| 1. |
Keisuke Yamashita, Daisuke Takahashi, Yuki Yamamoto, Shingo Kiyomoto, Toshio Shibata, Shun-ichiro Kawabata, Effects of Ca2+ ions on the horseshoe crab coagulation cascade triggered by lipopolysaccharide, The Journal of Biochemistry, 10.1093/jb/mvad018, 2023.02. |
| 2. |
Keisuke Yamashita, Naoki Takeshita, Aina Arita, Toshio Shibata, Yuki Kobayashi, Shun-ichiro Kawabata, A mutant equipped with a regenerated disulfide for the missing his loop of a serine protease zymogen in the horseshoe crab coagulation cascade., The Journal of Biochemistry, 10.1093/jb/mvab064, mvab064, 2021.05. |
| 3. |
Doshun Ito, Hinata Kawamura, Akira Oikawa, Yuta Ihara, Toshio Shibata, Nobuhiro Nakamura, Tsunaki Asano, Shun-Ichiro Kawabata, Takashi Suzuki, Shinji Masuda, ppGpp functions as an alarmone in metazoa., Communications Biology, 3, 1, article number 671, 2020.11. |
| 4. |
Keisuke Yamashita, Toshio Shibata, Toshiaki Takahashi, Yuki Kobayashi, Shun-ichiro Kawabata, Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs., Journal of Biological Chemistry, 10.1074/jbc.RA119.012452, 295, 26, 8857-8866, 2020.06. |
| 5. |
Shun-ichiro Kawabata, Toshio Shibata, Purification and Assays of Tachylectin-2, Methods in Molecular Biology, 10.1007/978-1-0716-0430-4_30, 309-316, 2020.04. |
| 6. |
Purification and Assays of Tachycitin. |
| 7. |
Purification and Assays of Tachylectin-5. |
| 8. |
Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide
Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, -factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversion to -factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site– uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules.. |
| 9. |
Seung Ah Lee, Seong Han Jang, Byung Hyun Kim, Toshio Shibata, Jinwook Yoo, Yunjin Jung, Shun-Ichiro Kawabata, Bok Luel Lee, Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity, Developmental and Comparative Immunology, 10.1016/j.dci.2017.11.014, 81, 116-126, 2018.04. |
| 10. |
Toshio Shibata, Jinki Hadano, Daichi Kawasaki, Xiaoqing, Shun-ichiro Kawabata, Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations, The Journal of Biological Chemistry, 10.1074/jbc.M117.779710, 292, 25, 10723-10734, 2017.06. |
| 11. |
Kouki Maki, Toshio Shibata, Shun-ichiro Kawabata, Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish, The Journal of Biological Chemistry, 10.1074/jbc.M117.779579, 292, 15, 6369-6380, 2017.04. |
| 12. |
Sanae Sekihara, Toshio Shibata, Mai Hyakkendani, Shun-ichiro Kawabata, RNA Interference Directed against the Transglutaminase Gene Triggers Dysbiosis of Gut Microbiota in Drosophila, The Journal of Biological Chemistry, 10.1074/jbc.M116.761791, 291, 48, 25077-25087, 2016.11. |
| 13. |
Toshio Shibata, Kouki Maki, Jinki Hadano, Takumi Fujikawa, Kazuki Kitazaki, Takumi Koshiba, Shun-ichiro Kawabata, Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins, PLOS Pathogens, 10.1371/journal.ppat.1005244, 11, 10, e1005244, e1005244, 2015.10. |
| 14. |
Yuki Kobayashi, Toshiaki Takahashi, Toshio Shibata, Shunsuke Ikeda, Takumi Koshiba, Hikaru Mizumura, Toshio Oda, Shun-ichiro Kawabata, Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade., The Journal of Biological Chemistry, 10.1074/jbc.M115.653196, 290, 31, 19379-19386, 2015.06. |
| 15. |
Toshio Shibata, Sanae Sekihara, Takumi Fujikawa, Ryuta Miyaji, Kouki Maki, Takeshi Ishihara, Takumi Koshiba, Shun-ichiro Kawabata, Transglutaminase-Catalyzed Protein-Protein Cross-Linking Suppresses the Activity of the NF-κB–Like Transcription Factor Relish, Science Signaling, 10.1126/scisignal.2003970, 6, 285, ra61, 2013.07. |
| 16. |
Yuki Kobayashi, Takafumi Shiga, Toshio Shibata, Miyuki Sako, Katsumi Maenaka, Takumi Koshiba, Hikaru Mizumura, Toshio Oda, Shun-ichiro Kawabata, The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen, The Journal of Biological Chemistry, 10.1074/jbc.M114.586933, 289, 37, 25987-25995, 2014.09. |
| 17. |
Toshio Shibata, Shigeru Ariki, Naoaki Shinzawa, Ryuta Miyaji, Haruka Suyama, Miyuki Sako, Nobuyuki Inomata, Takumi Koshiba, Hirotaka Kanuka, Shun-ichiro Kawabata, Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila, PLoS ONE, 10.1371/journal.pone.0013477, 5, 10, e13477-e13477, 2010.10. |
| 18. |
Keisuke Tagawa, Toyoki Yoshihara, Toshio Shibata, Kazuki Kitazaki, Yuichi Endo, Teizo Fujita, Takumi Koshiba, Shun-ichiro Kawabata, Microbe-Specific C3b Deposition in the Horseshoe Crab Complement System in a C2/Factor B-Dependent or -Independent Manner, PLoS ONE, 10.1371/journal.pone.0036783, 7, 5, e36783-e36783, 2012.05. |
| 19. |
Factor G Utilizes a Carbohydrate-Binding Cleft That Is Conserved between Horseshoe Crab and Bacteria for the Recognition of β-1,3-d-Glucans
Abstract
In the horseshoe crab, the recognition of β-1,3-d-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes β-1,3-d-glucans. To gain an insight into the recognition of β-1,3-d-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than ∼30 and 3 μM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble β-1,3-d-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble β-1,3-d-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a β-sheet in a predicted β-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for β-1,3-d-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.. |
| 20. |
Factor C Acts as a Lipopolysaccharide-Responsive C3 Convertase in Horseshoe Crab Complement Activation
Abstract
The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.. |
