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• W1998778934 abstract "One innate immune response pathway of insects is a serine protease cascade that activates prophenol oxidase (pro-PO) in plasma. However, details of this pathway are not well understood, including the number and order of proteases involved. Protease inhibitors from the serpin superfamily appear to regulate the proteases in the pathway. Manduca sexta serpin-4 and serpin-5 suppress pro-PO activation in plasma, apparently by inhibiting proteases upstream of the direct activator of pro-PO. To identify plasma proteases inhibited by these serpins, we used immunoaffinity chromatography with serpin antibodies to isolate serpin-protease complexes that formed after activation of the cascade by exposure of plasma to bacteria or lipopolysaccharide. Covalent complexes of serpin-4 with hemolymph proteases HP-1 and HP-6 appeared in plasma activated by Gram-positive or Gram-negative bacteria, whereas serpin-4 complexes with HP-21 and two unidentified proteases were unique to plasma treated with Gram-positive bacteria. HP-1 and HP-6 were also identified as target proteases of serpin-5, forming covalent complexes after bacterial activation of the cascade. These results suggest that HP-1 and HP-6 may be components of the pro-PO activation pathway, which are activated in response to infection and regulated by serpin-4 and serpin-5. HP-21 and two unidentified proteases may participate in a Gram-positive bacteria-specific branch of the pathway. Several plasma proteins that co-purified with serpin-protease complexes, most notably immulectins and serine protease homologs, are known to be components of the pro-PO activation pathway. Our results suggest that after activation by exposure to bacteria, components of the pro-PO pathway associate to form a large noncovalent complex, which localizes the melanization reaction to the surface of invading microorganisms. One innate immune response pathway of insects is a serine protease cascade that activates prophenol oxidase (pro-PO) in plasma. However, details of this pathway are not well understood, including the number and order of proteases involved. Protease inhibitors from the serpin superfamily appear to regulate the proteases in the pathway. Manduca sexta serpin-4 and serpin-5 suppress pro-PO activation in plasma, apparently by inhibiting proteases upstream of the direct activator of pro-PO. To identify plasma proteases inhibited by these serpins, we used immunoaffinity chromatography with serpin antibodies to isolate serpin-protease complexes that formed after activation of the cascade by exposure of plasma to bacteria or lipopolysaccharide. Covalent complexes of serpin-4 with hemolymph proteases HP-1 and HP-6 appeared in plasma activated by Gram-positive or Gram-negative bacteria, whereas serpin-4 complexes with HP-21 and two unidentified proteases were unique to plasma treated with Gram-positive bacteria. HP-1 and HP-6 were also identified as target proteases of serpin-5, forming covalent complexes after bacterial activation of the cascade. These results suggest that HP-1 and HP-6 may be components of the pro-PO activation pathway, which are activated in response to infection and regulated by serpin-4 and serpin-5. HP-21 and two unidentified proteases may participate in a Gram-positive bacteria-specific branch of the pathway. Several plasma proteins that co-purified with serpin-protease complexes, most notably immulectins and serine protease homologs, are known to be components of the pro-PO activation pathway. Our results suggest that after activation by exposure to bacteria, components of the pro-PO pathway associate to form a large noncovalent complex, which localizes the melanization reaction to the surface of invading microorganisms. In the hemolymph of insects and crustaceans, microbial infection initiates a serine protease cascade, resulting in proteolytic activation of a prophenol oxidase (pro-PO) 1The abbreviations used are: pro-PO, prophenol oxidase; PO, phenol oxidase; HP, hemolymph protease; PAP, pro-PO-activating protease; SPHs, serine protease homologs; LPS, lipopolysaccharide; IML, immulectin; PGRP, peptidoglycan recognition protein; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SPC, serpin-protease complexes. zymogen (1Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall Ltd., London1997: 135-172Google Scholar, 2Jiang H. Kanost M.R. Insect Biochem. Mol. Biol. 2000; 30: 95-105Crossref PubMed Scopus (325) Google Scholar, 3Kanost M.R. Jiang H. Wang Y. Yu X.-Q. Ma C. Zhu Y. Adv. Exp. Med. Biol. 2001; 484: 319-328Crossref PubMed Scopus (54) Google Scholar, 4Jiang H. Wang Y. Kanost M.R. Adv. Exp. Med. Biol. 2001; 484: 313-317Crossref PubMed Scopus (10) Google Scholar). Activated phenol oxidase (PO) hydroxylates monophenols to o-diphenols and oxidizes o-diphenols to quinones, which can polymerize to form melanin at the injury site or around invading organisms (1Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall Ltd., London1997: 135-172Google Scholar, 5Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1111) Google Scholar). Quinones may also be involved in the production of cytotoxic molecules such as superoxides and hydroxyl radicals that could participate in killing pathogens or parasites (5Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1111) Google Scholar, 6Nappi A.J. Vass E. Adv. Exp. Med. Biol. 2001; 484: 329-348Crossref PubMed Scopus (77) Google Scholar). Three pro-PO activating proteases (PAPs) from the tobacco hornworm, Manduca sexta (7Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar, 8Jiang H. Wang Y. Yu X.-Q. Kanost M.R. J. Biol. Chem. 2003; 278: 3552-3561Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Jiang H. Wang Y. Yu X.-Q. Zhu Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 1049-1060Crossref PubMed Scopus (190) Google Scholar), are similar to pro-PO-activating enzymes or factors identified from the silkworm, Bombyx mori (10Satoh D. Horij A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), a beetle, Holotrichia diomphalia (11Lee S.Y. Kwon T.H. Hyun J.H. Choi J.S. Kawabata S-I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 254: 50-57Crossref PubMed Scopus (119) Google Scholar, 12Lee S.Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K-I. Natori S. Kawabata S.-I. Iawanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (109) Google Scholar), and from a crayfish, Pacifastacus leniusculus (13Wang R. Lee S.Y. Cerenius L. Soderhall K. Eur. J. Biochem. 2001; 268: 895-902Crossref PubMed Scopus (158) Google Scholar). These enzymes contain one or two clip domains (2Jiang H. Kanost M.R. Insect Biochem. Mol. Biol. 2000; 30: 95-105Crossref PubMed Scopus (325) Google Scholar) at their amino terminus and a carboxyl-terminal serine protease domain. They are activated by a specific proteolytic cleavage between the clip domain and the protease domain by unknown upstream proteases. For efficient activation of pro-PO, M. sexta PAPs and H. diomphalia PPAF-I require the presence of serine protease homolog(s) (SPHs) that lack proteolytic activity but function as a co-factor (7Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar, 8Jiang H. Wang Y. Yu X.-Q. Kanost M.R. J. Biol. Chem. 2003; 278: 3552-3561Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Jiang H. Wang Y. Yu X.-Q. Zhu Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 1049-1060Crossref PubMed Scopus (190) Google Scholar, 11Lee S.Y. Kwon T.H. Hyun J.H. Choi J.S. Kawabata S-I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 254: 50-57Crossref PubMed Scopus (119) Google Scholar, 14Yu X.-Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (206) Google Scholar, 15Kwon T.H. Kim M.S. Choi H.W. Joo C.H. Cho M.Y. Lee B.L. Eur. J. Biochem. 2000; 267: 6188-6196Crossref PubMed Scopus (144) Google Scholar). The SPHs have domain organizations similar to PAPs except that the active site serine residue in the protease-like domain is replaced by glycine. SPHs may also require proteolytic activation of pro-forms to make them functional (14Yu X.-Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (206) Google Scholar, 16Lee K.Y. Zhang R. Kim M.S. Park J.W. Park H.Y. Kawabata S. Lee B.L. Eur. J. Biochem. 2002; 269: 4375-4383Crossref PubMed Scopus (95) Google Scholar, 17Kim M.S. Baek M.J. Lee M.H. Park J.W. Lee S.Y. Soderhall K. Lee B.L. J. Biol. Chem. 2002; 277: 39999-40004Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Initiation of the pro-PO activation cascade in response to microbial infection is mediated by pattern recognition proteins that recognize pathogen-associated molecular patterns such as lipopolysaccharide (LPS), peptidoglycan, and β-1,3-glucan (1Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall Ltd., London1997: 135-172Google Scholar, 18Yu X.-Q. Zhu Y.-F. Ma C. Fabrick J.A. Kanost M.R. Insect Biochem. Mol. Biol. 2002; 32: 1287-1293Crossref PubMed Scopus (190) Google Scholar, 19Medzhitov R. Janeway C.A. Science. 2002; 296: 298-300Crossref PubMed Scopus (1674) Google Scholar). Two M. sexta C-type lectins (immulectins) bind LPS from Gram-negative bacteria and stimulate pro-PO activation in plasma (20Yu X.-Q. Gan H. Kanost M.R. Insect Biochem. Mol. Biol. 1999; 29: 585-597Crossref PubMed Scopus (219) Google Scholar, 21Yu X.-Q. Kanost M.R. J. Biol. Chem. 2000; 275: 37373-37381Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 22Yu X.-Q. Kanost M.R. Dev. Comp. Immunol. 2003; 27: 189-196Crossref PubMed Scopus (105) Google Scholar). Two β-1,3-glucan recognition proteins have also been characterized from M. sexta (23Ma C. Kanost M.R. J. Biol. Chem. 2000; 275: 7505-7514Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 24Jiang H. Ma C. Lu Z.-Q. Kanost M.R. Insect Biochem. Mol. Biol. 2004; 34: 89-100Crossref PubMed Scopus (115) Google Scholar). They bind to β-1,3-glucan from fungal cell walls and lipoteichoic acid (a cell wall component of Gram-positive bacteria) and stimulate pro-PO activation (23Ma C. Kanost M.R. J. Biol. Chem. 2000; 275: 7505-7514Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 24Jiang H. Ma C. Lu Z.-Q. Kanost M.R. Insect Biochem. Mol. Biol. 2004; 34: 89-100Crossref PubMed Scopus (115) Google Scholar). A peptidoglycan recognition protein (PGRP) that binds to peptidoglycan and initiates pro-PO activation in plasma has been characterized in B. mori (25Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 26Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 11854-11858Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). PGRPs have also been identified in other insects and arthropods (18Yu X.-Q. Zhu Y.-F. Ma C. Fabrick J.A. Kanost M.R. Insect Biochem. Mol. Biol. 2002; 32: 1287-1293Crossref PubMed Scopus (190) Google Scholar, 27Lee M.H. Osaki T. Lee J.Y. Baek M.J. Zhang R. Park J.W. Kawabata S. Söderhäll K. Lee B.L. J. Biol. Chem. 2004; 279: 3218-3227Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 28Kurata S. Dev. Comp. Immunol. 2004; 28: 89-95Crossref PubMed Scopus (41) Google Scholar, 29Werner T. Liu G. Kang D. Ekengren S. Steiner H. Hultmark D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13772-13777Crossref PubMed Scopus (446) Google Scholar). Insect plasma contains serine protease inhibitors, including members of the serpin superfamily, which regulate the pro-PO activation pathway. Serpins are proteins of ∼400 amino acid residues, with an exposed reactive center loop near their carboxyl terminus (30Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1064) Google Scholar, 31Gettins P.G.W. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar, 32Li J. Wang Z. Canagarajah B. Jiang H. Kanost M.R. Goldsmith E.J. Structure (Lond.). 1999; 7: 103-109Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 33Elliott P.R. Pei X.Y. Dafforn T.R. Lomas D.A. Protein Sci. 2000; 9: 1274-1281Crossref PubMed Scopus (171) Google Scholar). Serpins function as suicide-substrate inhibitors by forming stable covalent complexes with proteases after the cleavage of a scissile bond in the reactive center loop (30Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1064) Google Scholar, 31Gettins P.G.W. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar, 34Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar, 35Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (512) Google Scholar). The P1 residue located at the amino-terminal side of the scissile bond determines primary specificity of inhibition. In M. sexta, six serpins have been identified so far (36Kanost M.R. Prasad S.V. Wells M.A. J. Biol. Chem. 1989; 264: 965-972Abstract Full Text PDF PubMed Google Scholar, 37Jiang H. Wang Y. Kanost M.R. J. Biol. Chem. 1994; 269: 55-58Abstract Full Text PDF PubMed Google Scholar, 38Jiang H. Wang Y. Huang Y. Mulnix A.B. Kadel J. Cole K. Kanost M.R. J. Biol. Chem. 1996; 271: 28017-28023Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 39Gan H. Wang Y. Jiang H. Mita K. Kanost M.R. Insect Biochem. Mol. Biol. 2001; 31: 887-898Crossref PubMed Scopus (53) Google Scholar, 40Zhu Y. Wang Y. Gorman M.J. Jiang H. Kanost M.R. J. Biol. Chem. 2003; 278: 46556-46564Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 41Wang Y. Jiang H. Insect Biochem. Mol. Biol. 2004; 34: 387-395Crossref PubMed Scopus (69) Google Scholar, 42Jiang H. Kanost M.R. J. Biol. Chem. 1997; 272: 1082-1087Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Serpin-1J, serpin-3, and serpin-6 inhibit PAPs to regulate the last step of the pro-PO activation pathway (9Jiang H. Wang Y. Yu X.-Q. Zhu Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 1049-1060Crossref PubMed Scopus (190) Google Scholar, 40Zhu Y. Wang Y. Gorman M.J. Jiang H. Kanost M.R. J. Biol. Chem. 2003; 278: 46556-46564Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 41Wang Y. Jiang H. Insect Biochem. Mol. Biol. 2004; 34: 387-395Crossref PubMed Scopus (69) Google Scholar). Two new immune-responsive serpins, serpin-4 and serpin-5, have recently been identified. They are able to inhibit pro-PO activation to different degrees but are not efficient inhibitors of PAPs, indicating that they inhibit serine proteases upstream of PAPs in the activation cascade (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). However, it is still not known how many proteases are involved in the pro-PO activation pathway, how they are regulated, or how microbial components trigger the cascade pathway. In this study, we used M. sexta serpin-4 and serpin-5 to probe functions of proteases in the pro-PO activation pathway. Insects—M. sexta larvae were reared as described previously (44Dunn P.E. Drake D. J. Invertebr. Pathol. 1983; 41: 77-85Crossref Scopus (183) Google Scholar) from a laboratory colony originally obtained from Carolina Biological Supply. Immunoaffinity Purification of Serpin-Protease Complexes—Antibody-coupled protein A-Sepharose CL-4B beads (Sigma) were prepared according to Harlow and Lane (45Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar), using rabbit antisera to M. sexta serpin-4 or serpin-5 (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Hemolymph (20–30 ml) was collected from day 3 fifth instar larvae 24 h after injection with Micrococcus luteus or Escherichia coli (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and hemocytes were removed by centrifugation at 9000 × g for 15 min at 4 °C. The plasma was warmed to room temperature and adjusted to contain 10 mm diethylthiocarbonate and 1 mm phenylthiourea. Bacteria or lipopolysaccharide (LPS) was then added to the plasma to stimulate activation of protease cascades. Dried M. luteus (Sigma) was added (0.5 μg/μl) to plasma from larvae previously injected with M. luteus. Formaldehyde-killed E. coli XL-1 (1 × 108 cells/ml) or LPS from E. coli 026/B6 (0.01 μg/μl, Sigma) was added to the plasma from larvae induced by E. coli. After incubation for 30 min at room temperature, diisopropyl fluorophosphate (Sigma; final concentration 5 mm) and a protease inhibitor mixture (Sigma, P8849; 1 ml for 30 ml of plasma) were added to inactivate proteases. After 10 min, the mixture was centrifuged at 5000 × g for 15 min at 4 °C. The supernatant was mixed with 1–2 ml of protein A-Sepharose CL-4B beads coupled with serpin antibody overnight at 4 °C, and the mixture was then packed into a column. The column was washed with 20 volumes of 1 m NaCl and then 10 volumes of 10 mm sodium phosphate, pH 6.5. For purification of serpin-5 complexes from E. coli-treated plasma, these washing steps were replaced with 10 volumes of phosphate-buffered saline (PBS, 4.3 mm Na2PO4, 1.4 mm KH2PO4, 137 mm NaCl, 2.7 mm KCl, pH 7.4) and then 10 volumes of 0.5 m NaCl. The columns were eluted with 10 volumes of 100 mm glycine, pH 2.5, 10% ethylene glycol. Fractions (0.5 or 1 ml each, equivalent to 0.5 column volume) were collected into 50 or 100 μl (0.1 fraction volume) of 1 m sodium phosphate, pH 8.0. The fractions were analyzed by SDS-PAGE and immunoblotting (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), using rabbit antisera to the serpins (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and to M. sexta hemolymph proteases (HPs) HP-1 (AAB94557), HP-2 (AAB94558), HP-6 (AAV91004), HP-8 (AAV91006), HP-9 (AAV91007), HP-10 (AAV91008), HP-12 (AAV91010), HP-13 (AAV91011), HP-14 (AAR29602), HP-15 (AAV91012), HP-16 (AAV91013), HP-17 (AAV91014), HP-18 (AAV91016), HP-19 (AAV91017), HP-21 (AAV91019), and HP-22 (AAV91020) (46Jiang H. Wang Y. Kanost M.R. Insect Mol. Biol. 1999; 8: 39-53Crossref PubMed Scopus (44) Google Scholar, 48Ji C. Wang Y. Guo X. Hartson S. Jiang H. J. Biol. Chem. 2004; 279: 34101-34106Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Antisera to M. sexta immulectins (20Yu X.-Q. Gan H. Kanost M.R. Insect Biochem. Mol. Biol. 1999; 29: 585-597Crossref PubMed Scopus (219) Google Scholar, 21Yu X.-Q. Kanost M.R. J. Biol. Chem. 2000; 275: 37373-37381Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), pro-PO (49Jiang H. Wang Y. Ma C. Kanost M.R. Insect Biochem. Mol. Biol. 1997; 27: 835-850Crossref PubMed Scopus (155) Google Scholar), and serine protease homologs (14Yu X.-Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (206) Google Scholar) were prepared previously. Determination of Amino-terminal Sequences—Protein samples were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, stained with 0.025% Coomassie Blue R-250 in 40% methanol, and destained with 50% methanol. The protein bands of interest were excised and subjected to automated Edman degradation sequencing. Serpin-protease complexes were sequenced by the HHMI/Keck Biotechnology Resource Laboratory, Yale University. Other proteins were sequenced by the Biotechnology Microchemical Core Facility, Kansas State University. Mass Spectrometry Analyses—To identify serpin-4- and serpin-5-protease complexes after separation by SDS-PAGE, the bands were excised, reduced with dithiothreitol, alkylated with iodoacetamide, and then subjected to in-gel digestion with modified l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated porcine trypsin (Promega). The tryptic peptide pools were evaporated to near-dryness and desalted on C18 Ziptips (Millipore). The eluted peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in the Proteomics Center, University of Missouri, Columbia. Spectra were acquired in the positive ion delayed extraction reflector mode on a Voyager DEPro mass spectrometer (Applied Biosystems, Inc.). Spectra were calibrated with a six-peptide calibration standard mixture. The peptide masses were used to search the NCBI nonredundant protein sequence data base with ProFound (prowl.rockefeller.edu/) or MASCOT Peptide Mass Fingerprint (www.matrixscience.com/) programs. The mass values were also compared with the masses calculated for tryptic peptides derived from M. sexta hemolymph proteases, using the MS-Digest program of ProteinProspector version 4.0.5 (prospector.ucsf.edu/). Inhibition of Pro-HP and Pro-SPH Activation by Serpin-4 or Serpin-5—Samples of 2 μl of plasma were incubated at room temperature for 5 min in the presence or absence of recombinant serpin-4 or serpin-5 (final concentration 0.4 μg/μl), then activated by addition of M. luteus in saline (0.4 μg/μl), and incubated at room temperature for 10 min (43Tong Y. Kanost M.R. J. Biol. Chem. 2005; 280: 14923-14931Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Samples were then analyzed by SDS-PAGE and immunoblotting with available HP or SPH antibodies. Identification and Purification of Serpin-Protease Complexes—Serpin-4 and serpin-5 antibodies detected bands in untreated plasma representing intact serpins (∼50 kDa) and a minor, slightly smaller protein consistent with cleaved serpin (Fig. 1). After incubation with M. luteus, E. coli, or LPS, the intensity of the 50-kDa band was reduced, and the 45-kDa band increased, suggesting that some intact serpins were converted to the cleaved form. At the same time, higher molecular weight bands (∼70 kDa) containing serpin-4 or serpin-5 appeared, consistent with the expected size for serpin-protease complexes (Fig. 1). Apparently, the exposure to bacteria/LPS led to the activation of plasma proteases, which formed covalent complexes with serpin-4 and serpin-5. Similar changes occurred in plasma from naive larvae (data not shown), although all of the serpin bands were less intense due to lower concentration of serpin-4 and serpin-5 in plasma from naive animals. Treatment of plasma with M. luteus consistently stimulated the formation of more intense serpin-4-protease complex bands than did treatment with LPS. To identify the proteases inhibited by serpin-4 or serpin-5, we purified the serpin-protease complexes by immunoaffinity chromatography by using the serpin antibodies. Five serpin-4-protease complexes (SPC4-1–5) were isolated from M. luteus-treated plasma (Fig. 2). Two serpin-4-protease complexes, which had the same electrophoretic mobility as SPC4-1 and SPC4-5, were isolated from LPS-treated plasma (Fig. 2). The appearance of the multiple serpin-4-protease complex bands suggests that serpin-4 inhibited several plasma proteases. Two serpin-5-protease complexes (SPC5H and SPC5L) were isolated by immunoaffinity chromatography using serpin-5 antibody (Fig. 3). These two SPCs were present in plasma treated with M. luteus or E. coli.Fig. 3Purification and identification of serpin-5-protease complexes from plasma. Immunoaffinity-purified serpin-5-protease complexes (SPC5s) were subjected to SDS-PAGE and detected by silver staining or immunoblotting. A, silver-stained gel of purified serpin-5-protease complex (SPC5) fractions. B, immunoblots of SPC5 fractions. Lane 1, SPC5 fraction purified from M. luteus-treated plasma; lane 2, SPC5 fraction purified from E. coli-treated plasma. SPC5H/SPC5L, serpin-5-protease complex of high or low Mr; C, cleaved serpin. An uncomplexed HP-1 band is indicated by an arrowhead. The heavy chain of rabbit IgG is marked with *.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Other plasma proteins that co-purified with the complexes and serpin-4 or serpin-5 were not recognized by antibodies to the serpins. Based on their apparent masses, we named the most abundant of these proteins P42, P40, P37, and P34 (Figs. 2A and 3A). The association of these proteins with serpin-4 and serpin-5 was apparently not due to incomplete washing of the columns, because the abundance of these proteins in plasma is much lower than major hemolymph proteins (e.g. hexamerin storage proteins and lipophorin) that did not bind to the antibody columns. In controls using columns coupled with antibodies from preimmune sera, these proteins did not bind (data not shown), further indicating their specific interaction with serpin-4 and serpin-5 or with plasma proteins that interact with the serpins. Identification of Proteases in Serpin-Protease Complexes—We have cloned cDNAs for 25 M. sexta hemolymph proteases, including three PAPs and other HPs synthesized in fat body or hemocytes (7Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar, 8Jiang H. Wang Y. Yu X.-Q. Kanost M.R. J. Biol. Chem. 2003; 278: 3552-3561Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Jiang H. Wang Y. Yu X.-Q. Zhu Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 1049-1060Crossref PubMed Scopus (190) Google Scholar, 46Jiang H. Wang Y. Kanost M.R. Insect Mol. Biol. 1999; 8: 39-53Crossref PubMed Scopus (44) Google Scholar), 2H. Jiang, Y. Wang, and M. R. Kanost, unpublished results. and we prepared antibodies to 16 of these proteases. To test whether these proteases were present in the isolated serpin-4 and serpin-5 complexes described above, we analyzed fractions containing the complexes by immunoblotting, using the HP antibodies. Antibodies to proteases HP-1, HP-6, and HP-21 bound to complex bands SPC4-1, SPC4-5, and SPC4-4, respectively (Fig. 2B). The SPC4-4 band recognized by antibody to HP-21 was present in M. luteus-treated plasma but not in plasma treated with LPS (Fig. 2B), consistent with the result using serpin-4 antibody. Likewise, a 31-kDa band detected by HP-21 antibody (consistent in size with the catalytic domain of active HP-21) was detected only in M. luteus-treated plasma. These results indicate that HP-21 might be involved in a response specific to Gram-positive bacteria. In contrast, the putative catalytic domains of both HP-1 and HP-6 and their complexes with serpin-4 were detected in fractions obtained after treatment with LPS or M. luteus (Fig. 2B). None of the available HP antibodies recognized SPC4-2 or SPC4-3, which were present only in plasma samples activated by treatment with M. luteus. SPC5H (∼85 kDa) was recognized by antibodies to serpin-5 and HP-1, whereas SPC5L (∼70 kDa) was recognized by serpin-5 and HP-6 antibodies (Fig. 3B). These data suggest that serpin-5 regulates HP-1 and HP-6, which are activated in the presence of Gram-positive or Gram-negative bacteria. HP-1, HP-6, and HP-21 belong to a family of proteases that contain an amino-terminal clip domain and a serine protease domain, linked by a disulfide bond. They are predicted to be activated by specific cleavage between the clip domain and the protease domain (2Jiang H. Kanost M.R. Insect Biochem. Mol. Biol. 2000; 30: 95-105Crossref PubMed Scopus (325) Google Scholar). In SDS-PAGE of a serpin-protease complex, it is expected that the catalytic domain of the protease will remain connected to the serpin through a covalent bond between the catalytic serine residue of the protease and the P1 residue of the serpin. The clip domain is released under reducing conditions due to reduction of the disulfide bond that links the clip and protease domains. In Edman degradation analysis of a serpin-protease complex, two amino-terminal sequences can be expected, one for the serpin and one for the catalytic domain of the protease. Amino-terminal sequencing of proteins in SPC4-1, SPC4-4, and SPC4-5 bands isolated from gels showed that all three complexes contained a sequence matching the amino-terminal sequence of serpin-4 (Table I). SPC4-5 also yielded phenylthiohydantoin-derivatives expected from the HP-6 catalytic chain, consistent with the immunoblot result (Fig. 2B) indicating that SPC4-5 is a complex of serpin-4 with HP-6. However, no second sequence was detected in SPC4-1 or SPC4-4, perhaps due to blocking of the amino termini of the proteases.Table IAmino-terminal sequencing by automated Edman degradation of serpin-4-protease complexesExperimental resultsExpected sequencesCycleSPC4-1 and SPC4-4SPC4-5Serpin-4HP-6 catalytic domainPhenoloxidase-11AspAsp, Ile, PheAspIlePhe2AspAsp, Leu, GlyAspLeuGly3LeuLeu, Gly, AsnLeuGlyAsn4ProPro, Gly, Gl" @default.
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• W1998778934 title "Identification of Plasma Proteases Inhibited by Manduca sexta Serpin-4 and Serpin-5 and Their Association with Components of the Prophenol Oxidase Activation Pathway" @default.
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