| 1. |
Yamashita, K., Takeshita, N., Arita, A., Shibata, T., Kobayashi, Y., and Kawabata, S., A mutant equipped with a regenerated disulfide for the missing His loop of a serine protease zymogen in the horseshoe crab coagulation cascade., Journal of Biochemistry, in press, 2021.12, The lipopolysaccharide-triggered coagulation cascade in horseshoe crabs is composed of three zymogens belonging to the trypsinogen family: prochelicerase C, prochelicerase B (proB), and the proclotting enzyme (proCE). Trypsinogen-family members contain three conserved disulfides located around the active site. While it is known that proB evolutionarily lost one of the disulfides, the His-loop disulfide, the roles of the missing His-loop disulfide in proB remain unknown. Here we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulfide. The activation rate by upstream α-chelicerase C for proB-murasame was indistinguishable from that for wild-type (WT) proB. The resulting protease chelicerase B-murasame exhibited an 8-fold higher kcat value for downstream proCE than WT chelicerase B, whereas the Km value of chelicerase B-murasame was equivalent to that of WT chelicerase B. WT serpins-1, -2, and -3, identified as scavengers for the cascade, had no reactivity against WT chelicerase B, whereas chelicerase B-murasame was inhibited by WT serpin-2, suggesting that WT chelicerae B may trigger as-yet-unsolved phenomena after performing its duty in the cascade. The reconstituted lipopolysaccharide-triggered cascade containing proB-murasame exhibited ~5-fold higher CE production than that containing WT proB. ProB-murasame might be used as a high value-adding reagent for lipopolysaccharide detection.. |
| 2. |
Keisuke Yamashita, Toshio Shibata, Toshiaki Takahashi, Shun-ichiro Kawabata, Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs, Journal of Biological Chemistry, 2020.06, The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs comprises three protease zymogens, prochelicerase C (proC), prochelicerase B (proB), and the proclotting enzyme (proCE). The presence of LPS results in autocatalytic activation of proC to α-chelicerase C, which, in turn, activates proB to chelicerase B, converting proCE to the clotting enzyme (CE). ProB and proCE contain an N-terminal clip domain, but the roles of these domains in this coagulation cascade remain unknown. Here, using recombinant proteins and kinetics and binding assays, we found that five basic residues in the clip domain of proB are required to maintain its LPS-binding activity and activation by α-chelicerase C. Moreover, an amino acid substitution at a potential hydrophobic cavity in proB’s clip domain (V55A-proB) reduced both its LPS-binding activity and activation rate. WT proCE exhibited no LPS-binding activity, and the WT chelicerase B–mediated activation of a proCE variant with a substitution at a potential hydrophobic cavity (V53A-proCE) was approximately 4-fold slower than that of WT proCE. The kcat/Km value of the interaction of WT chelicerase B with V53A-proCE was 7-fold lower than that of the WT chelicerase B–WT proCE interaction. The enzymatic activities of V55A-chelicerase B and V53A-CE against specific peptide substrates were indistinguishable from those of the corresponding WT proteases. In conclusion, the clip domain of proB recruits it to a reaction center composed of α-chelicerase C and LPS, where α-chelicerase C is ready to activate proB, leading to chelicerase B–mediated activation of proCE via its clip domain.. |
| 3. |
Shibata, T., Kobayashi, Y., Ikeda, Y., and Kawabata, S., Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide., Journal of Biological Chemistry, 10.1074/jbc.RA118.002311, 293, 11589-11599, 2018.05, タンパク質組換え体の技術で調製したカブトガニ凝固因子のひとつであるC因子の変異体を用いて、遷移状態のC*因子を捕らえることに成功しました。グラム陰性菌の細胞壁成分であるリポ多糖(LPS)は、過剰に感染すると発熱や多臓器不全、さらには致死性のショック症状を引き起こします。一方で、カブトガニ血球から分泌されるタンパク質分解酵素前駆体のC因子は、ごく微量のLPSに鋭敏に反応して活性型のα-C因子となるため、LPSの高感度検出試薬として利用されてきました。これまで、細菌表面のLPSに結合したC因子は、不安定な中間状態である遷移状態のC*因子となり、C*因子同士が接近して活性型のα-C因子に変換されると推定されていました。この活性化の過程をタンパク質分解酵素前駆体の自己触媒的活性化といいます。しかし、自己触媒的活性化の重要なステージである遷移状態は不安的で寿命が短く、その実態を捕らえることはできませんでした。今回の遷移状態を捕らえる研究手法は、自己触媒的活性化を介して活性化される他のタンパク質分解酵素前駆体の研究に応用されることが期待されます。. |
| 4. |
Shibata, T., Hadano, J., Kawasaki, D., Dong, X., and Kawabata, S., Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations, J. Biol. Chem., 2017.05, エクソソーム（exosome）は血液やリンパ液などに含まれる、直径が20nmから100nm程度のナノ小胞です。主に細胞同士のコミュニケーションやがん細胞の浸潤・転移などに関わっており、重要な研究課題です。申請者らの研究グループは、これまでにキイロショウジョウバエ（ハエ）のタンパク質架橋（糊付け）酵素であるトランスグルタミナーゼ（TG）の機能研究を進めてきました。今回、同グループはハエTGの一つであるTG-Aのアミノ末端側に見出された新たな分泌シグナルが脂質修飾を受けて、エクソソームとして分泌されることを明らかにしました。脂質修飾によるタンパク質のエクソソームを介した分泌はこれまでに知られていませんでした。同グループが行ったショウジョウバエを用いた実験においては、エクソソームは細菌や他の細胞に取り込まれ、殺菌や生体の恒常性維持に関わっていることが分かりました。これにより今後はハエの分野に限らず、哺乳類を用いた研究分野全般においてもタンパク質の分泌機構の研究が推進されるきっかけとなることが期待できます。. |
| 5. |
Maki, K., Shibata, T., and Kawabata, S., Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor relish., J. Biol. Chem., 292, 10723-10734, 2017.04, 免疫系は微生物に対する過剰反応を防ぐために、巧妙な制御機構を備えています。著者らの研究グループは、これまでショウジョウバエを用いて、架橋（糊付け）酵素であるトランスグルタミナーゼ（TG）が、免疫遺伝子のスイッチを入れるNF-κBと呼ばれるタンパク質（転写因子）のひとつであるレリッシュの分子の間を架橋することを報告していました。今回、細胞の核内において、トランスグルタミナーゼ（TG）の架橋反応を介して、レリッシュ分子内の特定のアミノ酸（グルタミン）にポリアミンが架橋されることで、レリッシュのDNA結合能力が阻害され、過剰な免疫遺伝子の発現が抑制されることが判明しました。哺乳類においては、NF-κBの過剰な活性化は炎症疾患の原因となります。また哺乳類と昆虫においては、免疫の情報伝達経路のひとつであるNF-κB経路は相互に類似しており、進化的に保存されています。今回の発見により炎症疾患の原因解明や治療に寄与すると期待されます。. |
| 6. |
Mizumura, H., Ogura, N., Aketagawa, J., Aizawa, M., Kobayashi, Y., Kawabata, S., and Oda, T., Genetic engineering approach to develop next-generation reagents for endotoxin quantification., Innate Immunity, DOI: 10.1177/1753425916681074, 23, 136-146, 2017.03, カブトガニの体液凝固因子群の組換えタンパク質を調製して、グラム陰性菌の細胞壁成分のリポ多糖（ＬＰＳ）を検出する方法を開発した。これまで、生きたカブトガニから血球細胞を回収して、体液凝固成分を抽出し、リムスル試薬として調製する必要があったが、今回の組換えタンパク質の調製法の確立により、将来的にも安定的にＬＰＳ検出試薬の提供が可能となった。. |
| 7. |
Sekihara, S., Shibata, T., Hyakkendani, M., and Kawabata, S., RNA interference directed against the transglutaminase gene triggers dysbiosis of gut microbiota in Drosophila. , J. Biol. Chem., doi:10.1074/jbc.M116.761791, 291, 25077-25087, 2016.11, 宿主の腸管は常に多種多様な感染微生物にさらされおり、宿主は巧妙に制御された免疫機構を発達させることで、腸内細菌叢の恒常性を保っています。著者らは、次世代シークエンサーによりショウジョウバエの腸内細菌の遺伝子解析を行い、野生型とトランスグルタミナーゼ遺伝子をノックダウンしたハエでは腸内細菌叢が大きく異なっていることを見出しました。また、腸管から単離した4種の細菌株（SK1～SK4）の抗菌ペプチドと活性酸素に対する耐性を比較したところ、常在菌が腸管内の環境に順応すると、試験管での培養した際の菌とは異なる性質を示すことが推定されました。さらに、無菌バエにSK1とSK4を1：1の比率で感染させると菌を単独で感染させたハエよりも短命になることが判明しました。今回の研究により、単離した細菌を無菌ハエに摂食させることで、腸内環境における細菌間、あるいは細菌と宿主間の相互作用研究に利用できることが判明しました。ハエだけでなく、ほ乳類やヒトに共生している腸内細菌叢の研究にも応用できる実験系であり、今後、宿主・細菌の共生の分子機構の理解に寄与することが期待されます。. |
| 8. |
Shun-ichiro Kawabata, Crosslinking of a peritrophic matrix protein protects gut epithelia from bacterial exotoxins., PLoS Pathog., 10.1371/journal.ppat.1005244, 11, e1005244, 2015.10, Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.. |
| 9. |
Shun-ichiro Kawabata, Crosslinking of a peritrophic matrix protein protects gut epithelia from bacterial exotoxins., PLoS Pathog., 10.1371/journal.ppat.1005244, 11, e1005244, 2015.10, Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.. |
| 10. |
Shun-ichiro Kawabata, Factor B is the second lipopolysaccharide-binding protease zymogen in the horseshoe crab coagulation cascade., J. Biol. Chem. , doi:10.1074/jbc.M115.653196, 290, 19379-19386, 2015.07. |
| 11. |
Shun-ichiro Kawabata, The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen, Journal of Biological Chemistry
, 10.1074/jbc.M114.586933, 289, 25987-25995, 2014.07, Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N-terminus and the tripartite of lipopolysaccharide-binding site are essential factors for the autocatalytic activation, and that the positive charge of the N-terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N-terminus is required to form a complex of the factor C molecules in sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.. |
| 12. |
Shun-ichiro Kawabata, Transglutaminase-Catalyzed Protein-Protein Cross-Linking Suppresses the Activity of the NF-κB-like Transcription Factor Relish, Science Signaling, DOI: 10.1126/scisignal.2003970, 6, 285, ra61, 2013.07, Cross-linking of proteins by mammalian transglutaminases (TGs) plays important roles in physiological
phenomena such as blood coagulation and skin formation. We show that Drosophila TG suppressed innate
immune signaling in the gut. RNA interference (RNAi) directed against TG reduced the life span of
flies reared under conventional nonsterile conditions but not of those raised under germ-free conditions. In
conventionally reared flies, TG RNAi enhanced the expression of genes encoding antimicrobial peptides in the
immune deficiency (IMD) pathway. Wild-type flies that ingested gut lysates prepared from conventionally
reared TG RNAi–treated flies had shorter life spans. In conventionally reared flies, TG RNAi triggered
apoptosis in the gut and induced the nuclear translocation of Relish, the NF-kB (nuclear factor kB)–like
transcription factor of the IMD pathway. Wild-type flies that ingested synthetic amine donors, which inhibit
the TG-catalyzed protein-protein cross-linking reaction, showed nuclear translocation of Relish and enhanced
expression of genes encoding IMD-controlled antimicrobial peptide genes in the gut. We conclude
that TG-catalyzed Relish cross-linking suppressed the IMD signaling pathway to enable immune tolerance
against commensal microbes.. |
| 13. |
Tagawa, K., Yoshihara, Y., Shibata, T., Kitazaki, K., Endo, Y., Fujita, T., Koshiba, T., and Kawabata, S., Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner., PLoS ONE, doi:10.1371/journal.pone.0036783, 7, e36783, 2012.05. |
| 14. |
Koshiba, T., Yasukawa, K., Yanagi, Y., and Kawabata, S., Mitochondrial membrane potential is involved in the MAVS-mediated antiviral signaling., Science Signaling, 4, ra7 , 2011.02, Mitochondria, dynamic organelles that undergo cycles of fusion and fission, are the powerhouses of eukaryotic cells and are also involved in cellular innate antiviral immunity in mammals. Mitochondrial antiviral immunity depends on activation of the cytoplasmic retinoic acid–inducible gene I (RIG-I)–like receptor (RLR) signaling pathway and the participation of a mitochondrial outer membrane adaptor protein called MAVS (mitochondrial antiviral signaling). We found that cells that lack the ability to undergo mitochondrial fusion as a result of targeted deletion of both mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) exhibited impaired induction of interferons and proinflammatory cytokines in response to viral infection, resulting in increased viral replication. In contrast, cells with null mutations in either Mfn1 or Mfn2 retained their RLR-induced antiviral responses. We also found that a reduced mitochondrial membrane potential (DYm) correlated with the reduced antiviral response. The dissipation in DYm did not affect the activation of the transcription factor interferon regulatory factor 3 downstream of MAVS, which suggests that DYm and MAVS are coupled at the same stage in the RLR signaling pathway. Our results provide evidence that the physiological function of mitochondria plays a key role in innate antiviral immunity.. |
| 15. |
Koshiba, T., Holman, H. A., Kubara, K., Yasukawa, K., Kawabata, S., Okamoto, K., Macfarlane, and Shaw, J. M., Structure-function analysis of the yeast Miro GTPase, Gem1p: implications for mitochondrial inheritance., J. Biol. Chem. , 10.1074/jbc.M110.180034, 286, 354-362, 2011.01. |
| 16. |
Haipeng, L., Wu, C., Matsuda, Y., Kawabata, S., Lee, B. L., Söderhäll, K., and Söderhäll, I., Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)? , Dev. Comp. Immunol., 35, 51-61, 2011.01. |
| 17. |
Shibata, T., Ariki, S., Shinzawa, N., Miyaji, R., Suyama, H., Sako, M., Inomata, N., Koshiba, T., Kanuka, H., and Kawabata, S., Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila., PLoS ONE, 10.1371/journal.pone.0013477, 5, e13477, 2010.10. |
| 18. |
Yasukawa, K., Oshiumi, H., Takeda, M., Ishihara, N., Yanagai, Y., Seya, T., Kawabata, S., and Koshiba, T, Mitofusin 2 inhibits mitochondorial antiviral signaling, Science Signaling , 2, ra47 , 2009.08. |
| 19. |
S. Ariki, S. Takahara, T. Shibata, T. Fukuoka, A. Ozaki, Y. Endo, T. Fujita, T. Koshiba, and S. Kawabata, Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation, Journal of Immunology, 181巻、7994-8001, 2008.12. |
| 20. |
Matusda, Y., Koshiba,T., Osaki, T., Suyama, H., Arisaka, F., Toh, Y., and Kawabata, S., An arthropod cuticular chitin-binding protein endows injured sites with transglutaminase-dependent mesh. , Journal of Biological Chemistry, 282巻、37316-37324 , 2007.12. |
| 21. |
Matsuda, Y., Osaki, T., Hashii, T., Koshiba, T., and Kawabata, S., A cysteine-rich protein from an arthropod stabilizes clotting mesh and immobilizes bacteria at injured sites., Journal of Biological Chemistry, 282巻, 37316-37324 , 2007.11. |
| 22. |
Takumi Koshiba, Tomoyuki Hashii, Shun-ichiro Kawabata, A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria., Journal of Biological Chemistry, 282巻3962-3967, 2007.02. |
| 23. |
Fujitani, N., Kouno, T., Nakahara, T., Takaya, K., Osaki, T., Kawabata, S., Mizuguchi, M., Aizawa, T., Demura, M., Nishimura, S., and Kawano, K., The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cysteine-knot motif., Journal of Peptide Science, 13巻、269-279, 2007.01. |
| 24. |
Friedrich, R., Bode, W R., Panizzi, P., I., Kawabata, S., Bock, P. E., and Fuentes-Prior, P, Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation., Journal of Biological Chemistry, 281巻、1188-1195, 2006.01. |
| 25. |
Manabu Iijima, Tomonori Hashimoto, Yasuyuki Matsuda, Taku Nagai, Youichiro Yamano, Tomohiko Ichi, Tsukasa Osaki, Shun-ichiro Kawabata, Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking, FEBS Journal, 10.1111/j.1742-4658.2005.04891.x, 272, 18, 4774-4786, 2005.08. |
| 26. |
Aya Osaki, Shigeru Ariki, Shun-ichiro Kawabata, An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis, FEBS Jounarl, 10.1111/j.1742-4658.2005.04800.x, 272, 15, 3863-3871, 272, 3863-3871, 2005.06. |
| 27. |
Ariki, S., Koori, K., Osaki, T., Motoyama, K., Inamori, K., and Kawabata, S, A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides., Proc. Natl. Acad. Sci. USA, 101, 953-958, 2004.01. |
| 28. |
Zhang, R., Cho, H. Y., Kim, H. S., Ma, Y. G., Osaki, T., Kawabata, S., Soderhall, K., and Lee, B. L., Characterization and properties of a 1,3-b-D-glucan pattern recognition protein of Tenebrio molitor larvae which is specifically degraded by serine protease during prophenoloxidase activation., J. Biol. Chem., 10.1074/jbc.M307475200, 278, 43, 42072-42079, 2003.01. |
| 29. |
Lee, M. H., Osaki, T., Lee, J. Y., Baek, M. J., Zhang, R., Park, J. W., Kawabata, S., Soderhall, K., and Lee, B. L., Peptidoglycan recognition proteins involved in 1,3-b-D-glucan-dependent prophenoloxidase activation system of insect., J. Biol. Chem., 10.1074/jbc.M309821200, 279, 5, 3218-3227, 2003.01. |
