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W2105663335 abstract "The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses. The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses. The Drosophila Toll signaling pathway is responsible for defending against Gram-positive bacteria and fungi by inducing the expression of antimicrobial peptides via NF-κB-like transcription factors (1Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (3020) Google Scholar, 2Ferrandon D. Imler J.L. Hetru C. Hoffmann J.A. Nat. Rev. Immunol. 2007; 7: 862-874Crossref PubMed Scopus (649) Google Scholar). The recognition of lysine-type peptidoglycan (PG) 2The abbreviations used are: PGpeptidoglycanproPOprophenoloxidasePOphenoloxidasePPAFproPO-activating factorPPAEproPO-activating enzymeTmT. molitorPGRP-SAPG recognition protein-SAMSPmodular serine proteaseSPHserine protease homologueSPESpätzle-processing enzyme. by the Drosophila PG recognition protein-SA and GNBP1 (Gram-negative binding protein 1) complex has been suggested to cause activation of the serine protease cascade, leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway (2Ferrandon D. Imler J.L. Hetru C. Hoffmann J.A. Nat. Rev. Immunol. 2007; 7: 862-874Crossref PubMed Scopus (649) Google Scholar, 3Leulier F. Lemaitre B. Nat. Rev. Genet. 2008; 9: 165-178Crossref PubMed Scopus (400) Google Scholar). The elegant genetic studies in Drosophila have been and remain very powerful for characterizing and arranging the components of the Drosophila Toll pathway (4Anderson K.V. Curr. Opin. Immunol. 2000; 12: 13-19Crossref PubMed Scopus (527) Google Scholar). Recently, we found that three serine proteases are involved in the activation of the Toll pathway in a large beetle, Tenebrio molitor, and we indicated the sequence in which they are activated in vitro (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This three-step proteolytic cascade linking the PG recognition complex and subsequent Spätzle processing is essential for the peptidoglycan-dependent Toll signaling pathway (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). peptidoglycan prophenoloxidase phenoloxidase proPO-activating factor proPO-activating enzyme T. molitor PG recognition protein-SA modular serine protease serine protease homologue Spätzle-processing enzyme. The prophenoloxidase (proPO) activation cascade is known to be one of the major innate immune responses in arthropods (6Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1116) Google Scholar, 7Cerenius L. Söderhäll K. Immunol. Rev. 2004; 198: 116-126Crossref PubMed Scopus (1280) Google Scholar, 8Matarese G. De Rosa V. La Cava A. Trends Immunol. 2008; 29: 12-17Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), even though the role of the proPO activation cascade remains controversial in Drosophila and mosquito innate immunity (9Leclerc V. Pelte N. El Chamy L. Martinelli C. Ligoxygakis P. Hoffmann J.A. Reichhart J.M. EMBO Rep. 2006; 7: 231-235Crossref PubMed Scopus (122) Google Scholar, 10Schnitger A.K. Kafatos F.C. Osta M.A. J. Biol. Chem. 2007; 282: 21884-21888Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Upon injury or infection, proPO in the blood plasma is activated to phenoloxidase (PO) by clip-domain serine proteases, which are called proPO-activating factors (PPAFs) or enzymes (PPAEs), or alternatively proPO-activating proteins (11Lee S.Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K.I. Natori S. Kawabata S.I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (109) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 14Wang R. Lee S.Y. Cerenius L. Söderhäll K. Eur. J. Biochem. 2001; 268: 895-902Crossref PubMed Scopus (159) Google Scholar). We recently determined the crystal structures of two PPAFs and the functional roles of the clip domains during the proPO activation cascade (15Piao S. Kim S. Kim J.H. Park J.-W. Lee B.L. Ha N.-C. J. Biol. Chem. 2007; 282: 10783-10791Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Piao S. Song Y.L. Kim J.H. Park S.Y. Park J.-W. Lee B.L. Oh B.H. Ha N.-C. EMBO J. 2005; 24: 4404-4414Crossref PubMed Scopus (114) Google Scholar). PO, the active form of proPO, catalyzes the production of quinones, which can nonspecifically cross-link neighboring molecules to form melanin at the injury site or all over the surface of invading microorganisms (17Kumar S. Christophides G.K. Cantera R. Charles B. Han Y.S. Meister S. Dimopoulos G. Kafatos F.C. Barillas-Mury C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14139-14144Crossref PubMed Scopus (243) Google Scholar, 18Galko M.J. Krasnow M.A. PLoS Biol. 2004; 2: e239Crossref PubMed Scopus (297) Google Scholar). Quinones may also be involved in the production of cytotoxic molecules such as superoxides and hydroxyl radicals, which could help to kill the invading microorganisms (6Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1116) Google Scholar, 19Nappi A.J. Ottaviani E. BioEssays. 2000; 22: 469-480Crossref PubMed Scopus (278) Google Scholar). PO-induced melanin synthesis is thought to be essential for defense and development, but it must be tightly controlled because systemic hyperactivation of the proPO system, excessive production of quinones, and excessive melanin synthesis are harmful to the host. This implies that proPO activation and melanin synthesis are tightly regulated by melanization regulatory molecules (6Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1116) Google Scholar, 7Cerenius L. Söderhäll K. Immunol. Rev. 2004; 198: 116-126Crossref PubMed Scopus (1280) Google Scholar). However, the molecular regulatory mechanism of melanin synthesis is unclear. Recently, we reported that a soluble fragment of Lys-type PG, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the Tenebrio proPO activation cascade (20Park J.-W. Kim C.-H. Kim J.H. Je B.R. Roh K.-B. Kim S.J. Lee H.H. Ryu J.H. Lim J.H. Oh B.H. Lee W.J. Ha N.-C. Lee B.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6602-6607Crossref PubMed Scopus (131) Google Scholar). The fact that the same elicitor activates both the proPO system and the Toll pathway and that the clustering of T. molitor (Tm) PG recognition protein-SA (PGRP-SA) molecules on the PG is followed by activation of the proPO cascade suggests that there are obvious possibilities for molecular cross-talk between these two innate immune responses. Consistent with this possibility, we showed that partial lysozyme digestion of highly cross-linked Lys-type PG dramatically increases the binding of Tm-PGRP-SA, presumably by inducing clustering of Tm-PGRP-SA, which then recruits Tm-GNBP1 and Tm-modular serine protease (MSP) (20Park J.-W. Kim C.-H. Kim J.H. Je B.R. Roh K.-B. Kim S.J. Lee H.H. Ryu J.H. Lim J.H. Oh B.H. Lee W.J. Ha N.-C. Lee B.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6602-6607Crossref PubMed Scopus (131) Google Scholar). In that study, we suggested that formation of the Lys-type PG ·Tm-PGRP-SA ·Tm-GNBP1 complex leads to the activation of downstream PPAEs or PPAFs. However, we did not investigate the detailed molecular cross-talk between the proPO and Toll cascades. We and other groups have reported the biochemical properties of clip-domain serine protease and clip-domain serine protease homologues (SPHs) that function as PPAEs or PPAFs (21Lee 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 (96) Google Scholar, 22Kwon 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, 23Yu X.Q. Jiang H. Wang Y. Kanost M.R. Insect Biochem. Mol. Biol. 2003; 33: 197-208Crossref PubMed Scopus (207) Google Scholar, 24Gupta S. Wang Y. Jiang H. Insect Biochem. Mol. Biol. 2005; 35: 241-248Crossref PubMed Scopus (95) Google Scholar, 25Barillas-Mury C. Trends Parasitol. 2007; 23: 297-299Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). All these serine proteases are known to consist of a trypsin-like domain at the C terminus and one or two clip domains at the N terminus, but all SPHs lack proteolytic activity due to substitution of the catalytic Ser residue with a Gly residue (16Piao S. Song Y.L. Kim J.H. Park S.Y. Park J.-W. Lee B.L. Oh B.H. Ha N.-C. EMBO J. 2005; 24: 4404-4414Crossref PubMed Scopus (114) Google Scholar). We reported the detailed characterization of three PPAFs purified from the larvae of a large beetle, Holotrichia diomphalia (26Kim M.S. Baek M.J. Lee M.H. Park J.-W. Lee S.Y. Söderhäll K. Lee B.L. J. Biol. Chem. 2002; 277: 39999-40004Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Two of these PPAFs have been crystallized, and these structural studies have provided details about the activation mechanism (15Piao S. Kim S. Kim J.H. Park J.-W. Lee B.L. Ha N.-C. J. Biol. Chem. 2007; 282: 10783-10791Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Piao S. Song Y.L. Kim J.H. Park S.Y. Park J.-W. Lee B.L. Oh B.H. Ha N.-C. EMBO J. 2005; 24: 4404-4414Crossref PubMed Scopus (114) Google Scholar). For example, they showed that proPO cleavage alone is insufficient to produce active PO. In fact, an activated SPH (reported to be PPAF-II) is needed to lead to enzymatically active PO. Furthermore, the structural studies showed that when an active PPAF cleaves an SPH, the activated SPH oligomerizes, and the clip domain of SPH acts as a module for binding PO (16Piao S. Song Y.L. Kim J.H. Park S.Y. Park J.-W. Lee B.L. Oh B.H. Ha N.-C. EMBO J. 2005; 24: 4404-4414Crossref PubMed Scopus (114) Google Scholar). This probably serves to ensure that the active PO is under tight control and does not spread through the hemolymph in an uncontrolled manner but rather remains in the vicinity of the original trigger of the activation cascade. These findings provided some of the clues about how the proPO cascade and melanin synthesis are tightly regulated, but they did not provide detailed insights. To provide compelling biochemical evidence for the regulatory control of melanin synthesis and to search for possibilities of molecular cross-talk between the Toll pathway and proPO activation, we purified Tm-proPO, Tm-Spätzle-processing enzyme (SPE), and two different SPH zymogens (Tm-SPH1 and Tm-SPH2) to homogeneity. By performing in vitro reconstitution experiments with these purified proteins, we provide clear biochemical evidence that the active form of Tm-SPE and a specific Tm-SPH1 tightly regulate the activation of proPO and melanin synthesis in the Tm-proPO system. Insect and Antibodies—T. molitor larvae (mealworm) were maintained on a laboratory bench in terraria containing wheat bran. Hemolymph was collected as described previously (27Zhang R. Cho H.Y. Kim H.S. Ma Y.G. Osaki T. Kawabata S. Söderhäll K. Lee B.L. J. Biol. Chem. 2003; 278: 42072-42079Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, to harvest the hemolymph, a larva was pricked using a 25-gauge needle, and then a 10-μl drop of hemolymph was collected in 50 μl of modified anticoagulation buffer (136 mm trisodium citrate, 26 mm citric acid, 20 mm EDTA, and 15 mm sodium chloride, pH 5.0). The collected crude hemolymph was centrifuged at 200,000 × g for 15 min at 4 °C. The supernatant was then stored at –80 °C until used. Rabbit antisera against Tm-proPO, Tm-SPE, and Tm-SPH1 raised previously (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 21Lee 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 (96) Google Scholar, 28Lee H.S. Cho M.Y. Lee K.M. Kwon T.H. Homma K. Natori S. Lee B.L. FEBS Lett. 1999; 444: 255-259Crossref PubMed Scopus (28) Google Scholar) were used after affinity purification as described previously. Determination of Melanin Synthesis—Estimation of PO-induced melanin synthesis was carried out according to our previously published method (29Lee K.M. Lee K.Y. Choi H.W. Cho M.Y. Kwon T.H. Kawabata S. Lee B.L. Eur. J. Biochem. 2000; 267: 3695-3703Crossref PubMed Scopus (35) Google Scholar). In brief, a mixture of the active form of Tm-SPE (150 ng), Tm-proPO (3 μg), and Tm-SPH1 zymogen (1 μg) in 50 μl of 20 mm Tris-HCl, pH 8.0, containing 10 mm CaCl2 was preincubated at 30 °C for 5 min, after which 150 μl of the substrate solution (20 mm Tris-HCl, pH 8.0, containing 1 mm dopamine and 10 mm CaCl2) were added, and the mixture was incubated at 30 °C for 30 min. The increase in absorbance at 400 nm, which occurred in parallel with the synthesis of melanin, was measured using a Shimadzu spectrophotometer. Purification of Tm-proPO, Active Tm-SPE, and Tm-SPH2 Zymogen—Detailed procedures used to purify Tm-proPO, active and zymogenic forms of Tm-SPE, and Tm-SPH2 from the hemolymph (insect blood) of T. molitor larvae are described under the supplemental “Experimental Procedures.” In Vitro Reconstitution Experiments and Peptide Sequencing—To determine the cleavage sites of Tm-proPO and Tm-SPH1 induced by active Tm-SPE, in vitro reconstitution experiments were performed as described previously (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). To map proteolytic cleavage sites, the reaction mixtures following enzyme treatment were analyzed by SDS-PAGE under reducing conditions, blotted onto a polyvinylidene difluoride membrane, and stained with a solution containing 0.1% Coomassie Brilliant Blue R-250 and 50% methanol. The membrane was destained with 50% methanol containing 10% (v/v) acetic acid until the protein bands became visible. The zymogen cleavage products were identified by their N-terminal sequences using a gas-phase protein sequencer (Applied Biosystems). Immunofluorescence Microscopy—Melanin-coated bacterial cells were fixed in ice-cold 100% methanol. After washing twice with ice-cold phosphate-buffered saline, bacteria were incubated in phosphate-buffered saline containing 0.3% Tween 20 (blocking buffer) containing 3% skim milk for 1 h. After washing, cells were sequentially incubated with mouse anti-His monoclonal antibody (H-3, Santa Cruz Biotechnology) and rabbit anti-Tm-proPO polyclonal antibody (1:100 in blocking buffer) for 2 h. Fluorescein isothiocyanate-conjugated goat anti-mouse antibody or rhodamine-conjugated goat anti-rabbit antibody (1:200 in blocking buffer; Santa Cruz Biotechnology) was then added and incubated for 1 h. After washing twice with blocking buffer, stained cells were observed under a Zeiss fluorescence microscope. Expression and Purification of Recombinant Tm-SPH1—A DNA fragment encoding Tm-SPH1 (21Lee 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 (96) Google Scholar) was amplified by PCR using primers 5′-CCCGGATTCGCAAAAAGATGTCGATGATGCT-3′ and 5′-CCCTCTAGATCATATCAGGTAAGAGGATGTACCA-3′ with BamHI and XbaI at the 5′ and 3′ termini, respectively. These sites were later used to add a C-terminal tobacco etch virus protease cleavage site and a hexahistidine tag. The PCR products were subcloned into the pFast-Bac-SEa vector. The resulting plasmid was transformed into DH10Bac cells, and the transformation mixture was spread on LB agar culture medium containing isopropyl β-d-thiogalactopyranoside, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), gentamicin, kanamycin, and tetracycline. The white colonies were selected and cultured for amplification, and the bacmid DNA was extracted. After bacmid verification, Sf9 cells were transfected with the bacmid DNAs, and the resulting viruses were harvested and amplified as recommended by Invitrogen (Bac-to-Bac baculovirus expression system manual). For protein expression, Sf9 cells in 1 liter of suspension culture were infected with recombinant baculovirus. For purification of the expressed protein, the medium was first concentrated to ∼100 ml by ultrafiltration through a membrane filter and then dialyzed at 4 °C against 10 liters of 20 mm Tris-HCl and 150 mm NaCl, pH 8.0. After centrifugation at 20,000 × g for 15 min at 4 °C to remove precipitates, the supernatant was mixed with 3 ml of precharged nickel-nitrilotriacetic acid-agarose (Qiagen Inc.) and gently rotated at 4 °C for 1 h. Unbound proteins were allowed to pass through the resin, which was then washed with 200 ml of the same buffer containing 20 mm imidazole. Bound proteins were eluted with 200 mm imidazole in the same buffer and analyzed by SDS-PAGE under reducing conditions. For His tag removal, the fractions containing expressed proteins were incubated with tobacco etch virus protease (Invitrogen). His-tagged removed recombinant Tm-SPH1 was purified using a size exclusion high pressure liquid chromatography column (TSK G3000SW) equilibrated with 50 mm Tris-HCl containing 150 mm NaCl, pH 8.0. The fractions containing expressed proteins were pooled and concentrated on Centricon YM-10 to a final concentration of 0.1 mg/ml. The N-terminal amino acid sequencing of the recombinant Tm-SPH1 protein was carried out to verify the identities of the purified proteins. Inhibition Experiments Using Tm-proPO—The reaction mixture containing Tm-proPO (3 μg), Tm-SPH1 zymogen (1 μg), and the active form of Tm-SPE (150 ng) in 50 μl of buffer (20 mm Tris-HCl, pH 8.0, containing 10 mm CaCl2) was incubated at 30 °C for 60 min, and the reaction mixtures were cooled on ice for 5 min and divided into three equal portions. One portion was mixed with 200 μl of substrate solution (20 mm Tris-HCl, pH 8.0, containing 1 mm dopamine and 10 mm CaCl2) at 30°C for 15 min, and the melanin synthesis activity was estimated. Benzamidine was added to the second portion to a final concentration of 5 mm; the mixture was incubated at 37 °C for 1 h; and the melanin synthesis activity was examined. Benzamidine to a final concentration of 5 mm and 30 μg of Tm-proPO were simultaneously added to the third portion; the mixture was incubated at 37 °C for 1 h; and the melanin synthesis activity was estimated as described above. Assay of Antibacterial Activity—Antibacterial activity was assayed essentially as described previously (30Moon H.J. Lee S.Y. Kurata S. Natori S. Lee B.L. J. Biochem. (Tokyo). 1994; 116: 53-58Crossref PubMed Scopus (85) Google Scholar). Briefly, the bactericidal activity of melanin-concentrated bacteria was assayed using Staphylococcus aureus (strain Cowan 1) and Escherichia coli (strain K12). Bacteria grown in antibiotic medium were collected during the exponential phase of growth and suspended in 10 mm sodium phosphate buffer containing 130 mm NaCl, pH 6.0. The bacterial cells (106) were incubated with a mixture of active Tm-SPE (150 ng), Tm-proPO (3 μg), and Tm-SPH1 (1 μg) in the presence of 1 mm l-dopamine in 200 μl of assay buffer (20 mm Tris-HCl, pH 8.0, and 10 mm CaCl2) for 40 min at 30 °C. Melanin-coated bacteria were diluted 500-fold with assay buffer, and aliquots of 50 μl were spread on Bactoagar (Difco). The plates were incubated for 18 h at 37 °C, and colony numbers on test and control plates were compared. Active Tm-SPE Induces Melanin Synthesis in Vivo—Recently, we reported that the active form of Tm-SPE cleaves the 24-kDa Spätzle proprotein between Arg124 and Phe125 residues (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Cleaved 14-kDa Spätzle, a ligand of the Toll receptor, induces strong antimicrobial activity when injected into Tenebrio larvae (5Kim C.-H. Kim S.J. Kan H. Kwon H.-M. Roh K.-B. Jiang R. Yang Y. Park J.-W. Lee H.H. Ha N.-C. Kang H.J. Nonaka M. Söderhäll K. Lee B.L. J. Biol. Chem. 2008; 283: 7599-7607Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In addition, we observed previously that an unidentified serine protease specifically cleaved 79-kDa Tm-proPO between Arg50 and Phe51, and the resulting molecule of cleaved Tm-76-kDa PO was concentrated in the residues of the melanized cell clump/adhesion region (28Lee H.S. Cho M.Y. Lee K.M. Kwon T.H. Homma K. Natori S. Lee B.L. FEBS Lett. 1999; 444: 255-259Crossref PubMed Scopus (28) Google Scholar). To examine the possibility that active Tm-SPE can cleave Tm-proPO between Arg50 and Phe51 and thereby induce melanin synthesis in vivo, we injected the active form of Tm-SPE into Tenebrio larvae. The active form of Tm-SPE induced high levels of melanin synthesis after 4 days (Fig. 1A). These results suggest that the active form of Tm-SPE may convert Tm-proPO to Tm-PO, and the activated Tm-PO then induces melanin synthesis in vivo. To confirm that active Tm-SPE cleaves Tm-proPO between Arg50 and Phe51 and then induces melanin synthesis in vitro, we incubated purified Tm-proPO (Fig. 1B, lane 2) with purified active Tm-SPE (lane 1) and subjected the reaction samples to SDS-PAGE under reducing conditions (lanes 3 and 4). Surprisingly, active Tm-SPE generated three new bands (bands a–c). N-terminal sequencing of these three newly generated bands (Fig. 1C) revealed sequences identical to those deduced for Tm-proPO zymogen (28Lee H.S. Cho M.Y. Lee K.M. Kwon T.H. Homma K. Natori S. Lee B.L. FEBS Lett. 1999; 444: 255-259Crossref PubMed Scopus (28) Google Scholar). This result suggests that 79-kDa Tm-proPO was cleaved by active Tm-SPE at two different sites: one at Arg51 and another at Arg281 (Fig. 1D). To examine whether the cleaved products of Tm-proPO induce melanin synthesis in the presence of active Tm-SPE, the mixture was incubated with l-dopamine. This mixture did not induce any melanin production (Fig. 1E, bar 2); given that we observed melanin synthesis in vivo, this result indicates that some other molecule(s) may be required to induce melanin synthesis in vitro. Because an SPH is required to form an active PO complex in Holotrichia (16Piao S. Song Y.L. Kim J.H. Park S.Y. Park J.-W. Lee B.L. Oh B.H. Ha N.-C. EMBO J. 2005; 24: 4404-4414Crossref PubMed Scopus (114) Google Scholar), we selected Tenebrio SPHs. We reported previously that Tenebrio SPHs, such as 56-kDa Tm-SPH1 (referred to as Tm-mas (21Lee 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 (96) Google Scholar)) and 52-kDa Tm-SPH2 (referred to as Tm-45 (22Kwon 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)), are necessary for showing PO activity in the insect T. molitor. However, the biological functions of these SPHs during proPO activation were not determined. When we co-incubated Tm-proPO, active Tm-SPE, and purified Tm-SPH1 zymogen, the mixture induced strong melanin synthesis (Fig. 1E, bar 3). However, if the reaction mixture contained Tm-SPH2 instead of Tm-SPH1, no melanin synthesis was induced (bar 4). These results suggest that a specific SPH is required for proper activation of Tenebrio proPO because SPH1 but not SPH2 triggers melanin synthesis. Tm-SPE Proteolytically Cleaves Tm-SPH1—To compare the roles of Tm-SPH1 and Tm-SPH2 in melanin synthesis, we first analyzed the cleavage patterns of Tm-SPHs after incubation with active Tm-SPE in vitro (Fig. 2A). Active Tm-SPE rapidly cleaved Tm-SPH1 zymogen between Arg96 and Ile97, generating the 43-kDa active form of Tm-SPH1 (Fig. 2A, lane 2, band d). Under the same conditions, however, Tm-SPH2 was not cleaved (lane 4). This result supports the idea that active Tm-SPE specifically cleaves Tm-SPH1 but not Tm-SPH2. To examine whether Tm-SPH1 is involved in melanin synthesis, we compared SDS-PAGE patterns when active Tm-SPE and Tm-proPO were mixed in the presence or absence of Tm-SPH1 zymogen (Fig. 2B, lanes 1 and 2). Interestingly, only a 76-kDa band was generated in the presence of Tm-SPH1; performing SDS-PAGE under reducing conditions led to the appearance of an additional high molecular mass band (band e) at the top of the gel (Fig. 2B, lane 1). However, this band was not generated in the presence of Tm-SPH2 (lane 2). These results suggest that the active form of Tm-SPE completely converts 79-kDa Tm-proPO to 76-kDa Tm-PO by cleaving at the first site (Arg50), but not at the second one (Arg281). This cleavage subsequently led to production of the chemically cross-linked high molecular mass band (band e). To explore what kind of proteins are engaged in this high molecular mass band (band e), we performed Western blot analysis using anti-Tm-proPO, anti-Tm-SPE, and anti-Tm-SPH1 antibodies (Fig. 2C). Band e was recognized by antibodies against both Tm-proPO and Tm-SPH1 (Fig. 2C, lanes 3 and 6), but not by an antibody against Tm-SPE (lane 9). This result supports the idea that band e is a covalently cross-linked protein complex consisting of active Tm-PO and active Tm-SPH1. Chemically Cross-linked Adducts Are Generated by the Melanization Complex—To conduct further tests of whether covalently cross-linked high molecular mass complexes consist of active Tm-PO and active Tm-SPH1, we tried to isolate this complex by size exclusion column chromatography. First, as a control, the mixture of Tm-proPO and active Tm-SPE was loaded onto a size exclusion column, and each fraction of peak 1 was analyzed by SDS-PAGE under reducing conditions (Fig. 3, A and B). As shown previously in Fig. 2A, the mixture of Tm-proPO cleavage products co-eluted in peak 1 (Fig. 3B, lanes 2–4). However, when we loaded the mixture of Tm-proPO, active Tm-SPE, and Tm-SPH1 zymogen onto the same column, the proteins eluted as two peaks (Fig. 3C). When we examined the melanin synthesis activities of the two peaks, peak 2 was quite active in synthesizing melanin, but peak 3 was not under the same conditions (Fig. 3D). This result suggests that peak 2 contains protein components capable of melanin synthesis. To identify the protein components involved in melanin synthesis, we performed SDS-PAGE analysis and Western blotting on fractions from peak 2. The high molecular mass band (band e) and the 76-kDa PO band (band a) were observed mainly in peak 2 by SDS-PAGE analysis (Fig. 3E, lane 1), and both bands were strongly recognized by antibodies against" @default.
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W2105663335 title "Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect" @default.
W2105663335 cites W1501361836 @default.
W2105663335 cites W1514768711 @default.
W2105663335 cites W1603962084 @default.
W2105663335 cites W1626760783 @default.
W2105663335 cites W1644457456 @default.
W2105663335 cites W1785424492 @default.
W2105663335 cites W1977891171 @default.
W2105663335 cites W1978014890 @default.
W2105663335 cites W1988314455 @default.
W2105663335 cites W1998278278 @default.
W2105663335 cites W2003156936 @default.
W2105663335 cites W2005166322 @default.
W2105663335 cites W2017482741 @default.
W2105663335 cites W2029478181 @default.
W2105663335 cites W2044714805 @default.
W2105663335 cites W2047267173 @default.
W2105663335 cites W2048644187 @default.
W2105663335 cites W2052129538 @default.
W2105663335 cites W2060656097 @default.
W2105663335 cites W2060687174 @default.
W2105663335 cites W2063599549 @default.
W2105663335 cites W2064003574 @default.
W2105663335 cites W2069339162 @default.
W2105663335 cites W2070793039 @default.
W2105663335 cites W2072090415 @default.
W2105663335 cites W2080485420 @default.
W2105663335 cites W2089931734 @default.
W2105663335 cites W2102006028 @default.
W2105663335 cites W2102549002 @default.
W2105663335 cites W2112994024 @default.
W2105663335 cites W2133031337 @default.
W2105663335 cites W2133148204 @default.
W2105663335 cites W2136457322 @default.
W2105663335 cites W2137590150 @default.
W2105663335 cites W2139898583 @default.
W2105663335 cites W2151364245 @default.
W2105663335 cites W2167668177 @default.
W2105663335 cites W2169554406 @default.
W2105663335 cites W2325708085 @default.
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