 5 mm dithiothreitol, 5% glycerol (v/v), and 1 mm p-aminobenzamidine. The reaction was carried out at 4 °C for 18 h followed by 25 °C for 4 h. The proteins in the mixture were precipitated by trichloroacetic acid at a final concentration of 10%. After centrifugation, the pellet was washed with cold anhydrous acetone and dissolved in 20 μl of the SDS sample buffer at 95 °C for 5 min. After gel separation and transfer, the first six residues of the 25-kDa band were determined as described above. λ phage DNA (0.1 μg) isolated from a bacteria-induced fat body cDNA library was used as a template in a PCR to amplify a PAP-2 cDNA fragment. Two degenerate primers were designed based on the amino-terminal sequence of PAP-2 catalytic domain: 660 (5′-ACA GCC ATC GAY CAR TAY CCN TGG-3′) and 661 (5′-G CTG GCG CTG ATH GAR TAY CAY AA-3′), which encode TAIDQYPW and LALIEYHK, respectively. Primer 625 (5′-CAT GAG SGG RCC RCC SGA RTC NCC-3′) is the reverse complement of the sequence encoding GDSGGPLM, a highly conserved sequence around the active site serine residue in the chymotrypsin family of serine proteinases. In a first PCR, primer 660 was used with primer T7, which anneals with sequence in the cloning vector near the 3′-end of the inserted cDNA, under the following conditions: 94 °C, 30 s; 53 °C, 40 s; and 72 °C, 80 s for 30 cycles. The reaction product (1 μl) was used directly as a template for a second, nested PCR by using primers 661 and 625 under the same cycling conditions. After electrophoresis of the resulting products on a 1% agarose gel, a DNA band of the expected size (about 0.6 kb) was recovered and cloned into the pGem-T vector (Promega). After this clone was confirmed by DNA sequence analysis to encode PAP-2, the PCR-derived cDNA fragment was labeled by 32P and used as a probe to screen the induced M. sexta fat body cDNA library in λZAP II (Stratagene) (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 2.90-2.100Google Scholar). Positive clones were purified to homogeneity and subcloned by in vivo excision of pBluescript phagemids. Nucleotide sequence analysis was carried out using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Fat body and hemocyte total RNA samples were prepared according to Wang et al. (20Wang Y. Willott E. Kanost M.R. Insect Mol. Biol. 1995; 4: 113-123Crossref PubMed Scopus (41) Google Scholar), separated by electrophoresis in agarose gels containing formamide, transferred to nitrocellulose, and hybridized with 32P-labeled PAP-2 cDNA. A duplicate blot was hybridized with a cDNA for M. sexta ribosomal protein S3 (21Jiang H. Wang Y. Kanost M.R. Insect Mol. Biol. 1996; 5: 31-38Crossref PubMed Scopus (55) Google Scholar) as a loading control. To detect a possible change of proPAP-2 in larval hemolymph upon bacterial challenge,Manduca larvae were injected with buffer, Escherichia coli, or M. luteus. Cell-free hemolymph samples collected at 24 h after injection were resolved by electrophoresis on an SDS-polyacrylamide gel (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Immunoblot analysis was performed using the polyclonal antiserum to proPAP-2 as the first antibody. 2C. Ji, Y. Wang, J. Ross, and H. Jiang, manuscript in preparation. All procedures for purification of PAP-2 were carried out at 4 °C. A frozen bar stage hemolymph sample (40 ml) was thawed, and the protein precipitate was collected by centrifugation at 12,000 × g for 25 min. The pellet was resuspended in 80 ml of HT buffer (pH 6.8, 10 mm potassium phosphate, 0.5 m NaCl), supplemented with 0.001% 1-phenyl-2-thiourea and 0.5 mm p-aminobenzamidine. To remove the β-1,3-glucan recognition proteins in the hemolymph (22Ma C. Kanost M.R. J. Biol. Chem. 2000; 275: 7505-7514Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), curdlan (0.2 g) was incubated with the protein solution with gentle agitation for 10 min on ice. The reaction mixture was centrifuged at 15,000 × g for 30 min to remove the flocculent materials including curdlan. Saturated ammonium sulfate solution was slowly added to the supernatant to a final saturation of 35%. After centrifugation at 15,000 × gfor 30 min, the pellet was collected and dissolved in 20 ml of HT buffer. The fractionated plasma sample was dialyzed against the same buffer (1.0 liter for 8 h, twice), and the resulting particulate substances were removed by passing the sample through a syringe filter (0.45 μm, low protein binding; Fisher). The protein solution was diluted with equal volume of H2O and applied to a hydroxylapatite column (2.5-cm inner diameter × 7 cm; Bio-Rad) equilibrated with 0.5 × HT buffer. Following a washing step with 60 ml of 0.5× HT and 50 ml of 1× HT buffers, bound proteins were eluted at 0.5 ml/min for 4 h with a linear gradient of 20–150 mm potassium phosphate (pH 6.8), 0.5m NaCl. Fractions were analyzed by immunoblotting and an IEARpNa amidase assay. Active fractions containing PAP-2 were combined and precipitated with ammonium sulfate (60% saturation). The precipitate was collected by centrifugation at 15,000 ×g for 30 min, dissolved in 3.0 ml, 20 mmTris-HCl, 0.5 m NaCl, pH 7.5 (S100 buffer), and then immediately applied to a Sephacryl S100-HR column (2.5-cm inner diameter × 100 cm; Amersham Biosciences) equilibrated with the same buffer. The column was eluted with S100 buffer at a flow rate of 0.7 ml/min, and fractions were collected at 2.5 ml/tube after the first 100 ml. Fractions containing PAP-2 were pooled and supplemented with CaCl2 and MgCl2 at a final concentration of 1 mm each. The sample was loaded onto a ConA-Sepharose 4B column (5.0 ml) equilibrated with 20 mm Tris-HCl, 0.5m NaCl, 1 mm CaCl2, 1 mm MgCl2, pH 7.4 and washed with the same buffer until A 280 was lower than 0.05. The flow-through fraction was dialyzed against Q buffer (25 mm Tris-HCl, pH 7.5), supplemented with 0.5 mm p-aminobenzamidine (2 liters each time for 8 h, twice). After passing through a syringe filter, the dialyzed sample (45 ml) was applied to a UNO-Q6 column (6 ml; Bio-Rad) equilibrated by Q buffer. Following a washing step, the bound proteins were eluted with a gradient of 0–0.2 M NaCl in Q buffer at a flow rate of 1.0 ml/min for 30 min. This step was performed in a cold chamber using a Biologic Duo-Flow Protein Purification System (Bio-Rad). Fractions (0.5 ml/tube) were collected and analyzed by electrophoresis on a SDS-polyacrylamide gel (10%) followed by silver staining (23Switzer R.C. Merril C.R. Shifrin S. Anal. Biochem. 1979; 98: 231-237Crossref PubMed Scopus (840) Google Scholar) or immunoblot analysis. Affinity labeling with [3H]DFP and fluorographic detection of PAP-2 were carried out according to Skinner and Griswold (24Skinner M.K. Griswold M.D. Biochem. J. 1983; 209: 281-284Crossref PubMed Scopus (190) Google Scholar). Purified PAP-2 was stored at −70 °C for characterization and activity assays. The purified PAP-2 was desalted using a C18 zip tip (Millipore) and eluted with 70% acetonitrile, 0.1% trichloroacetic acid. The sample was mixed with an equal volume of saturated sinapinic acid matrix on a MALDI plate, air-dried, and subjected to mass determination on a Voyager Elite mass spectrometer (PerkinElmer Life Sciences) with delayed extraction. The spectra were calibrated using bovine serum albumin as an external standard. To determine the site at which PAP-2 cleaves proPO, 1.0 μg of proPO was incubated with 0.2 μg of PAP-2 on ice for 60 min, and the reaction mixture was desalted and eluted as described above. Molecular masses of peaks that were not present in the control spectra of proPO and PAP-2 were compared with calculated values of the amino-terminal peptides to determine the cleavage site in proPO. The SPH-1 and -2 were isolated from induced larval hemolymph according to Yu et al. (13Yu, X-Q., Jiang, H., Wang, Y., and Kanost, M. R. (2003)Insect Biochem. Mol. Biol., in pressGoogle Scholar). The protein preparation (10 μl) was incubated with 40 μl of 5 mmphenylmethylsulfonyl fluoride (Sigma) in 5 mmCaCl2, 25 mm Tris-HCl (pH 7.5) at 4 °C for 16 h to inactivate serine proteinases that might be associated with the SPHs. The treated sample was dialyzed against the same buffer without phenylmethylsulfonyl fluoride (100 ml/8 h each time, twice). To test the effect of M. sexta SPH-1 and SPH-2 on the activation of proPO, purified proPO was incubated with buffer, PAP-2, SPH-1 and -2, or a mixture of PAP-2 and the SPHs at 4 °C for 60 min. PO activity in the reaction mixtures was assayed, using dopamine as a substrate. As described in the legend to Fig. 6, the reaction mixtures were also subjected to electrophoresis followed by silver staining or immunoblot analysis using antiserum againstManduca proPO (diluted 1:2000) as the first antibody. To study whether PO activity is involved in the formation of SDS-stable PO polymers, we included 0.001% 1-phenyl-2-thiourea (PTU), a PO inhibitor, in some of the reaction mixtures and analyzed the samples similarly. Preliminary experiments indicated that active PO and proteinases are already present in the hemolymph of bar stage prepupalM. sexta larvae in the absence of microbial challenge. As a step toward understanding the prophenoloxidase-activating system inM. sexta hemolymph, we attempted to purify a PAP from plasma of bar stage insects. To preserve the enzyme activities and minimize protein cross-linking caused by active PO, we kept the plasma and column fractions at high salt concentrations in the presence of inhibitors of phenoloxidase and proteases. Based on our previous experience with the cuticular PAP (PAP-1) (9Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar), we employed three different assays to follow each purification step: proPO activation, [3H]DFP labeling, and an amidase assay using acetyl-Ile-Glu-Ala-Arg-p-nitroanilide as a substrate. Among the commercially available peptidyl-p-nitroanilide substrates, IEARpNa is most similar to the amino-terminal side of the putative activation site in M. sexta proPO-p1 (Leu-Ser-Asn-Arg51) and proPO-p2 (Leu-Asn-Asn-Arg51) (17Jiang H. Wang Y., Ma, C. Kanost M.R. Insect Biochem. Mol. Biol. 1997; 27: 835-850Crossref PubMed Scopus (156) Google Scholar). When the 15–40% ammonium sulfate fraction of the cell-free hemolymph was separated on a hydroxylapatite column, the proPO-activating activity roughly coincided with one of the IEARase peaks (data not shown). These fractions were pooled, concentrated, and then resolved by gel filtration chromatography on a Sephacryl S100-HR column (Fig. 1 A). We detected a major IEARase activity peak in fractions 30–34 as well as low levels of IEARpNa hydrolysis in some other column fractions. This amidase peak did not correspond to the proPO-activating activity found in fractions 10–16 (data not shown). The proPO-activating activity in all of the fractions accounted for only 5–10% of the total activity loaded onto the column. One possibility is that, like PAP-1 (9Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar), proPO activation is mediated by a complex of a proteinase and a protein cofactor, and separation of this complex led to the low activity of PAP-2. To test this hypothesis, we incubated aliquots of fraction 32 with a small amount of other column fractions for 1 h along with purified proPO. Fraction 32 did not contain PO activity, and it generated little active PO when incubated with proPO (Fig. 1 B). After fraction 32 was incubated with proPO in the presence of fraction 10–16, which contained only low levels of PO and proPO-activating activities, a large increase in PO activity was observed. This result supported the hypothesis that proPO activation requires PAP and a cofactor. Because high amidase activity was detected in fraction 32 but not in fraction 14, we concluded that the PAP is probably present in fraction 32 and that the cofactor is in fraction 14. Their molecular masses are estimated to be about 50 and 200–500 kDa, respectively, based on their elution volume from the Sephacryl S-100 column. The pooled fractions 30–34 from the gel filtration column were passed through a ConA-Sepharose column and a Jacalin-agarose column to remove some plasma glycoproteins. The flow-through fractions from the lectin affinity columns were concentrated and separated on an anion exchange HPLC column. A single small IEARase peak was present in fractions 26–28, and a [3H]DFP labeling experiment indicated that these fractions contained a single radioactive band at 35 kDa (data not shown). As shown on an SDS-polyacrylamide gel stained with Coomassie Blue, this band was prominent and well separated from other proteins (Fig. 1 C). This 35-kDa band and a 25-kDa band were shifted to a 48-kDa position under nonreducing conditions (see Fig. 5), which suggests that the proteinase, named PAP-2, is composed of a catalytic heavy chain (35 kDa) and a regulatory light chain (25 kDa) linked by a disulfide bond. The first 29 residues of the 35-kDa polypeptide were determined to be Ile-Leu-Gly-Gly-Glu-Ala-Thr-Ala-Ile-Asp-Gln-Tyr-Pro-Trp-Leu-Ala-Leu-Ile-Glu-Tyr-His-Lys-Leu-Ala-Glu-Ile-Lys-Leu-Met (Fig. 2). This sequence is similar to a region at the beginning of the catalytic domain in PPAE from the silkworm Bombyx mori (12Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The amino terminus of the 25-kDa light chain starts with a pyro-Glu residue, since, after deblocking with pyroglutamate aminopeptidase, the newly exposed sequence was determined to be Ala-(Cys)-Thr-Leu-Pro-Asn (Fig. 2).Figure 2Nucleotide and deduced amino acid sequence of M. sexta PAP-2. Amino acid residues, shown in one-letter codes, are aligned with the first nucleotide of each codon. The secretion signal peptide isunderlined. The double underlinedsequence of the mature protein was confirmed by deblocking and sequencing of the PAP-2 light chain (Fig. 1 C). The proteolytic activation site is indicated (∥), and the double-underlined sequence after that was determined by Edman degradation sequencing of the 36-kDa PAP-2 heavy chain. PutativeN- and O-linked glycosylation sites are marked (+). The catalytic residues at the active site are indicated withasterisks, and residues determining the primary specificity of PAP-2 are labeled (@). A polyadenylation signal (AATAAA,underlined) near the 3′-end of the coding region is probably used for generating a short form of the mRNA, which ends at nucleotide 1365 followed by a 19-nucleotide poly(A) tail.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We designed two degenerate primers encoding part of the amino-terminal sequence of the 35-kDa polypeptide. Using these primers along with a vector-specific primer (T7) and a degenerate primer based on a conserved sequence around the active site serine, we obtained a 0.6-kb cDNA fragment in a nested polymerase chain reaction and cloned it into a pGem-T plasmid vector. Sequence analysis confirmed that the cDNA encoded a sequence starting with Leu-Ala-Leu-Ile-Glu-Tyr-His-Lys-Leu-Ala-Glu-Ile-Lys-Leu-Met. The rest of the sequence is typical of a serine proteinase from the S1 family (25Rawlings R.D. Barrett A.J. Biochem. J. 1993; 290: 205-218Crossref PubMed Scopus (715) Google Scholar). Using the 32P-labeled cDNA fragment as a probe, we screened a bacteria-induced M. sexta larval fat body cDNA library. Approximately 1.0 × 103 positives were found in the first round screening of 6.0 × 105plaques, indicating that PAP-2 mRNA is abundant at 24 h after immune challenge. Plaque purification and in vivo excision of phagemids were carried out with 16 of the putative positive clones. Sequence analysis of their 3′ termini indicated that these clones are all identical except for clone 4. Therefore, we determined the complete nucleotide sequences of clone 2 (the longest) and clone 4. Clone 2 encompasses a 2299-nucleotide cDNA, which includes a complete open reading frame spanning nucleotides 34–1359 (Fig. 2). The same coding region was found in clone 4 with a few nucleotide substitutions, most of which do not alter the amino acid sequence. A significant difference occurs in the 3′-untranslated regions of the two sequences; clone 2 contains 915 nucleotides between the stop codon and poly(A) tail, but clone 4 has only six. Apparently, in clone 4 the AATAAA sequence near the en