腸内細菌に対する免疫寛容による腸管恒常性維持機構解明に対する功績

ショウジョウバエの腸内細菌に対する免疫応答と寛容の分子機構解明に対する功績

架橋酵素による腸管上皮の情報伝達制御と腸内細菌叢の維持機構解明に対する功績

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.RA119.012452

Language：English  

Purification and Assays of Tachylectin-2

DOI： 10.1007/978-1-0716-0430-4_30

Language：Others  

Purification and Assays of Tachylectin-5

DOI： 10.1007/978-1-0716-0430-4_27

Language：Others  

Purification and Assays of Tachycitin

DOI： 10.1007/978-1-0716-0430-4_31

Language：Others   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1074/jbc.RA118.002311

Language：English   Publishing type：Research paper (scientific journal)  

Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity
The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses.

DOI： 10.1016/j.dci.2017.11.014

Language：English   Publishing type：Research paper (scientific journal)  

Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish
In Drosophila, the final immune deficiency (IMD) pathwaydependent signal is transmitted through proteolytic conversion of the nuclear factor-B (NF-B)-like transcription factor Relish to the active N-Terminal fragment Relish-N. Relish-N is then translocated from the cytosol into the nucleus for the expression of IMD-controlled genes. We previously demonstrated that transglutaminase (TG) suppresses the IMD pathway by polymerizing Relish-N to inhibit its nuclear translocation. Conversely, we also demonstrated that orally ingested synthetic amines, such as monodansylcadaverine (DCA) and biotin-labeled pentylamine, are TG-dependently incorporated into Relish-N, causing the nuclear translocation of modified Relish-N in gut epithelial cells. It remains unclear, however, whether polyamine- containing Relish-N retains transcriptional activity. Here, we used mass spectrometry analysis of a recombinant Relish- N modified with DCA by TG activity after proteolytic digestion and show that the DCA-modified Gln residues are located in the DNA-binding region of Relish-N. TG-catalyzed DCA incorporation inhibited binding of Relish-N to the Rel-responsive element in the NF-B-binding DNA sequence. Subcellular fractionation of TG-expressing Drosophila S2 cells indicated that TG was localized in both the cytosol and nucleus. Of note, natural polyamines, including spermidine and spermine, competitively inhibited TG-dependentDCAincorporation into Relish- N. Moreover, in vivo experiments demonstrated that Relish- N was modified by spermine and that this modification reduced transcription of IMD pathway-controlled cecropin A1 and diptericin genes. These findings suggest that intracellular TG regulates Relish-N-mediated transcriptional activity by incorporating polyamines into Relish-N and via protein-protein cross-linking.

DOI： 10.1074/jbc.M117.779579

Language：English   Publishing type：Research paper (scientific journal)  

RNA Interference Directed against the Transglutaminase Gene Triggers Dysbiosis of Gut Microbiota in Drosophila
We recently reported that transglutaminase (TG) suppresses immune deficiency pathway-controlled antimicrobial peptides (IMD-AMPs), thereby conferring immune tolerance to gut microbes, and that RNAi of the TG gene in flies decreases the lifespan compared with non-TG-RNAi flies. Here, analysis of the bacterial composition of the Drosophila gut by next-generation sequencing revealed that gut microbiota comprising one dominant genus of Acetobacter in non-TG-RNAi flies was shifted to that comprising two dominant genera of Acetobacter and Providencia in TG-RNAi flies. Four bacterial strains, including Acetobacter persici SK1 and Acetobacter indonesiensis SK2, Lactobacillus pentosus SK3, and Providencia rettgeri SK4, were isolated from the midgut of TG-RNAi flies. SK1 exhibited the highest resistance to the IMD-AMPs Cecropin A1 and Diptericin among the isolated bacteria. In contrast, SK4 exhibited considerably lower resistance against Cecropin A1, whereas SK4 exhibited high resistance to hypochlorous acid. The resistance of strains SK1-4 against IMD-AMPs in in vitro assays could not explain the shift of the microbiota in the gut of TG-RNAi flies. The lifespan was reduced in gnotobiotic flies that ingested both SK4 and SK1, concomitant with the production of reactive oxygen species and apoptosis in the midgut, whereas the survival rate was not altered in gnotobiotic flies that monoingested either SK4 or SK1. Interestingly, significant amounts of reactive oxygen species were detected in the midgut of gnotobiotic flies that ingested SK4 and SK2, concomitant with no significant apoptosis in the midgut. In gnotobiotic flies that co-ingested SK4 and SK1, an additional unknown factor(s) may be required to cause midgut apoptosis.

DOI： 10.1074/jbc.M116.761791

Language：English   Publishing type：Research paper (scientific journal)  

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.
Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to alpha-factor C on LPS and is artificially converted to beta-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by alpha-factor C, but not by beta-factor C, in an LPS-dependent manner and that beta-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by alpha-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by alpha-factor C.

DOI： 10.1074/jbc.M115.653196

Language：English   Publishing type：Research paper (scientific journal)  

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen
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 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 a 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.

DOI： 10.1074/jbc.M114.586933

Language：English   Publishing type：Research paper (scientific journal)  

Microbe-Specific C3b Deposition in the Horseshoe Crab Complement System in a C2/Factor B-Dependent or -Independent Manner

DOI： 10.1371/journal.pone.0036783

Language：English   Publishing type：Research paper (scientific journal)  

Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

DOI： 10.1371/journal.pone.0013477

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.4049/jimmunol.0900430

Language：Others   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.4049/jimmunol.181.11.7994

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡市   Country：Japan  

Language：Japanese  

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Event date： 2023.9

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Type：Competition, symposium, etc. 

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