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Tomoyasu Aizawa
Faculty of Advanced Life Science　Advanced Transdisciplinary Science　Molecular Life Science
Professor
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Last Updated :2024/05/09

Researcher Profile and Settings

Affiliation

  • Faculty of Advanced Life Science　Advanced Transdisciplinary Science　Molecular Life Science

Job Title

  • Professor

Degree

  • March , 2001, Doctor of Philosophy (Hokkaido University)（Hokkaido University）

Contact information

  • aizawasci.hokudai.ac.jp

URL

Research funding number

  • 40333596

ORCID ID

J-Global ID

Research Interests

  • 蛋白質科学   構造生物学   蛋白質工学   NMR   metabolomics   innate immunity   antimicrobial peptide   アレルゲン   腸内細菌叢   

Research Areas

  • Life sciences / Structural biochemistry
  • Life sciences / Biophysics
  • Life sciences / Food sciences
  • Nanotechnology/Materials / Biochemistry
  • Nanotechnology/Materials / Analytical chemistry
  • Natural sciences / Bio-, chemical, and soft-matter physics

Educational Organization

Academic & Professional Experience

  • 2019/01 - Today 北海道大学（大学院先端生命科学研究院） 教授
  • 2007 - 2018 北海道大学（大学院先端生命科学研究院） 准教授
  • 2006 - 2007 北海道大学（大学院先端生命科学研究院） 助手
  • 2001 - 2006 北海道大学（大学院理学研究科生物科学専攻） 助手
  • 2001 生物系特定産業技術研究推進機構（富山医科薬科大学） 研究員

Education

  •        - 2001  Hokkaido University
  •        - 1998  Hokkaido University
  •        - 1996  Hokkaido University  School of Science

Association Memberships

  • 日本農芸化学会   日本蛋白質科学会   日本生物物理学会   日本生化学会   日本核磁気共鳴学会   日本ペプチド学会   

Research Activities

Published Papers

  • Hiroaki Ishida, Andrew G Woodman, Naoya Kitada, Tomoyasu Aizawa, Hans J Vogel
    Scientific reports 13 (1) 16208 - 16208 2023/09/27 [Refereed]
     
    Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory 'switch helix' that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.
  • Lorenzo Cecchi, Pascal Poncet, Lucia Maltagliati, Giulia Carli, Donatella Macchia, Laura Maggi, Elisa Meucci, Paola Parronchi, Alessio Mazzoni, Lorenzo Salvati, Enrico Scala, Hélène Sénéchal, Tomoyasu Aizawa, Danilo Villalta, Francesco Annunziato, Lorenzo Cosmi, Alessandro Farsi
    Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology 2023/09/26 [Refereed]
     
    BACKGROUND: Pru p 7 was the first gibberellin-regulated protein (GRP) to be identified as a food allergen as the basis of a pollen food allergy syndrome. OBJECTIVE: To clinically and biologically characterize a group of patients with suspected allergy to Pru p 7 to optimize the diagnostic workup of GRP sensitization. METHODS: Allergy to Pru p 7 was suspected in the presence of a systemic allergic reaction to plant food, positive skin prick test results for cypress pollen and lipid-transfer protein-enriched peach extract, and absence of Pru p 3-specific immunoglobulin E. Controls were patients with food allergies, patients sensitized to Pru p 3, and patients with cypress allergy without food allergy. Diagnostic workup included skin tests, basophil activation test, Western blot, and single and multiplex assays. RESULTS: In total, 23 patients and 14 controls were enrolled. The most implicated food was peach (91.3%). Approximately 70% of patients reacted to multiple foods. Mueller 4 reactions were 8.7%. In 26.1% of cases, a cofactor triggered the reaction. The basophil activation test results were positive for rPru p 7 in 87% of the patients. Specific immunoglobulin E to Pru p 7 was detected in 95.7% by singleplex and in 73.9% by multiplex assays in patients with suspected allergies; 73.9% of them also reacted to cypress pollen GRP (Cup s 7) in Western blot analysis. CONCLUSION: Patients with Pru p 7-Cup s 7 allergy in our cohort confirm a mild-to-severe clinical syndrome characterized by pollen and food allergy. The diagnosis may benefit from the proposed selection criteria that can be used as preliminary steps to further characterize the cross-reactive GRP sensitization.
  • Li Gan, Yuga Inamura, Yu Shimizu, Yuki Yokoi, Yuki Ohnishi, Zihao Song, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Masaaki Ito, Tokiyoshi Ayabe, Kiminori Nakamura, Tomoyasu Aizawa
    Metabolites 13 (9) 1003 - 1003 2023/09/10 [Refereed]
     
    Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01–0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.
  • Miu Sakagami, Yuki Ohnishi, Yasuhiro Kumaki, Tomoyasu Aizawa, Akira Inoue
    Fisheries Science 2023/09 [Refereed][Not invited]
  • Kazuma Kishi, Masaki Kuwatani, Yuki Ohnishi, Yasuhiro Kumaki, Hiroyuki Kumeta, Hajime Hirata, Yunosuke Takishin, Ryutaro Furukawa, Kosuke Nagai, Hiroki Yonemura, Shunichiro Nozawa, Ryo Sugiura, Kazumichi Kawakubo, Tomoyasu Aizawa, Naoya Sakamoto
    Cancers 15 (17) 4370 - 4370 2023/09/01 [Refereed]
     
    The poor prognosis of malignant biliary diseases is partially caused by their difficult early diagnosis. Therefore, many patients are only diagnosed at advanced stages. This study aimed to improve diagnosis by clarifying the differences in the duodenal juice metabolomes of benign and malignant biliary diseases. From October 2021 to January 2023, duodenal juice was obtained from 67 patients with suspected biliary diseases who required endoscopic ultrasonography and endoscopic retrograde cholangiography for diagnosis/treatment. The samples metabolomes were analyzed via nuclear magnet resonance spectroscopy using an 800-MHz spectrometer. Metabolomes of malignant and benign diseases were then compared, and multivariate analysis was performed to determine the relevant factors for malignancy/benignancy. For benignancy, no significant predictors were observed. For malignancy, acetone was a significant predictor, with higher concentrations in the malignant group than in the benign group. Regarding the receiver operating characteristic curve analysis for biliary tract carcinoma diagnosis, the predictive value of acetone in duodenal juice was comparable with serum CA19-9 levels (area under the curve: 0.7330 vs. 0.691, p = 0.697). In conclusion, duodenal juice metabolomics is a feasible method that is available for differential diagnosis in the biliary disease field.
  • Yi Wang, Yuchi Song, Shaonan Yan, Rina Hiramine, Yuki Ohnishi, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Antibiotics 12 (6) 1047 - 1047 2023/06/14 [Refereed]
     
    Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.
  • Zihao Song, Yuki Ohnishi, Seiji Osada, Li Gan, Jiaxi Jiang, Zhiyan Hu, Hiroyuki Kumeta, Yasuhiro Kumaki, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe, Kazuo Yamauchi, Tomoyasu Aizawa
    Metabolites 13 (5) 611 - 611 2023/04/28 [Refereed][Not invited]
     
    Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.
  • 和泉 裕久, 両角 麻衣, 田畑 風華, 川上 智美, 武田 安弘, 宮地 一裕, 中村 公則, 綾部 時芳, 中岡 慎治, 相沢 智康, 中村 宝弘, 木村 尚史, 中村 幸志, 玉腰 暁子
    腸内細菌学雑誌 (公財)腸内細菌学会 37 (2) 122 - 122 1343-0882 2023/04
  • 両角 麻衣, 和泉 裕久, 田畑 風華, 川上 智美, 武田 安弘, 宮地 一裕, 中村 公則, 綾部 時芳, 中岡 慎治, 相沢 智康, 中村 宝弘, 木村 尚史, 中村 幸志, 玉腰 暁子
    腸内細菌学雑誌 (公財)腸内細菌学会 37 (2) 123 - 123 1343-0882 2023/04
  • Shunta Nakamura, Kiminori Nakamura, Yuki Yokoi, Yu Shimizu, Shuya Ohira, Mizu Hagiwara, Zihao Song, Li Gan, Tomoyasu Aizawa, Daigo Hashimoto, Takanori Teshima, Andre J. Ouellette, Tokiyoshi Ayabe
    Scientific Reports 13 (1) 2023/03/09 [Refereed]
     
    Abstract Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.
  • Yuchi Song, Yi Wang, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Microbial cell factories 22 (1) 9 - 9 2023/01/13 [Refereed]
     
    BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.
  • Lareno L. Villones, Anna-Kristin Ludwig, Hiroyuki Kumeta, Seiya Kikuchi, Rika Ochi, Tomoyasu Aizawa, Shin-Ichiro Nishimura, Hans-Joachim Gabius, Hiroshi Hinou
    Scientific Reports 12 (1) 2022/10/23 [Refereed]
     
    Abstract Dystroglycan (DG), which constitutes a part of the dystrophin–glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans. Therefore, based on the presence of N-acetylLactosamine (LacNAc) in this glycan following the core extension, the binding interactions with adhesion/growth-regulatory galectins were explored. To elucidate this process, the interaction between galectin (Gal)-1, -3, -4 and -9 with α-DG fragment 372TRGAIIQTPTLGPIQPTRV390 core M1-based glycopeptide library were profiled, using glycan microarray and nuclear magnetic resonance studies. The binding of galectins was revealed irrespective of its modular architecture, adding galectins to the list of possible binding partners of α-DG core M1 glycoconjugates by cis-binding (via peptide- and carbohydrate-protein interactions), which can be abrogated by α2,3-sialylation of the LacNAc units. The LacNAc-terminated α-DG glycopeptide interact simultaneously with both the S- and F-faces of Gal-1, thereby inducing oligomerization. Furthermore, Gal-1 can trans-bridge α-DG core M1 structures and laminins, which proposed a possible mechanism by which Gal-1 ameliorates muscular dystrophies; however, this proposal warrants further investigation.
  • Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa
    ACS Omega 2470-1343 2022/09/02 [Refereed]
  • Mari Takei, Charles Nin, Tomona Iizuka, Marine Pawlikowski, Marie-Ange Selva, Yannick Chantran, Yurie Nakajima, Jingkang Zheng, Tomoyasu Aizawa, Motohiro Ebisawa, Hélène Sénéchal, Pascal Poncet
    Allergy, asthma & immunology research 14 (3) 328 - 338 2022/05 [Refereed]
     
    The Capsicum genus belongs to the Solanaceae family. Bell or chili peppers are consumed worldwide, but allergy to Capsicum is rare. It is involved in the celery-birch-mugwort-spice syndrome and cross-reactivities were reported with latex. Several allergens have been described, but only 2 are referenced in the World Health Organization/International Union of Immunological Societies allergen data bank, a thaumatin-like protein and a profilin. A patient allergic to bell/chili pepper, peach, orange and Japanese cedar pollen was clinically and biologically analyzed including direct and competitive immunoblots and basophil activation tests (BATs) with allergenic source extracts and recombinant gibberellin-regulated proteins (GRPs). The patient was shown to be sensitized to Cap a 7, the GRP of Capsicum annuum newly described herein. Cross-reactivities were demonstrated between various GRPs from bell/chili pepper, peach, orange and Japanese cedar pollen either in native form in the different extracts or as recombinant allergens. A similar immunoglobulin E reactivity was found also in Capsicum chinense and against snakin-1, the GRP from potato. The patient showed a positive BAT with recombinant Cry j 7, Pru p 7 and Cap a 7, but not with recombinant snakin-1. Despite the ubiquitous nature of GRPs in plants and the immunochemical cross-reactivity observed between different GRPs, clinically relevant sensitization to this protein family seems restricted to some allergenic sources, often associated with Cupressaceae pollen allergy, and to some patients, therefore reflecting very specific and peculiar mechanisms of conditional sensitization.
  • Kenji Kubo, Hikaru Watanabe, Hiroyuki Kumeta, Tomoyasu Aizawa, Chigusa Seki, Hiroto Nakano, Kiyotaka Tokuraku, Koji Uwai
    Bioorganic & Medicinal Chemistry 68 116840 - 116840 0968-0896 2022/05 [Refereed]
     
    Amyloid β (Aβ) aggregates in the brains of patients with Alzheimer's disease (AD) and accumulates via oligomerization and subsequent fiber elongation processes. These toxicity-induced neuronal damage and shedding processes advance AD progression. Therefore, Aβ aggregation-inhibiting substances may contribute to the prevention and treatment of AD. We screened for Aβ42 aggregation inhibitory activity using various plant extracts and compounds, and found high activity for a Geranium thunbergii extract (EC50 = 18 μg/mL). Therefore, we screened for Aβ42 aggregation inhibitors among components of a G. thunbergii extract and investigated their chemical properties in this study. An active substance was isolated from the ethanol extract of G. thunbergii based on the Aβ42 aggregation inhibitory activity as an index, and the compound was identified as geraniin (1) based on spectral data. However, although geraniin showed in vitro aggregation-inhibition activity, no binding to Aβ42 was observed via saturation transfer difference-nuclear magnetic resonance (STD-NMR). In contrast, the hydrolysates gallic acid (2) and corilagin (5) showed aggregation-inhibiting activity and binding was observed via STD-NMR. Therefore, the hydrolysates produced under the conditions of the activity test may contribute to the Aβ42 aggregation-inhibition activity of G. thunbergii extracts. Geraniin derivatives may help prevent and treat AD.
  • Yuji Sato, Yi Wang, Yuchi Song, Weiming Geng, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Makoto Demura, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Amino acids 2022/01/17 [Refereed]
     
    Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.
  • Ryuji Nishiyama, Takao Ojima, Yuki Ohnishi, Yasuhiro Kumaki, Tomoyasu Aizawa, Akira Inoue
    Communications Biology 4 (1) 2021/12 [Refereed]
     
    AbstractAlginate-assimilating bacteria degrade alginate into an unsaturated monosaccharide, which is converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEHU). DEHU is reduced to 2-keto-3-deoxy-D-gluconate by a DEHU-specific reductase using NAD(P)H. This is followed by pyruvate production via the Entner-Doudoroff pathway. Previously, we identified FlRed as a DEHU reductase in an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. Here, we showed that FlRed can also catalyze the oxidation of DEHU with NAD+, producing 2-keto-3-deoxy-D-glucarate (KDGR). FlRed showed a predilection for NADH and NAD+ over NADPH and NADP+, respectively, and the Km value for NADH was approximately 2.6-fold less than that for NAD+. Furthermore, we identified two key enzymes, FlDet and FlDeg, for KDGR catabolism. FlDet was identified as an enzyme of the ribonuclease activity regulator A family, which converts KDGR to α-ketoglutaric semialdehyde (α-KGSA). FlDeg, a type II α-KGSA dehydrogenase, generated α-ketoglutaric acid by oxidizing the aldehyde group of α-KGSA using NAD(P)+. Consequently, unlike the conventional DEHU reduction pathway, DEHU can be directly converted to α-ketoglutaric acid without consuming NAD(P)H. Alginate upregulated the expression of not only FlRed and two enzymes of the DEHU-reduction pathway, but also FlDet and FlDeg. These results revealed dual pathways of DEHU metabolism involving reduction or oxidation by FlRed.
  • Tomona Iizuka, Mari Takei, Yukiko Saito, Farhana Rumi, Jingkang Zheng, Xiaoshuang Lu, Philippe Chafey, Cédric Broussard, Laurence Guilloux-Assalet, Denis Charpin, Motohiro Ebisawa, Hélène Sénéchal, Tomoyasu Aizawa, Pascal Poncet
    Allergy 76 (7) 2297 - 2302 2021/07 [Refereed]
  • Kosuke Suzuki, Kiminori Nakamura, Yu Shimizu, Yuki Yokoi, Shuya Ohira, Mizu Hagiwara, Yi Wang, Yuchi Song, Tomoyasu Aizawa, Tokiyoshi Ayabe
    Scientific reports 11 (1) 9915 - 9915 2021/05/10 [Refereed]
     
    Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.
  • Yosuke Komatsu, Yasuaki Wada, Hirohisa Izumi, Takashi Shimizu, Yasuhiro Takeda, Tomoyasu Aizawa
    The British journal of nutrition 125 (6) 633 - 643 2021/03/28 [Refereed]
     
    Protein undernutrition contributes to the development of various diseases in broad generations. Urinary metabolites may serve as non-invasive biomarkers of protein undernutrition; however, this requires further investigation. We aimed to identify novel urinary metabolites as biomarker candidates responsive to protein undernutrition. Adult rats were fed control (CT; 14 % casein) or isoenergetic low-protein (LP; 5 % casein) diets for 4 weeks. 1H NMR metabolomics was applied to urine, plasma and liver samples to identify metabolites responsive to protein undernutrition. Liver samples were subjected to mRNA microarray and quantitative PCR analyses to elucidate the mechanisms causing fluctuations in identified metabolites. Urinary taurine levels were significantly lower in the LP group than in the CT group at week 1 and remained constant until week 4. Hepatic taurine level and gene expression level of cysteine dioxygenase type 1 were also significantly lower in the LP group than in the CT group. Urinary trimethylamine N-oxide (TMAO) levels were significantly higher in the LP group than in the CT group at week 2 and remained constant until week 4. Hepatic TMAO level and gene expression levels of flavin-containing mono-oxygenase 1 and 5 were also significantly higher in the LP group than in the CT group. In conclusion, urinary taurine and TMAO levels substantially responded to protein undernutrition. Furthermore, changes in hepatic levels of these metabolites and gene expressions associated with their metabolic pathways were also reflected in their fluctuating urinary levels. Thus, taurine and TMAO could act as non-invasive urinary biomarker candidates to detect protein undernutrition.
  • Yosuke Komatsu, Daiki Kumakura, Namiko Seto, Hirohisa Izumi, Yasuhiro Takeda, Yuki Ohnishi, Shinji Nakaoka, Tomoyasu Aizawa
    Frontiers in nutrition 8 813690 - 813690 2021 
    Background: The gut microbiome and fecal metabolites of breastfed infants changes during lactation, and are influenced by breast milk components. This study aimed to investigate dynamic associations of milk components with the infant gut microbiome and fecal metabolites throughout the lactation period in a mother-infant model. Methods: One month after delivery, breast milk and subsequent infant feces were collected in a pair for 5 months from a mother and an exclusively breastfed infant. Composition of the fecal microbiome was determined with 16S rRNA sequencing. Low-molecular-weight metabolites, including human milk oligosaccharides (HMOs), and antibacterial proteins were measured in feces and milk using 1H NMR metabolomics and enzyme-linked immunosorbent assays. The association of milk bioactive components with the infant gut microbiome and fecal metabolites was determined with Python clustering and correlation analyses. Results: The HMOs in milk did not fluctuate throughout the lactation period. However, they began to disappear in infant feces at the beginning of month 4. Notably, at this time-point, a bifidobacterium species switching (from B. breve to B. longum subsp. infantis) occurred, accompanied by fluctuations in several metabolites including acetate and butyrate in infant feces. Conclusions: Milk bioactive components, such as HMOs, might play different roles in the exclusively breastfed infants depending on the lactation period.
  • Kazuya Kusama, Mayuko Miyagawa, Koichiro Ota, Naoko Kuwabara, Kaori Saeki, Yuki Ohnishi, Yasuhiro Kumaki, Tomoyasu Aizawa, Toyokazu Nakasone, Shigemi Okamatsu, Hiroaki Miyaoka, Kazuhiro Tamura
    Nutrients 13 (1) 2020/12/26 
    The androgens testosterone and dihydrotestosterone (DHT) are essential for a variety of systemic functions in mature males. Alteration of these hormones results in late-onset hypogonadism (LOH) and benign prostate hyperplasia (BPH). The fruit bodies of fungi of the genus Cordyceps have been regarded as folk medicine or health food with tonic and antifatigue effects. The extract from the fruit body of Cordyceps militaris parasitizing Samia cynthia ricini (CM) was evaluated as a novel-candidate natural product for ameliorating male andropause symptoms. To explore the effects of CM on LOH and BPH, CM was applied to rat models and cultured testicular cells and prostate cells. The concentrations of androgens in the serum and culture media were determined by ELISA. Expression of steroidogenic enzymes and androgen-related genes was evaluated by qPCR, and prostatic cell proliferation was assessed with the cell-viability assay. CM maintained the serum levels of testosterone and DHT, but inhibited testosterone-induced prostate hypertrophy. CM also increased the secretion of testosterone and DHT by primary testicular cells, with no changes in the mRNA expression of steroidogenic enzymes, but decreased the growth of prostatic cell lines. Our data suggest that CM could improve both LOH and BPH in males.
  • Keisuke Murabe, Takashi Tsukamoto, Tomoyasu Aizawa, Makoto Demura, Takashi Kikukawa
    Journal of the American Chemical Society 142 (37) 16023 - 16030 2020/09/16 
    For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle.
  • Yosuke Komatsu, Yu Shimizu, Megumi Yamano, Mani Kikuchi, Kiminori Nakamura, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Metabolomics : Official journal of the Metabolomic Society 16 (4) 48 - 48 2020/04/10 
    INTRODUCTION: Crohn's disease (CD) is a chronic, relapsing inflammatory bowel disease affecting the gastrointestinal tract. Although its precise etiology has not been fully elucidated, an imbalance of the intestinal microbiota has been known to play a role in CD. Fecal metabolites derived from microbiota may be related to the onset and progression of CD OBJECTIVES: This study aimed to clarify the transition of gut microbiota and fecal metabolites associated with disease progression using SAMP1/YitFc mice, a model of spontaneous CD METHODS: The ileum tissues isolated from SAMP1/YitFc mice at different ages were stained with hematoxylin-eosin for histologic characterization with CD progression. Feces from control, Institute of Cancer Research (ICR; n = 6), and SAMP1/YitFc (n = 8) mice at different ages were subjected to microbial analysis and 1H nuclear magnetic resonance (NMR) analysis to investigate fluctuations in gut microbiota and fecal metabolites with CD progression RESULTS: Relative abundance of the Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, and Bacteroidales S24-7 at family-level gut microbiota and fecal metabolites, such as short-chain fatty acids, lactate, glucose, xylose, and choline, dramatically fluctuated with histologic progression of intestinal inflammation in SAMP1/YitFc mice. Unlike the other metabolites, fecal taurine concentration in SAMP1/YitFc mice was higher than ICR mice regardless of age CONCLUSION: The fecal metabolites showing characteristic fluctuations may help to understand the inflammatory mechanism associated with CD, and might be utilized as potential biomarkers in predicting CD pathology.
  • 宋 子豪, 包 克非, 北田 直也, 清水 由宇, 菊池 摩仁, 熊木 康裕, 大西 裕季, 塚本 卓, 菊川 峰志, 出村 誠, 中村 公則, 綾部 時芳, 山村 凌大, 中村 幸志, 玉腰 暁子, 相沢 智康
    腸内細菌学雑誌 34 (2) 148 - 148 1343-0882 2020/04
  • Tomoyasu Aizawa, Makoto Demura, Kazutoshi Gohara, Hisashi Haga, Koichiro Ishimori, Masataka Kinjo, Tamiki Komatsuzaki, Katsumi Maenaka, Min Yao
    Biophysical reviews 12 (2) 233 - 236 2020/04
  • Ryodai Yamamura, Koshi Nakamura, Naoya Kitada, Tomoyasu Aizawa, Yu Shimizu, Kiminori Nakamura, Tokiyoshi Ayabe, Takashi Kimura, Akiko Tamakoshi
    Bioscience of microbiota, food and health 39 (1) 11 - 17 2020 
    In recent years, short-chain fatty acids (SCFAs) have been reported to play an important role in maintaining human health. Fecal SCFA concentrations correlate well with colonic SCFA status and gut microbiota composition. However, the associations with the gut microbiota functional pathway, dietary intake, blood SCFAs, and fecal SCFAs remain uncertain. To clarify these relationships, we collected fecal samples, blood samples, and dietary habit data from 12 healthy adults aged 22-51 years. The relative abundance of several SCFA-producing bacteria, gut microbiota diversity, and functional pathways related to SCFA biosynthesis were positively associated with fecal SCFAs even after adjusting for age and sex. Furthermore, fecal acetate was likely to be positively associated with serum acetate. By contrast, dietary intake was not associated with fecal SCFAs. Overall, the present study highlights the potential usefulness of fecal SCFAs as an indicator of the gut microbiota ecosystem and dynamics of SCFAs in the human body.
  • Watsana Penkhrue, Dieter Jendrossek, Chartchai Khanongnuch, Wasu Pathom-Aree, Tomoyasu Aizawa, Rachel L Behrens, S Lumyong
    PloS one 15 (3) e0230443  2020 
    Polyhydroxybutyrate (PHB) is a biodegradable biopolymer which is useful for various applications including packing, medical and coating materials. An endospore-forming bacterium (strain BP17) was isolated from composted soil and evaluated for PHB production. Strain BP17, taxonomically identified as Bacillus drentensis, showed enhanced PHB accumulation and was selected for further studies. To achieve maximum PHB production, the culture conditions for B. drentensis BP17 were optimized through response surface methodology (RSM) employing central composite rotatable design (CCRD). The final optimum fermentation conditions included: pineapple peel solution, 11.5% (v/v); tryptic soy broth (TSB), 60 g/L; pH, 6.0; inoculum size, 10% (v/v) and temperature, 28°C for 36 h. This optimization yielded 5.55 g/L of PHB compared to the non-optimized condition (0.17 g/L). PHB accumulated by B. drentensis BP17 had a polydispersity value of 1.59 and an average molecular weight of 1.15x105 Da. Thermal analyses revealed that PHB existed as a thermally stable semi-crystalline polymer, exhibiting a thermal degradation temperature of 228°C, a melting temperature of 172°C and an apparent melting enthalpy of fusion of 83.69 J/g. It is evident that B. drentensis strain BP17 is a promising bacterium candidate for PHB production using agricultural waste, such as pineapple peel as a low-cost alternative carbon source for PHB production.
  • Yukiko Hirabayashi, Kiminori Nakamura, Tsuyoshi Sonehara, Daisuke Suzuki, Satoru Hanzawa, Yu Shimizu, Tomoyasu Aizawa, Koshi Nakamura, Akiko Tamakoshi, Tokiyoshi Ayabe
    Mass spectrometry (Tokyo, Japan) 9 (1) A0081  2020 
    Serotonin, an important neurotransmitter, is produced mainly in intestines, and serotonin levels in feces can be an indicator of the intestinal environment. Human feces, however, contain a large amount of contaminants, which vary widely owing to food contents and the intestinal environment, and these contaminants would be expected to interfere with the determination of serotonin levels in human feces. To remove these contaminants and determine serotonin levels, we developed a new method using solid phase extraction (SPE) and column-switching LC-MS/MS. Serotonin, labeled with a stable isotope, was added to human feces samples prior to SPE as an internal standard to correct for individual differences in matrix effects. The recovery rate for SPE was 55.9-81.0% (intraday) and 56.5-78.1% (interday) for feces from two subjects. We analyzed 220 fecal samples from 96 subjects including 76 pregnant and post-delivery women. The endogenous serotonin content per unit weight of dried feces was 0.09-14.13 ng/mg for pregnant and post-delivery women and 0.30-9.93 ng/mg for the remaining subjects.
  • クローン病自然発症モデルSAMP1/YitFcマウスの病状進行に伴う腸内細菌叢および糞便メタボライトの変動
    小松 陽介, 清水 由宇, 山野 めぐみ, 中村 公則, 綾部 時芳, 相沢 智康
    日本生物工学会大会講演要旨集 (公社)日本生物工学会 2019年 180 - 180 2019/08
  • Pascal Poncet, Tomoyasu Aizawa, Hélène Sénéchal
    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 49 (8) 1163 - 1166 2019/08
  • Azusa Iizuka, Kousuke Kajimoto, Tomotsumi Fujisawa, Takashi Tsukamoto, Tomoyasu Aizawa, Naoki Kamo, Kwang-Hwan Jung, Masashi Unno, Makoto Demura, Takashi Kikukawa
    Scientific reports 9 (1) 10711 - 10711 2019/07/24 
    Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the "monomerization effects": Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.
  • Yukiko Horie, Hideyuki Nemoto, Jin Fujita, Shigeo Ikegawa, Yasuhiro Kumaki, Yuki Ohnishi, Hiroyuki Kumeta, Makoto Demura, Tomoyasu Aizawa
    Nippon Shokuhin Kagaku Kogaku Kaishi 66 (4) 139 - 146 1341-027X 2019/04/15
  • Takatoshi Hasemi, Takashi Kikukawa, Yumi Watanabe, Tomoyasu Aizawa, Seiji Miyauchi, Naoki Kamo, Makoto Demura
    Biochimica et biophysica acta. Bioenergetics 1860 (2) 136 - 146 2019/02/01 
    Mastigocladopsis repens halorhodopsin (MrHR) is a Cl--pumping rhodopsin that belongs to a distinct cluster far from other Cl- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I- similar to Cl- but cannot transport it. I--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl--pumping photocycle, Cl- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl--binding affinity nor the absorption spectrum, but completely eliminated the Cl--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H+ release was detected for photolyzed MrHR by using an indium‑tin oxide electrode, which acts as a good time-resolved pH sensor. This H+ release disappeared in the I--bound form of the wild-type and Cl--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl- movement to the CP channel.
  • Hélène Sénéchal, Sanaz Keykhosravi, Rémy Couderc, Marie Ange Selva, Youcef Shahali, Tomoyasu Aizawa, Jean Marc Busnel, Rihane Arif, Inna Mercier, Nhan Pham-Thi, Denis André Charpin, Pascal Poncet
    Allergy, asthma & immunology research 11 (1) 143 - 151 2019/01 
    A pollen/food-associated syndrome (PFAS) has been described between peach and cypress pollen. Cross-reactive allergens were characterized which belong to the Gibberellin-regulated protein (GRP) family, BP14 in cypress pollen and Pru p 7 in peach. GRP are small cationic protein with anti-microbial properties. A patient suffering from a peach/cypress syndrome was explored clinically and biologically using 2 types of immunoglobulin E (IgE) multiarray microchip, immunoblots and a basophil activation test to assess the clinical relevance of various extracts and purified allergens from fruits or cypress pollen. In addition to PR10 sensitization, the patient showed specific IgE to Pru p 7, BP14 and allergen from pomegranate. These last 3 allergens and allergenic sources are able to induce ex vivo basophil activation characterized by the monitoring of the expression of CD63 and CD203c, both cell surface markers correlated with a basophil mediator release. Up to 100% of cells expressed CD203c at 50 ng/mL of BP14 protein. In contrast, snakin-1, a GRP from potato sharing 82% sequence identity with Pru p 7 did not activate patient's basophils. These results strongly suggest that, like Pru p 7, BP14 is a clinically relevant allergenic GRP from pollen. Allergen members of this newly described protein family are good candidates for PFAS where no cross-reactive allergens have been characterized.
  • Takashi Tsukamoto, Chihiro Kikuchi, Hiromu Suzuki, Tomoyasu Aizawa, Takashi Kikukawa, Makoto Demura
    Scientific reports 8 (1) 13445 - 13445 2018/09/07 
    Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl- dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl- in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl- concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl- non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl- conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl-.
  • 平林 由紀子, 中村 公則, 曽根原 剛志, 半澤 悟, 清水 由宇, 相沢 智康, 中村 幸志, 玉腰 暁子, 綾部 時芳
    JSBMS Letters (一社)日本医用マススペクトル学会 43 (Suppl.) 70 - 70 1881-5464 2018/08
  • Watsana Penkhrue, Kanaporn Sujarit, Takuji Kudo, Moriya Ohkuma, Kazuo Masaki, Tomoyasu Aizawa, Wasu Pathom-Aree, Chartchai Khanongnuch, Saisamorn Lumyong
    International journal of systematic and evolutionary microbiology 68 (5) 1448 - 1454 2018/05 
    A novel bioplastic-degrading actinomycete, strain SCM_MK2-4T, was isolated from paddy soil in Thailand. The 16S rRNA gene sequence showed that strain SCM_MK2-4T belonged to the genus Amycolatopsis, with the highest sequence similarity to Amycolatopsisazurea JCM 3275T (99.4 %), and was phylogenetically clustered with this strain along with Amycolatopsislurida JCM 3141T (99.3 %), A. japonica DSM 44213T (99.2 %), A. decaplanina DSM 44594T (99.0 %), A. roodepoortensis M29T (98.9 %), A. keratiniphilasubsp. nogabecina DSM 44586T (98.8 %), A. keratiniphilasubsp. keratiniphila DSM 44409T (98.5 %), A. orientalis DSM 40040T (98.4 %) and A. regifaucium GY080T (98.3 %). A combination of DNA-DNA hybridization results ranging from 42.8±3.2 to 66.2±1.4 % with the type strains of A. azurea and A. lurida and some different phenotypic characteristics indicated that the strain could be distinguished from its closest phylogenetic neighbours. Whole-cell hydrolysates of the strain were shown to contain meso-diaminopimelic acid, arabinose, galactose, glucose, ribose, mannose, rhamnose and xylose. The predominant menaquinone was MK-9(H4). The major cellular fatty acid profile consisted of iso-C15 : 0, iso-C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2OH) and C16 : 0. The polar lipid composition of the strain consisted of phosphatidyl-N-methylethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol, aminophospholipids, an unidentified phospholipid and two unidentified glycolipids. The G+C content of the genomic DNA was 68.2 mol%. On the basis of phylogenetic analyses, DNA-DNA hybridization experimentation and the phenotypic characteristics, it was concluded that strain SCM_MK2-4T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis oliviviridis sp. nov. is proposed. The type strain is SCM_MK2-4T (=TBRC 7186T=JCM 32134T).
  • Hélène Sénéchal, Jiří Šantrůček, Magdalena Melčová, Petr Svoboda, Jarmila Zídková, Denis Charpin, Laurence Guilloux, Youcef Shahali, Marie-Ange Selva, Rémy Couderc, Tomoyasu Aizawa, Pascal Poncet
    The Journal of allergy and clinical immunology 141 (1) 411 - 414 2018/01
  • Eiko Hayase, Daigo Hashimoto, Kiminori Nakamura, Clara Noizat, Reiki Ogasawara, Shuichiro Takahashi, Hiroyuki Ohigashi, Yuki Yokoi, Rina Sugimoto, Satomi Matsuoka, Takahide Ara, Emi Yokoyama, Tomohiro Yamakawa, Ko Ebata, Takeshi Kondo, Rina Hiramine, Tomoyasu Aizawa, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Mori, Ken Kurokawa, Kazuma Tomizuka, Tokiyoshi Ayabe, Takanori Teshima
    The Journal of experimental medicine 214 (12) 3507 - 3518 2017/12/04 
    The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.
  • Md Ruhul Kuddus, Megumi Yamano, Farhana Rumi, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
    Biotechnology progress 33 (6) 1520 - 1528 2017/11 
    Snakin-1 (SN-1) is a cysteine-rich plant antimicrobial peptide and the first purified member of the snakin family. SN-1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN-1 in Escherichia coli by a previously developed coexpression method using an aggregation-prone partner protein. Our goal was to increase the productivity of SN-1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN-1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN-1, the identity of which was verified by MALDI-TOF MS and NMR studies. The purified recombinant SN-1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation-prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN-1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520-1528, 2017.
  • Shintaro Nakamura, Takashi Kikukawa, Jun Tamogami, Masakatsu Kamiya, Tomoyasu Aizawa, Martin W Hahn, Kunio Ihara, Naoki Kamo, Makoto Demura
    Biochimica et biophysica acta 1857 (12) 1900 - 1908 2016/12 
    Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed λmax at 534nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+ pumps. In contrast to conventional O, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P620, while the other first releases H+ prior to H+ uptake during P620 formation.
  • Hiroaki Ishida, Leonard T Nguyen, Ramamourthy Gopal, Tomoyasu Aizawa, Hans J Vogel
    Journal of the American Chemical Society 138 (35) 11318 - 26 2016/09/07 
    In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells.
  • Md Ruhul Kuddus, Farhana Rumi, Motosuke Tsutsumi, Rika Takahashi, Megumi Yamano, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
    Protein expression and purification 122 15 - 22 2016/06 
    Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris.
  • Mi-Hwa Baek, Masakatsu Kamiya, Takahiro Kushibiki, Taichi Nakazumi, Satoshi Tomisawa, Chiharu Abe, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
    Journal of peptide science : an official publication of the European Peptide Society 22 (4) 214 - 21 2016/04 
    Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed (15) N-labeled and (13) C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed (15) N-edited and (13) C-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A.
  • Motosuke Tsutsumi, Hideki Muto, Shohei Myoba, Mai Kimoto, Akira Kitamura, Masakatsu Kamiya, Takashi Kikukawa, Shigeharu Takiya, Makoto Demura, Keiichi Kawano, Masataka Kinjo, Tomoyasu Aizawa
    FEBS open bio 6 (2) 106 - 25 2016/02 
    Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and
  • Hajime Tamaki, Ayako Egawa, Kouki Kido, Tomoshi Kameda, Masakatsu Kamiya, Takashi Kikukawa, Tomoyasu Aizawa, Toshimichi Fujiwara, Makoto Demura
    Journal of biomolecular NMR 64 (1) 87 - 101 2016/01 
    Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn(2+) mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.
  • Satoshi Tomisawa, Yuji Sato, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Kiminori Nakamura, Tokiyoshi Ayabe, Tomoyasu Aizawa
    Protein expression and purification 112 21 - 8 2015/08 
    Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.
  • Takashi Kikukawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Kunio Ihara, Naoki Kamo, Makoto Demura
    Biochimica et biophysica acta 1847 (8) 748 - 58 2015/08 
    Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR'. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR' still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR' transitions.
  • Taro Miyoshi, Yuhei Nagai, Tomoyasu Aizawa, Katsuki Kimura, Yoshimasa Watanabe
    Water science and technology : a journal of the International Association on Water Pollution Research 72 (6) 844 - 9 2015 
    In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs.
  • Takahiro Kushibiki, Masakatsu Kamiya, Tomoyasu Aizawa, Yasuhiro Kumaki, Takashi Kikukawa, Mineyuki Mizuguchi, Makoto Demura, Shun-ichiro Kawabata, Keiichi Kawano
    Biochimica et biophysica acta 1844 (3) 527 - 34 2014/03 
    Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.
  • Hashimoto Arata, Tomisawa Satoshi, Kamiya Masakatsu, Kikukawa Takashi, Kumaki Yasuhiro, Nakamura Kiminori, Ayabe Tokiyoshi, Aizawa Tomoyasu, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S261  2014
  • Akitaya Tatsuo, Makita Naoko, Tsumura Naomi, Mayama Hiroyuki, Hazemoto Norio, Kanbe Toshio, Yamaguchi Hideaki, Kubo Koji, Zinchenko Anatoly, Murata Shizuaki, Yoshikawa Kenichi, Aizawa Tomoyasu, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S269  2014
  • Abe Chiharu, Nakazumi Taichi, Kamiya Masakatsu, Kikukawa Takashi, Kawano Keiichi, Demura Makoto, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S262  2014
  • Tomisawa Satoshi, Kamiya Masakatsu, Kikukawa Takashi, Demura Makoto, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S192  2014
  • Tamaki Hajime, Egawa Ayako, Kido Kouki, Kameda Tomoshi, Kamiya Masakatsu, Kikukawa Takashi, Aizawa Tomoyasu, Fujiwara Toshimichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S197  2014
  • Naho Toyama, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Kwang-Hwan Jung, Naoki Kamo, Makoto Demura
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S290  2014
  • Nakamura Shintaro, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Hahn Martin W, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S181  2014
  • Goto Kazuki, Kikukawa Takashi, Hasemi Takatoshi, Saito Yuta, Kamiya Masakatsu, Aizawa Tomoyasu, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S182  2014
  • Hasemi Takatoshi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Jung Kwang-Hwan, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S235  2014
  • Kushibiki Takahiro, Kamiya Masakatsu, Aizawa Tomoyasu, Kumaki Yasuhiro, Kikukawa Takashi, Mizuguchi Mineyuki, Demura Makoto, Kawabata Syunichiro, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S153  2014
  • Yuhara Kosuke, Kamiya Masakatsu, Kumaki Yasuhiro, Takiya Shigeharu, Kikukawa Takashi, Kawano Keiichi, Demura Makoto, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S160  2014
  • Tsutsumi Motosuke, Muto Hideki, Kimoto Mai, Kamiya Masakatsu, Kikukawa Takashi, Takiya Shigeharu, Demura Makoto, Kawano Keiichi, Kinjo Masataka, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 54 (1) S160  2014
  • Kousuke Shibasaki, Hiroaki Shigemura, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    Biochemistry 52 (51) 9257 - 68 2013/12/23 
    Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (P(i), i = 1-4) were adequate to describe the photocycle. The third component, P₃, was a quasi-equilibrium complex between the N and O intermediates, where a N ↔ O + Cl⁻ equilibrium was attained. The K(d,N↔O) values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, ΔV(N→O), were estimated from the pressure dependence of K(d,N↔O). A comparison between K(d,N↔O) and ΔV(N→O) led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K(d,N↔O). For some mutants, however, large ΔV(N→O) values were found, whereas the K(d,N↔O) values were small. This suggests that the special coordination of a water molecule with the OH group of Thr is necessary for the increase in K(d,N↔O). Mutants with a small K(d,N↔O) showed low pumping activities in the presence of inside negative membrane potential, while the mutant activities were not different in the absence of membrane potential. The effect of the mutation on the pumping activities is discussed.
  • Satoshi Tomisawa, Eri Hojo, Yoshitaka Umetsu, Shinya Ohki, Yusuke Kato, Mitsuhiro Miyazawa, Mineyuki Mizuguchi, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Tomoyasu Aizawa
    AMB Express 3 (1) 45 - 45 2013/08/15 
    Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.
  • Taro Yonekita, Ryuji Ohtsuki, Eri Hojo, Naoki Morishita, Takashi Matsumoto, Tomoyasu Aizawa, Fumiki Morimatsu
    Journal of microbiological methods 93 (3) 251 - 6 2013/06 
    The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 10(4)CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6 CFU per 25 g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.
  • Takashi Nakamura, Tomoyasu Aizawa, Ryusho Kariya, Seiji Okada, Makoto Demura, Keiichi Kawano, Koki Makabe, Kunihiro Kuwajima
    The Journal of biological chemistry 288 (20) 14408 - 14416 2013/05/17 
    Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
  • Baek Mihwa, Kamiya Masakatsu, Nakazumi Taichi, Tomisawa Satoshi, Kumaki Yasuhiro, Kikukawa Takashi, Demura Makoto, Kawano Keiichi, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S213  2013
  • Kushibiki Takahiro, Kamiya Masakatsu, Aizawa Tomoyasu, Kumiki Yasuhiro, Kikukawa Takashi, Demura Makoto, Kawabata Shun-ichiro, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S215  2013
  • Shibasaki Kousuke, Shigemura Hiroaki, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S199  2013
  • Kikukawa Takashi, Kusakabe Chikara, Kokubo Asami, Tsukamoto Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Ihara Kunio, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S199  2013
  • Tomisawa Satoshi, Abe Chiharu, Kamiya Masakatsu, Kikukawa Takashi, Demura Makoto, Kawano Keiichi, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 53 (1) S119  2013
  • Satoshi Tomisawa, Chiharu Abe, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
    Biophysics (Nagoya-shi, Japan) 9 79 - 83 2013 
    Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that peptide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method.
  • Takashi Tsukamoto, Xianglan Li, Hiromi Morita, Takashi Minowa, Tomoyasu Aizawa, Nobutaka Hanagata, Makoto Demura
    PloS one 8 (9) e75831  2013 
    Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.
  • Taro Miyoshi, Tomoyasu Aizawa, Katsuki Kimura, Yoshimasa Watanabe
    INTERNATIONAL BIODETERIORATION & BIODEGRADATION 75 15 - 22 0964-8305 2012/11 [Refereed][Not invited]
     
    The proteins that caused membrane fouling in a continuous operation of membrane bioreactors (MBRs) treating real municipal wastewater were investigated in detail. We continuously operated two identical pilot-scale MBRs under different solid retention times (SRTs) and extracted the foulants at the end of the operation. Regardless of the operating conditions, proteins were dominant components in the foulants extracted from the fouled membranes. The extracted proteins were subjected to the separation with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the identification through the N-terminal amino acid sequencing analysis. The proteins concentrated by the combination of the crude concentration using an ultra-filtration (UF) membrane and trichloroacetic acid (TCA) precipitation were separated and visualized well on 2D-PAGE gels. The results of 2D-PAGE analysis indicated that the compositions of proteins that caused membrane fouling significantly differed depending on the SRT, although such differences cannot be seen in the amino acid composition analysis. Analyzing selected 2D-PAGE spots by N-terminal amino acid sequencing analysis led to the identification of two well-characterized outer membrane proteins originating from Pseudomonas genus, namely OprF and OprD. To our knowledge, this is the first successful identification of proteins that have caused membrane fouling in continuous operations of MBRs treating real wastewater. (C) 2012 Elsevier Ltd. All rights reserved.
  • Mineyuki Mizuguchi, Makoto Takeuchi, Shinya Ohki, Yuko Nabeshima, Takahide Kouno, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Katsuhide Yutani
    Biochemistry 51 (31) 6089 - 96 2012/08/07 
    The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.
  • Takashi Tsukamoto, Takanori Sasaki, Kazuhiro J Fujimoto, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    Biophysical journal 102 (12) 2906 - 15 2012/06/20 
    Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.
  • Sakai Chihiro, Hojo Eri, Nakazumi Taichi, Tomisawa Satoshi, Kikukawa Takashi, Kumaki Yasuhiro, Kamiya Masakatsu, Demura Makoto, Kawano Keiichi, Ohtsuki Ryuji, Yonekita Taro, Morishita Naoki, Matsumoto Takashi, Morimatsu Fumiki, Aizawa Tomoyasu
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 52 S22 - S23 2012
  • Tsukamoto Takashi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Jung Kwang-Hwan, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 52 S136  2012
  • Hasemi Takatoshi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Jung Kwang-Hwan, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 52 S135 - S136 2012
  • Yasutaka Yamashita, Takashi Kikukawa, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Seiji Miyauchi, Naoki Kamo, Makoto Demura
    Biochimica et biophysica acta 1808 (12) 2905 - 12 2011/12 
    Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.
  • Takahide Kouno, Nobuhisa Watanabe, Naoki Sakai, Takashi Nakamura, Yuko Nabeshima, Masashi Morita, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Tsuneo Imanaka, Isao Tanaka, Keiichi Kawano
    Journal of molecular biology 405 (2) 560 - 9 2011/01/14 
    Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.
  • Taro Miyoshi, Tomoyasu Aizawa, Katsuki Kimura, Yoshimasa Watanabe
    Desalination and Water Treatment Taylor & Francis 34 (1) 150 - 155 1944-3994 2011/01 [Refereed][Not invited]
     
    We investigated the relationship between the nature of proteins and their fouling propensities in pilot-scale membrane bioreactors (MBRs) treating real municipal wastewater with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In 2D-PAGE, proteins are separated based on their isoelectric points and molecular weights, and therefore, information can be obtained on the nature of the proteins. Foulants extracted from fouled membranes at the end of continuous operation and organic matter contained in mixed liquor suspension were analyzed by 2D-PAGE, and the results were compared. This analysis was performed for two identical MBRs operated under different solid retention times (SRT) to investigate the effect of the operating condition on the types of proteins with high fouling propensities. In the MBR operated with a long SRT, the presence of proteins that appeared in the neutral pH range was more pronounced in the extracted foulant. Because the pH of the mixed liquor suspension was in the range of 6-7 throughout the continuous operation, the difference in protein profiles between the extracted foulant and the organic matter contained in the mixed liquor suspension in the MBR operated with a long SRT can be explained by a decrease in solubility of a protein at its isoelectric point. In contrast, in the MBR operated with a short SRT, the presence of proteins that appeared in the acidic region (pH 3-5) was more prominent in the extracted foulant compared with the organic matter contained in the mixed liquor suspension. The results obtained in this study imply that dominant fouling mechanisms differed depending on the operating condition. © 2011 Desalination Publications. All rights reserved.
  • Nakamura Takashi, Makabe Koki, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto, Kuwajima Kunihiro
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S136  2011
  • Ogata Yumi, Aizawa Tomoyasu, Sakai Naoki, Kamiya Masakatsu, Kikukawa Takashi, Kumaki Yasuhiro, Demura Makoto, Ayabe Tokiyoshi, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S81  2011
  • Takahashi Rika, Aizawa Tomoyasu, Kamiya Masakatsu, Kikukawa Takashi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S85  2011
  • Ito Kanako, Aizawa Tomoyasu, Kamiya Masakatsu, Umetsu Yoshitaka, Kato Yusuke, Kumaki Yasuhiro, Kikukawa Takashi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S85  2011
  • Tsukamoto Takashi, Terada Kota, Nakagawa Marie, Tamaki Hajime, Kamiya Masakatsu, Kikukawa Takashi, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S32  2011
  • Shibasaki Kousuke, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keichi, Kamo naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 51 S32  2011
  • Wei Wang, Soichiro Itoh, Tomoyasu Aizawa, Atsushi Okawa, Katsuyoshi Sakai, Tsuneo Ohkuma, Makoto Demura
    Biomedical materials (Bristol, England) 5 (6) 065009 - 065009 2010/12 
    A chitosan/marine-originated collagen composite has been developed. This composite gel was characterized and its biocompatibility, as well as an inflammatory reaction, was observed. The chitosan gel including N-3-carboxypropanoil-6-O-(carboxymethyl) chitosan of 3 mol%, 6-O-(carboxymethyl) chitosan of 62 mol% and 6-O-(carboxymethyl) chitin of 35 mol% was prepared and compounded with the salmon atelocollagen (SA) gel at different mixture ratios. The composite gels were injected subcutaneously in to the back of rats. The specimens were harvested for a histological survey as well as a tumor necrosis factor-alpha (TNF-α) assay by ELISA. The inflammatory cell infiltration and release of TNF-α were successively controlled low with the ratio of SA to chitosan at 10:90 or 20:80. The SA gel first, within 2 weeks, and then chitosan in the composite gel were slowly absorbed after implantation, followed by soft tissue formation. It is expected that this composite gel will be available as a carrier for tissue filler and drug delivery systems.
  • Yasuhiro Nonaka, Hideki Muto, Tomoyasu Aizawa, Etsuro Okabe, Shohei Myoba, Takuya Yokoyama, Shin Saito, Fumie Tatami, Yasuhiro Kumaki, Masakatsu Kamiya, Takashi Kikukawa, Mineyuki Mizuguchi, Shigeharu Takiya, Masataka Kinjo, Makoto Demura, Keiichi Kawano
    Biochemistry 49 (38) 8367 - 75 2010/09/28 
    The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.
  • Masakatsu Kamiya, Keisuke Oyauchi, Yoshinori Sato, Takuya Yokoyama, Mofei Wang, Tomoyasu Aizawa, Yasuhiro Kumaki, Mineyuki Mizuguchi, Kunio Imai, Makoto Demura, Koichi Suzuki, Keiichi Kawano
    Journal of peptide science : an official publication of the European Peptide Society 16 (5) 242 - 8 2010/05 
    We previously reported that yamamarin, a pentapeptide with an amidated C-terminus (DILRG-NH(2)) isolated from larvae of the silkmoth, and its palmitoylated analog (C16-DILRG-NH(2)) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure-activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C-terminal part (-RG-NH(2)) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG-NH(2) and C16-DILRG-NH(2) revealed that the N-terminal palmitoyl group of C16-DILRG-NH(2) did not affect the conformation of the C-terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs.
  • Kamiya Masakatsu, Yoshida Tomoshi, Kikukawa Takashi, Aizawa Tomoyasu, Imai Kunio, Suzuki Koichi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S23  2010
  • Tsukamoto Takashi, Sasaki Takanori, Fujimoto Kazuhiro, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S66  2010
  • Sugita Kumi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S194  2010
  • Sakai Chihiro, Aizawa Tomoyasu, Kamiya Masakatsu, Kikukawa Takashi, Kumaki Yasuhiro, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S97  2010
  • Nakamura Takashi, Makabe Koki, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto, Kuwajima Kunihiro
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S92  2010
  • Kushibiki Takahiro, Kamiya Masakatsu, Aizawa Tomoyasu, Kumaki Yasuhiro, Kikukawa Takashi, Demura Makoto, Kawabata Syun-ichiro, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S145 - S146 2010
  • Tamaki Hajime, Higuchi Marika, Egawa Ayako, Fujiwara Toshimichi, Yokoyama Jun, Kigawa Takanori, Shimono Kazumi, Someya Tomomi, Yokoyama Shigeyuki, Kamiya Masakatsu, Kikukawa Takashi, Aizawa Tomoyasu, Kawano Keichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S145  2010
  • Aizawa Tomoyasu, Tomisawa Satoshi, Kamiya Masakatsu, Kikukawa Takashi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S162  2010
  • OKABE Etsuro, NONAKA Yasuhiro, MUTO Hideki, AIZAWA Tomoyasu, MYOBA Shohei, KAMIYA Masakatsu, KIKUKAWA Takashi, TAKIYA Shigeharu, KINJO Masataka, DEMURA Makoto, KAWANO Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 50 (2) S33 - S34 2010
  • Takanori Sasaki, Tomoyasu Aizawa, Masakatsu Kamiya, Takashi Kikukawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    Biochemistry 48 (51) 12089 - 95 2009/12/29 
    Halorhodopsin from Natronomonas pharaonis (NpHR) acts an inward-directed, light-driven chloride pump and forms a homotrimer. To evaluate effect of trimeric assembly, that is, intermolecular interaction, on the control or modulation of light-driven chloride pumping activity of individual HRs, it is important to understand the thermal and chloride sensitivity of trimer dissociation and the structural stability of HR. In this study, the thermal dissociation of NpHR trimer to monomer in a dodecyl beta-d-maltoside-solubilized system was investigated, using size-exclusion chromatography and visible absorption. In the absence of Cl(-), NpHR retained the trimer assembly at 25 degrees C but dissociated to the monomer with an increase in temperature to >40 degrees C. On the other hand, in the presence of Cl(-), the trimer assembly was maintained at 40 degrees C. The dissociation of the trimer to the monomer after incubation at 40 degrees C, which was determined via size-exclusion chromatography, depended on the Cl(-) concentration and showed a sigmoidal isotherm. From this isotherm, the apparent dissociation constant for Cl(-) was estimated to be 22 mM with a Hill coefficient of 2.2. A similar isotherm was obtained when SO(4)(2-) was used instead of Cl(-) with a dissociation constant of 94 mM. On the other hand, thermal dissociation of the NpHR trimer to the monomer in the absence of Cl(-) proceeded by two components: the fast component is susceptible to the changes in temperature and detergent concentration, and the slow component is accompanied by bleaching at the same time. Activation energies of the fast and slow dissociation components and bleaching were 57.8, 35.3, and 40.5 kcal/mol, respectively. The presence of a second chloride-binding site with a Hill coefficient of approximately 2 at the surface of NpHR to control the trimer-monomer conversion was discussed.
  • Yoshitaka Umetsu, Tomoyasu Aizawa, Kaori Muto, Hiroko Yamamoto, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano
    The Journal of biological chemistry 284 (43) 29625 - 34 2009/10/23 
    Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.
  • Takahide Kouno, Mineyuki Mizuguchi, Tomoyasu Aizawa, Hiroyuki Shinoda, Makoto Demura, Shun-ichiro Kawabata, Keiichi Kawano
    Biochemistry 48 (32) 7629 - 35 2009/08/18 
    Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. The amino acid sequence of big defensin is divided into an N-terminal hydrophobic domain and a C-terminal cationic domain, which are responsible for antimicrobial activities against Gram-positive and -negative bacteria, respectively. The N-terminal domain of big defensin forms a unique globular conformation with two alpha-helices and a parallel beta-sheet, while the C-terminal domain adopts a beta-defensin-like fold. Although our previous study implied that big defensin changes its N-terminal structure in a micellar environment, due to the poor quality of the NMR spectra it remained to be resolved whether the N-terminal domain adopts any structure in the presence of micelles. In this analysis, we successfully determined the structure of the N-terminal fragment of big defensin in a micellar solution, showing that the fragment peptide forms a single alpha-helix structure. Moreover, NMR experiments using paramagnetic probes revealed that the N-terminal domain of big defensin penetrates into the micelle with a dipping at the N-terminal edge of the alpha-helix. Here, we propose a model for how big defensin associates with the target membrane.
  • Shigeharu Takiya, Shin Saito, Takuya Yokoyama, Daisuke Matsumoto, Tomoyasu Aizawa, Masakatsu Kamiya, Makoto Demura, Keiichi Kawano
    Journal of biochemistry 146 (1) 103 - 11 2009/07 
    The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino-acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.
  • Shin-ichi Nakatogawa, Yasunori Oda, Masakatsu Kamiya, Tatsuro Kamijima, Tomoyasu Aizawa, Kevin D Clark, Makoto Demura, Keiichi Kawano, Michael R Strand, Yoichi Hayakawa
    Current biology : CB 19 (9) 779 - 85 2009/05/12 
    Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.
  • Yasuhiro Nonaka, Daisuke Akieda, Tomoyasu Aizawa, Nobuhisa Watanabe, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Takashi Kikukawa, Makoto Demura, Keiichi Kawano
    The FEBS journal 276 (8) 2192 - 200 2009/04 
    In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.
  • Yasuhiro Nonaka, Daisuke Akieda, Nobuhisa Watanabe, Masakatsu Kamiya, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano
    Biophysical Journal 96 (3) 67a - 68a 0006-3495 2009/02
  • Oyauchi Keisuke, Kamiya Masakatsu, Aizawa Tomoyasu, Demura Makoto, Suzuki Koichi, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S155  2009
  • Yamashita Yasutaka, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S185  2009
  • Nonaka Yasuhiro, Myoba Shohei, Okabe Etsuro, Muto Hideki, Kamiya Masakatsu, Kikukawa Takashi, Aizawa Tomoyasu, Kinjo Masataka, Takiya Shigeharu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S38  2009
  • Tomisawa Satoshi, Aizawa Tomoyasu, Kamiya Masakatsu, Kikukawa Takashi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S75  2009
  • Kawano Keiichi, Kouno Takahide, Fujitani Naoki, Mizuguchi Mineyuki, Osaki Tsukasa, Kawabata Shun-ichiro, Aizawa Tomoyasu, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S68  2009
  • Nakamura Takashi, Makabe Koki, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto, Kuwajima Kunihiro
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S67  2009
  • Myoba Shohei, Muto Hideki, Kimoto Mai, Kamiya Masakatsu, Kikukawa Takashi, Aizawa Tomoyasu, Kinjo Masataka, Takiya Shigeharu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S167  2009
  • Kamiya Masakatsu, Nakatogawa Shin-ichi, Oda Yasunori, Kamijima Tatsuro, Aizawa Tomoyasu, Demura Makoto, Hayakawa Yoichi, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S161  2009
  • Tsukamoto Takashi, Sasaki Takanori, Fujimoto Kazuhiro, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 49 S119  2009
  • Tatsuro Kamijima, Ayaka Ohmura, Toshiya Sato, Kaoru Akimoto, Miki Itabashi, Mineyuki Mizuguchi, Masakatsu Kamiya, Takashi Kikukawa, Tomoyasu Aizawa, Masayuki Takahashi, Keiichi Kawano, Makoto Demura
    Biochemical and biophysical research communications 376 (1) 211 - 4 2008/11/07 
    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.
  • Takahide Kouno, Mineyuki Mizuguchi, Masakiyo Sakaguchi, Eiichi Makino, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Nam-Ho Huh, Keiichi Kawano
    Journal of peptide science : an official publication of the European Peptide Society 14 (10) 1129 - 38 2008/10 
    S100A11 protein is a member of the S100 family containing two EF-hand motifs. It undergoes phosphorylation on residue T10 after cell stimulation such as an increase in Ca(2+) concentration. Phosphorylated S100A11 can be recognized by its target protein, nucleolin. Although S100A11 is initially expressed in the cytoplasm, it is transported to the nucleus by the action of nucleolin. In the nucleus, S100A11 suppresses the growth of keratinocytes through p21(CIP1/WAF1) activation and induces cell differentiation. Interestingly, the N-terminal fragment of S100A11 has the same activity as the full-length protein; i.e. it is phosphorylated in vivo and binds to nucleolin. In addition, this fragment leads to the arrest of cultured keratinocyte growth. We examined the solution structure of this fragment peptide and explored its structural properties before and after phosphorylation. In a trifluoroethanol solution, the peptide adopts the alpha-helical structure just as the corresponding region of the full-length S100A11. Phosphorylation induces a disruption of the N-capping conformation of the alpha-helix, and has a tendency to perturb its surrounding structure. Therefore, the phosphorylated threonine lies in the N-terminal edge of the alpha-helix. This local structural change can reasonably explain why the phosphorylation of a residue that is initially buried in the interior of protein allows it to be recognized by the binding partner.
  • Shin Saito, Takuya Yokoyama, Tomoyasu Aizawa, Kyosuke Kawaguchi, Takeshi Yamaki, Daisuke Matsumoto, Tatsuro Kamijima, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Sigeharu Takiya, Makoto Demura, Keiichi Kawano
    Proteins 72 (1) 414 - 26 2008/07 
    Fibroin-modulator-binding protein 1 (FMBP-1) is a predicted transcription factor of the silkworm fibroin gene. The DNA-binding domain of FMBP-1 consists of four almost perfect tandem repeats of 23 amino acids each (R1-R4), and is referred to as the score and three amino acid peptide repeat (STPR) domain. This characteristic domain is conserved in eukaryotes, but the DNA-binding mode is not known. In this study, the structural properties of the DNA-bound form of the STPR domain were characterized. The combined experiments indicated that the STPR domain bound to the DNA duplex with a 1:1 binding ratio. The specific DNA caused considerable changes in the thermal unfolding profile and the digestion pattern of the STPR domain. These data suggested that the domain adapts a quite rigid helix-rich structure in the DNA-bound state, even though it moves flexibly in the absence of DNA. Furthermore, mutual induced-fit conformational change was also observed in DNA. Finally, we determined the DNA-binding surface of the STPR third repeat (R3) by alanine scanning mutagenesis; a particular site, composed of hydrophobic and hydrophilic residues, was identified. Notably, the substitution of Arg-9 in R3 with alanine residue, which is located in the middle of the surface, drastically abolished the alpha-helix-inducing and DNA-binding abilities. From these results, we predicted the DNA-binding mode of the STPR domain.
  • Mineyuki Mizuguchi, Ayumi Hayashi, Makoto Takeuchi, Mizuki Dobashi, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano
    Proteins 72 (1) 261 - 9 2008/07 
    Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wild-type transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C-terminal fragments starting near amino acid residue 50 as well as full-length transthyretin. Although a number of previous studies suggest the importance of the C-terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C-terminal fragments of transthyretin. To understand the role of C-terminal fragments in SSA, we examined the effects of the truncation of the N-terminal portions on the structure and aggregation properties of wild-type transthyretin. The deletion mutant lacking 50 N-terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self-assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N-terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self-assembly into aggregations in SSA.
  • Yasuhiro Nonaka, Tomoyasu Aizawa, Daisuke Akieda, Masanori Yasui, Masahiro Watanabe, Nobuhisa Watanabe, Isao Tanaka, Masakatsu Kamiya, Mineyuki Mizuguchi, Makoto Demura, Keiichi Kawano
    Proteins 72 (1) 313 - 22 2008/07 
    Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.
  • Wei Wang, Soichiro Itoh, Atsushi Matsuda, Tomoyasu Aizawa, Makoto Demura, Shizuko Ichinose, Kenichi Shinomiya, Junzo Tanaka
    Journal of biomedical materials research. Part A 85 (4) 919 - 28 2008/06/15 
    We have developed a novel bilayered chitosan tube that comprises an outer layer of chitosan film and an inner layer of chitosan nonwoven nano/microfiber mesh. The tube is fabricated with an electrospinning method. We characterized the microstructure and mechanical properties of this material. We introduced glycine spacers into the CYIGSR sequence, domain of laminin-1 that enhances Schwann cells migration and attachment, as well as neural outgrowth, resulting in the amino acid sequences CGGYIGSR and CGGGGGGYIGSR. These peptides were covalently bound to the nano/microfiber mesh surface of the chitosan tube to examine the effects of peptide mobility on nerve regeneration. Scaffolds constructed from these bilayered chitosan tubes were grafted to bridge injured sciatic nerve. Isografting was performed as a control. These scaffolds were removed 5 and 10 weeks after implantation for histological analysis. Nerve regeneration into chitosan tubes, on which the CGGGGGGYIGSR peptide was immobilized, exhibited efficacy similar to that of the isograft and represent a promising candidate for promoting peripheral nerve repair.
  • D. Akieda, Y. Nonaka, N. Watanabe, I. Tanaka, M. Kamiya, T. Aizawa, K. Nitta, M. Demura, K. Kawano
    2008/05/27
  • Hamadate Tsukasa, Nodzumi Takuya, Tatami Fumie, Kamiya Masakatsu, Aizawa Tomoyasu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S139  2008
  • Tatami Fumie, Hojo Eri, Tomisawa Satoshi, Kamiya Masakatsu, Aizawa Tomoyasu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S138  2008
  • Tsukamoto Takashi, Sasaki Takanori, Kikukawa Takashi, Miyauchi Seiji, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S142  2008
  • Oyauchi Keisuke, Kamiya Masakatsu, Yokoyama Takuya, Wang Mofei, Aizawa Tomoyasu, Demura Makoto, Suzuki Koichi, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S83  2008
  • Aizawa Tomoyasu, Saito Shin, Yokoyama Takuya, Wang Mofei, Myoba Shohei, Kamiya Masakatsu, Kumaki Yasuhiro, Takiya Sigeharu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S27  2008
  • Nonaka Yasuhiro, Akieda Daisuke, Watanabe Nobuhisa, Tanaka Isao, Kamiya Masakatsu, Aizawa Tomoyasu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S36  2008
  • Kubo Megumi, Kikukawa Takashi, Miyauchi Seiji, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S115  2008
  • Yamashita Yasutaka, Kikukawa Takashi, Miyauchi Seiji, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 48 S115 - S116 2008
  • Yu Kitago, Shuichi Karita, Nobuhisa Watanabe, Masakatsu Kamiya, Tomoyasu Aizawa, Kazuo Sakka, Isao Tanaka
    The Journal of biological chemistry 282 (49) 35703 - 11 0021-9258 2007/12/07 
    The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 A. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a beta-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu(186) and Glu(359), are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a (1)H NMR experiment using the reduced cellooligosaccharide as a substrate.
  • Takahide Kouno, Mineyuki Mizuguchi, Hiromasa Tanaka, Ping Yang, Yoshihiro Mori, Hiroyuki Shinoda, Kana Unoki, Tomoyasu Aizawa, Makoto Demura, Koichi Suzuki, Keiichi Kawano
    Biochemistry 46 (48) 13733 - 41 0006-2960 2007/12/04 
    Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.
  • Y. Nonaka, D. Akieda, N. Watanabe, I. Tanaka, M. Kamiya, T. Aizawa, K. Nitta, M. Demura, K. Kawano
    2007/11/27
  • Hiroyasu Nakatani, Kosuke Maki, Kimiko Saeki, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Shuji Tomoda, Kunihiro Kuwajima
    Biochemistry 46 (17) 5238 - 51 0006-2960 2007/05/01 
    The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.
  • Naoki Fujitani, Takahide Kouno, Taku Nakahara, Kenji Takaya, Tsukasa Osaki, Shun-Ichiro Kawabata, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Shin-Ichiro Nishimura, Keiichi Kawano
    Journal of peptide science : an official publication of the European Peptide Society 13 (4) 269 - 79 1075-2617 2007/04 
    Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.
  • Shin Saito, Tomoyasu Aizawa, Kyosuke Kawaguchi, Takeshi Yamaki, Daisuke Matsumoto, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Sigeharu Takiya, Makoto Demura, Keiichi Kawano
    Biochemistry 46 (7) 1703 - 13 0006-2960 2007/02/20 
    Fibroin-modulator-binding protein 1 (FMBP-1) is a factor that binds the transcriptional activation elements of the fibroin gene. It has a novel structure, consisting of four tandem repeats (R1-R4) of 23 amino acids each in the C-terminal half. This region is referred to as the STPR (score and three amino acid peptide repeat) domain and acts as a DNA-binding domain in FMBP-1. Interestingly, the homology among the four repeats is remarkably high. Here, we have determined the three-dimensional structures of the four repeats by NMR. All four repeat units have basically the same structure: a short alpha-helix in the N-terminal half maintained by a salt bridge and an N-capping box. CD studies showed that the full-length STPR domain was 31% helical in solution. This is explained by the connections among the four short helices that were determined separately by NMR. From the thermal-denaturation study, it can be deduced that these four helices in the full-length STPR domain moved flexibly with no interaction among them. However, the specific DNA caused a distinct increase, of up to 76%, in the alpha-helical content of the full-length STPR domain. This finding suggests that the binding of the full-length STPR domain to specific DNA causes an induced-fit conformational change that increases alpha-helicity; the poorly structured regions of the protein may form a regular secondary structure. Furthermore, the mutation analysis showed that the four repeats of the STPR domain raise the possibility of interaction with DNA in different ways.
  • Makoto Takeuchi, Mineyuki Mizuguchi, Takahide Kouno, Yoshinori Shinohara, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Keiichi Kawano
    Proteins 66 (3) 716 - 25 2007/02/15 
    Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin.
  • 加茂 直樹, 相沢 智康, 永山 昌史
    Seibutsu Butsuri The Biophysical Society of Japan General Incorporated Association 47 (1) 62 - 63 0582-4052 2007/01/25
  • Masanori Yasui, Taku Miyahara, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    The protein journal 25 (7-8) 475 - 82 1572-3887 2006/12 
    In order to investigate the thermodynamics of the unfolding of metalloproteins, the thermal denaturation of bovine alpha-lactalbumin (BLA), a typical calcium-binding protein, was investigated under a wide variety of calcium ion activities by means of differential scanning calorimetry. The excess heat capacity obtained as above is composed of those of the following three reactions: (i) the release of a calcium ion from holo-BLA; (ii) the capture of the released calcium ion by the chelating reagent; and (iii) the denaturation of native apo-BLA. The results indicated that the presence of the chelating reagent had a remarkable effect on the apparent enthalpy change for the denaturation of holo-BLA. On the other hand, the influence of the chelator on the heat capacity change was shown to be negligible. Because the denaturation reaction of holo-BLA includes Reactions (i) and (iii), it had to be handled as a three-state reaction. Such an investigation of the unfolding has been scarcely found that the activity of the metal ion is controlled precisely in wide range.
  • Ken Arita, Masashi Akiyama, Tomoyasu Aizawa, Yoshitaka Umetsu, Ikuo Segawa, Maki Goto, Daisuke Sawamura, Makoto Demura, Keiichi Kawano, Hiroshi Shimizu
    The American journal of pathology 169 (2) 416 - 23 0002-9440 2006/08 
    Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient's keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional (1)H nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication.
  • Masahito Tada, Yoshinori Shinohara, Ichiro Kato, Koichi Hiraga, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Mineyuki Mizuguchi, Keiichi Kawano
    Acta histochemica et cytochemica 39 (2) 31 - 4 0044-5991 2006/04/22 
    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.
  • Nodzumi Takuya, Shimamoto Satoshi, Akieda Daisuke, Nonaka Yasuhiro, Aizawa Tomoyasu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S301  2006
  • Yokoyama Takuya, Saito Shin, Aizawa Tomoyasu, Takiya Shigeharu, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S301  2006
  • Hojo Eri, Kamiya Masakatsu, Aizawa Tomoyasu, Miyazawa Mitsuhiro, Kato Yusuke, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S181  2006
  • Nakatani Hiroyasu, Maki Kosuke, Saeki Kimiko, Aizawa Tomoyasu, Demura Makoto, Kawano Keiichi, Kuwajima Kunihiro
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S173  2006
  • Kamijima Tatsuro, Sato Toshiya, Akimoto Kaoru, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S165  2006
  • Sugita Keitaro, Ohkubo Tomoyuki, Kamiya Masakatsu, Kamijima Tatsuro, Shimamoto Satoshi, Tatami Fumie, Aizawa Tomoyasu, Mizuguchi Mineyuki, Kawabata Shun-ichiro, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S147  2006
  • Kamiya Masakatsu, Nakatogawa Shin-ichi, Oda Yasunori, Kamijima Tatsuro, Aizawa Tomoyasu, Kumaki Yasuhiro, Hayakawa Yoichi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S147  2006
  • Umetsu Yoshitaka, Aizawa Tomoyasu, Muto Kaori, Yamamoto Hiroko, Kamiya Masakatsu, Kumaki Yasuhiro, Mizuguchi Mineyuki, Demura Makoto, Hayakawa Yoichi, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S147  2006
  • Saito Yuko, Sasaki Takanori, Kubo Megumi, Kikukawa Takashi, Seki Akiteru, Miyauchi Seiji, Aizawa Tomoyasu, Kawano Keiichi, Kamo Naoki, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S373  2006
  • Matsunami Masatoshi, Tatami Fumie, Kubo Megumi, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S373  2006
  • Sasaki Takanori, Aizawa Tomoyasu, Kamiya Masakatsu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S374  2006
  • Habara Mina, Kubo Megumi, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S374  2006
  • Kubo Megumi, Sasaki Takanori, Saito Yuko, Kikukawa Takashi, Kamo Naoki, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S374  2006
  • Higuchi Marika, Sasaki Takanori, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S374  2006
  • Tanji Takahiro, Umetsu Yoshitaka, Kamiya Masakatsu, Aizawa Tomoyasu, Kumaki Yasuhiro, Arita Ken, Akiyama Masashi, Shimizu Hiroshi, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S337  2006
  • Bak Seongmu, Kamiya Masakatsu, Umetsu Yoshitaka, Aizawa Tomoyasu, Kouno Takahide, Mizuguchi Mineyuki, Kumaki Yasuhiro, Demura Makoto, Kawano Keiichi
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S274  2006
  • YOSHIDA Teruaki, NODZUMI Takuya, AKIEDA Daisuke, SIMAMOTO Satoshi, AIZAWA Tomoyasu, KAWANO Keiichi, DEMURA Makoto
    Seibutsu Butsuri 一般社団法人 日本生物物理学会 46 (2) S228  2006
  • Satoshi Watanabe, Masahito Tada, Tomoyasu Aizawa, Masanobu Yoshida, Tadamasa Sugaya, Makoto Taguchi, Takahide Kouno, Takashi Nakamura, Mineyuki Mizuguchi, Makoto Demura, Yoichi Hayakawa, Keiichi Kawano
    Protein and peptide letters 13 (8) 815 - 22 0929-8665 2006 
    GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.
  • Mineyuki Mizuguchi, Atsushi Matsuura, Yuko Nabeshima, Kazuo Masaki, Masahiro Watanabe, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta, Yoshihiro Mori, Hiroyuki Shinoda, Keiichi Kawano
    Proteins 61 (2) 356 - 65 2005/11/01 
    The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.
  • Megumi Kubo, Maki Sato, Tomoyasu Aizawa, Chojiro Kojima, Naoki Kamo, Mineyuki Mizuguchi, Keiichi Kawano, Makoto Demura
    Biochemistry 44 (39) 12923 - 31 0006-2960 2005/10/04 
    Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-d-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-d-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 mM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <--> OG-NpHR (disassembled) <--> intermediate --> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.
  • Takashi Nakamura, Hiroshi Takasugi, Tomoyasu Aizawa, Masanobu Yoshida, Mineyuki Mizuguchi, Yoshihiro Mori, Hiroyuki Shinoda, Yoichi Hayakawa, Keiichi Kawano
    Journal of biotechnology 116 (3) 211 - 9 0168-1656 2005/03/30 
    Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.
  • Maki Sato, Megumi Kubo, Tomoyasu Aizawa, Naoki Kamo, Takashi Kikukawa, Katsutoshi Nitta, Makoto Demura
    Biochemistry 44 (12) 4775 - 84 0006-2960 2005/03/29 
    Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.
  • Makoto Demura, Tsutomu Yoshida, Takatsugu Hirokawa, Yasuhiro Kumaki, Tomoyasu Aizawa, Katsutoshi Nitta, Isván Bitter, Klára Tóth
    Bioorganic & medicinal chemistry letters 15 (5) 1367 - 70 0960-894X 2005/03/01 
    The molecular recognition of neurotransmitters, dopamine and acetylcholine with an amphiphilic resorcinarene receptor was investigated in an aqueous sodium dodecylsulfate (SDS) micelle system by 1H NMR measurements. The interaction distances of these neurotransmitters from the hydrophilic cavity of the amphiphilic receptor were estimated based on the calculation of the ring current shift using the atomic coordinates obtained from molecular dynamics calculation.
  • Masanobu Yoshida, Tomoyasu Aizawa, Takashi Nakamura, Kunio Shitara, Yoichi Hayakawa, Kimiaki Matsubara, Kazunori Miura, Takahide Kouno, Kevin D Clark, Michael R Strand, Mineyuki Mizuguchi, Makoto Demura, Katsutoshi Nitta, Keiichi Kawano
    The Journal of biological chemistry 279 (49) 51331 - 7 0021-9258 2004/12/03 
    Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.
  • Masahiro Watanabe, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    Biochimica et biophysica acta 1702 (2) 129 - 36 0006-3002 2004/11/01 
    The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degrees C. The DeltaV values at 25 degrees C were obtained for the N-MG (-46 cm3/mol) and MG-unfolded-state (U) transition (-40 cm3/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm3/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate.
  • Masahiro Watanabe, Yoshihiro Kobashigawa, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    Protein and peptide letters 11 (4) 325 - 30 0929-8665 2004/08 
    The effect of pressure on the unfolding of the molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet spectroscopy. The volume changes of the MG-unfolded-state transition were observed at pH 2.0 and around 20 to 60 degrees C, but no volume change has been found for bovine alpha-lactalbumin, which is homologous to CML. Our results suggest that the MG state of CML possesses a tightly packed hydrophobic core.
  • Masahiro Watanabe, Yoshihiro Kobashigawa, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    The protein journal 23 (5) 335 - 42 1572-3887 2004/07 
    The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.
  • Katsuharu Sato, Takashi Nakamura, Mineyuki Mizuguchi, Kazunori Miura, Masahito Tada, Tomoyasu Aizawa, Tomoharu Gomi, Kaoru Miyamoto, Keiichi Kawano
    FEBS letters 553 (3) 232 - 8 0014-5793 2003/10/23 
    Epiregulin (EPR), a novel member of epidermal growth factor (EGF) family, is a ligand for ErbB-1 and ErbB-4 receptors. The binding affinity of EPR for the receptors is lower than those of other EGF-family ligands. The solution structure of EPR was determined using two-dimensional nuclear magnetic resonance spectroscopy. The secondary structure in the C-terminal domain of EPR is different from other EGF-family ligands because of the lack of hydrogen bonds. The structural difference in the C-terminal domain may provide an explanation for the reduced binding affinity of EPR to the ErbB receptors.
  • Masahito Tada, Tomoyasu Aizawa, Yoshinori Shinohara, Kimiaki Matsubara, Kazunori Miura, Masanobu Yoshida, Kunio Shitara, Takahide Kouno, Mineyuki Mizuguchi, Katsutoshi Nitta, Yoichi Hayakawa, Keiichi Kawano
    The Journal of biological chemistry 278 (12) 10778 - 83 0021-9258 2003/03/21 
    Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.
  • Naoki Fujitani, Motoi Kanagawa, Tomoyasu Aizawa, Tadayasu Ohkubo, Shunji Kaya, Makoto Demura, Keiichi Kawano, Shin-ichiro Nishimura, Kazuya Taniguchi, Katsutoshi Nitta
    Biochemical and biophysical research communications 300 (1) 223 - 9 0006-291X 2003/01/03 
    It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.
  • Kazunori Miura, Manabu Kamimura, Tomoyasu Aizawa, Makoto Kiuchi, Yoichi Hayakawa, Mineyuki Mizuguchi, Keiichi Kawano
    Peptides 23 (12) 2111 - 6 0196-9781 2002/12 
    Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of "ENF" is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second beta-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.
  • Atsushi Matsuura, Min Yao, Tomoyasu Aizawa, Nozomi Koganesawa, Kazuo Masaki, Mitsuhiro Miyazawa, Makoto Demura, Isao Tanaka, Keiichi Kawano, Katsutoshi Nitta
    Biochemistry 41 (40) 12086 - 92 0006-2960 2002/10/08 
    Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.
  • Tetsuya Suetake, Tomoyasu Aizawa, Nozomi Koganesawa, Tsukasa Osaki, Yoshihiro Kobashigawa, Makoto Demura, Shun-Ichiro Kawabata, Keiichi Kawano, Sakae Tsuda, Katsutoshi Nitta
    Protein engineering 15 (9) 763 - 9 0269-2139 2002/09 
    Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.
  • Tomoyasu Aizawa, Yoichi Hayakawa, Katsutoshi Nitta, Keiichi Kawano
    Molecules and cells 14 (1) 1 - 8 1016-8478 2002/08/31 
    Growth-blocking peptide (GBP) is an insect cytokine that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells. GBP is a 25-amino acid peptide with one disulfide bond. It has been revealed that the tertiary structure of GBP consists of an N- and C-terminal disordered region and a well-structured core. Although there is only a slight similarity between the primary structures of GBP and EGF and the molecular weight of GBP is about half that of EGF, GBP directly binds and activates the EGF receptor of human keratinocyte cells. Furthermore, the tertiary structure of the well-defined region of GBP is similar to that of the C-terminal domain of EGF. This review will focus on the tertiary structure of GBP and its activities, as compared with those of EGF.
  • Nozomi Koganesawa, Tomoyasu Aizawa, Hiroshi Shimojo, Kazunori Miura, Atsushi Ohnishi, Makoto Demura, Yoichi Hayakawa, Katsutoshi Nitta, Keiichi Kawano
    Protein expression and purification 25 (3) 416 - 25 1046-5928 2002/08 
    A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.
  • Kazunori Miura, Hideki Doura, Tomoyasu Aizawa, Hiroko Tada, Masaharu Seno, Hidenori Yamada, Keiichi Kawano
    Biochemical and biophysical research communications 294 (5) 1040 - 6 0006-291X 2002/06/28 
    The solution structure of the EGF-like domain of betacellulin (BTCe), a newly discovered member of the epidermal growth factor (EGF) family, has been determined using two-dimensional nuclear magnetic resonance spectroscopy. This is the first report to identify the solution structure of the EGF-family ligand monomers that interact with both ErbB-1 and ErbB-4. The solution structure of BTCe was calculated using 538 NMR-derived restraints. The overall structure of BTCe was stabilized by three disulfide bonds, a hydrophobic core, and 23 hydrogen bonds. It appears that BTCe is comprised of five beta-strands and one short 3(10) helical turn. The secondary structural elements of BTCe are basically similar to those of the other EGF-family proteins, except that several significant variations of the structural properties were found. It is suggested that the structural variations between BTCe and the other EGF-family ligands may affect the specific receptor-recognition properties of EGF-family ligands.
  • Yusuke Kato, Tomoyasu Aizawa, Hirokazu Hoshino, Keiichi Kawano, Katsutoshi Nitta, Hong Zhang
    The Biochemical journal 361 (Pt 2) 221 - 30 0264-6021 2002/01/15 
    Two genes encoding the ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptide, abf-1 and abf-2, were identified in Caenorhabditis elegans. Recombinant ABF-2 exhibited potent microbicidal activity against Gram-positive and Gram-negative bacteria, and yeasts. The tissue-specific distribution estimated by immunofluorescence staining and transgenic analysis of a gfp fusion gene (where GFP corresponds to green fluorescent protein) suggested that ABF-2 contributes to surface defence in the pharynx. abf-1 contains a single intron at a conserved position, suggesting that asabf and abf originated from a common ancestor. Both transcripts for abf-1 and abf-2 were detected as two distinct forms, i.e. spliced leader (SL)1-trans-spliced with a long 5'-untranslated region (UTR) and SL-less with a short 5'-UTR. A polycistronic precursor RNA encoding ABF-1 and ABF-2 was detected, suggesting that these genes form an operon. An 'opportunistic operon' model for regulation of abf genes, including the generation of short SL-less transcripts, is proposed. In conclusion, C. elegans should have an immune defence system due to the antimicrobial peptides. C. elegans can be a novel model for innate immunity. Furthermore, the combination of biochemical identification in Ascaris suum and homologue hunting in C. elegans should be a powerful method of finding rapidly evolved proteins, such as some immune-related molecules in C. elegans.
  • Matsuura A, Yao M, Aizawa T, Koganesawa N, Masaki K, Miyazawa M, Demura M, Tanaka I, Kawano K, Nitta K
    Biochemistry 44 (36) 12086 - 12093 0006-2960 2002 [Refereed][Not invited]
  • N Koganesawa, T Aizawa, K Masaki, A Matsuura, T Nimori, H Bando, K Kawano, K Nitta
    Protein engineering 14 (9) 705 - 10 0269-2139 2001/09 
    Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.
  • K Masaki, T Aizawa, N Koganesawa, T Nimori, H Bando, K Kawano, K Nitta
    Journal of protein chemistry 20 (2) 107 - 13 0277-8033 2001/02 
    Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26 degrees C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.
  • T Aizawa, N Fujitani, Y Hayakawa, A Ohnishi, T Ohkubo, Y Kumaki, K Kawano, K Hikichi, K Nitta
    The Journal of biological chemistry 274 (4) 1887 - 90 0021-9258 1999/01/22 
    Growth-blocking peptide (GBP) is an insect growth factor consisting of 25 amino acid residues that retards the development of lepidopteran larvae at high concentration while it stimulates larval growth at low concentration. In this study, we determined the solution structure of GBP by two-dimensional 1H NMR spectroscopy. The structure contains a short segment of double-stranded beta-sheet involving residues 11-13 and 19-21 and a type-II beta-turn in the loop region (residues 8-11), whereas the N and C termini are disordered. This is the first report of the three-dimensional structure of the peptiderigic insect growth factor, and the structure of the well defined region of GBP was found to share similarity with that of the C-terminal domain of the epidermal growth factor (EGF). Because GBP has been reported to stimulate DNA synthesis of not only insect cells but also human keratinocyte cells at the same level with EGF, the structural similarity between GBP and EGF may lead to the interaction of GBP to EGF receptor.
  • T Aizawa, N Koganesawa, A Kamakura, K Masaki, A Matsuura, H Nagadome, Y Terada, K Kawano, K Nitta
    FEBS letters 422 (2) 175 - 8 0014-5793 1998/01/30 
    To elucidate hydroxyapatite-protein interaction, mutant human lysozymes in which the surface charge was modified by site-directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys-13 and Arg-10 are located around Lys-1 and Arg-14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X-ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg-14, Lys-1, Arg-10 and Lys-13 play important roles in binding.

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Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : ISHIZUKA Satoshi
     
    Fatty liver is associated with development of noncommunicable diseases. inflammation and fibrosis occur based on lipid accumulation in the development of fatty liver. Although a high-fat-fed experimental animals are a well-known fatty liver model, those animals frequently have both lipid accumulation and inflammation in the liver, and no reasonable model is reported to separate lipid accumulation and inflammation with fibrosis. We observed an enhancement of secretion of 12α-hydroxylated bile acids from the liver in response to dietary energy and those bile acids induces liver lipid accumulation without inflammation and fibrosis. Also, induction of inflammation was found to aggravate the symptom substantially. This can be used as a presymptomatic model of fatty liver.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/04 -2021/03 
    Author : AIZAWA Tomoyasu
     
    We proceeded with the study of an allergenic protein, BP14 derived from European cypress. BP14 is thought to be the cause of pollen-food allergy syndrome (PFAS). We succeeded in identifying the BP14 gene by analysis with a next-generation sequencer. This sequence was registered as an allergen Cup s 7 in the WHO / IUIS database. By using recombinant Cup s 7 protein, the reactivity with patient serum was examined, and the three-dimensional structure was analyzed by the NMR method.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014 -2016 
    Author : AIZAWA TOMOYASU
     
    Antimicrobial peptides are considered an essential part of the innate immune response of plants, invertebrates and vertebrates as they provide host defenses and can target a wide range of pathogenic microorganisms, including bacteria, fungi, yeast, parasites and viruses. Antimicrobial peptides are thought to kill bacteria by breaking their cell membranes, although the exact mechanisms are still unclear. In this study, we investigated efficient way to prepare isotopically labelled antimicrobial peptides for NMR studies. By using an isotopically labelled antimicrobial peptide, we obtained structural information for LPS-bound form.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : KIMURA Katsuki, HINOU Hiroshi, AIZAWA Tomoyasu
     
    Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically purified via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was demonstrated that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was limited, implying that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) play an important role in the evolution of membrane fouling in MBRs.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/04 -2015/03 
    Author : HAYAKAWA Yoichi, AIZAWA Tomoyasu, MATSUMOTO Hitoshi, FURIHATA Shunsuke
     
    Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that GBP overexpression in Drosophila elevated expression of AMPs. GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential, indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.
  • 文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2011 -2012 
    Author : 相沢 智康
     
    カイコの絹の主成分であるフィプロインの時期特異的かつ部位特異的な転写を制御する因子FMBP-1から発見されたDNA結合ドメインであるSTPRドメインは、9塩基対からなるATリッチの特異的配列に結合する。このドメインは、23残基の極めて配列相同性の高いリピート配列が4回繰り返した特徴的なタンデムリピート構造を有しており、代表者の研究成果から、遊離状態では揺らぎの大きな天然変性構造をとるにもかかわらず、DNA結合状態ではα-helixリッチな構造を形成することが明らかになった。そこで、このDNA結合ドメインのDNAとの相互作用に伴う立体構造形成に関する、動的認識メカニズムを明らかにすることが本研究の目的である。まず、遊離状態のSTPRドメインのNMRスペクトルは天然変性蛋白質に特徴的なものであるが、安定同位体標識試料により帰属が可能であると考えられるため、本年度はこのNMRスペクトルの解析を重点的に進めた。今後、さらに帰属作業を進め、各種のスペクトルを詳細に解析することで、STPRドメインの揺らぎとDNA認識について残基レベルでの解析が期待できる。さらに細胞、絹糸腺でのFMBP-1の動的挙動についてFCSを用いた解析を進めた。STPRドメインを含むFMBP-1が核内でDNAを認識し転写活性化を行う際のFMBP-1とその変異体の振る舞いについて、Hela細胞を用いた系と絹糸腺でのFCS解析を進め、実際の生体内でのDNA認識に関する動的挙動に関していくつかの有用な知見を得ることに成功した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2009 
    Author : HAYAKAWA Yoichi, AIZAWA Tomoyasu
     
    Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to each other and a foreign surface. Although structure-activity studies have been extensively performed in GBP and its mutants of Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified a novel adaptor protein (P77) with a molecular mass of 77kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C-terminus. Although P77 did not have the capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after addition of GBP to a plasmatocyte suspension. Tyrosine phosphorylation of P77 was also observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. We further detected tyrosine phosphorylation of the integrin β subunit in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin β subunit, but also abolished the GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae following infection with Serratia marcescens, thus indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrated that GBP signaling in plasmatocytes requires the adaptor protein P77 and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))
    Date (from‐to) : 2007 -2008 
    Author : Tomoyasu AIZAWA
     
    チョウやガの仲間である鱗翅目昆虫、アワヨトウから発見された多機能サイトカインGBP は、血球の活性化などの生体防御や幼虫の成長に関連して重要な役割を担っていると推定されるが、その分子レベルでの作用機構については未知の点が多く残されている。GBP の各種変異体を調整し、その立体構造と活性の関係について検討を行うことで、昆虫の寄生に伴う成長抑制や、創傷や感染と関連した血球の働き等に関して興味深い成果を得ることに成功した。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2006 -2007 
    Author : 出村 誠, 河野 敬一, 相沢 智康, 神谷 昌克
     
    リゾチーム・αラクトアルブミンのフォールディングに関する基礎データを活かし、水溶性タンパク質のフォールディング挙動と、その構造中間体が示す細胞膜結合機能との関連を分子レベルで解明することを目的としている。昨年度までに、新規複合体調製法の開発、酸処理法で得られた脂肪酸結合型αラクトアルブミンのアポトーシス誘導活性等に関する成果が得られ、今年度新たに以下の成果が得られた。(1)酸処理法と熱処理法で得られた脂肪酸結合型ウシαラクトアルブミンの生理的条件下での近紫外CDスペクトルは、オレイン酸濃度に依存して三次構造崩壊を示唆する楕円率低下が起こる。NMRでも脂肪酸結合型ウシαラクトアルブミンは非天然構造型スペクトルパターンを示した。脂肪酸結合型ウシαラクトアルブミンの1H NMRスペクトルおよび15N HSQCスペクトルでは、脂肪酸結合による化学シフト変化はαラクトアルブミンのαドメインで大きく、しかもβドメイン側に面するアミノ酸残基であることがわかった。(2)αβαの3つのドメインからなるヒトとウシ由来のαラクトアルブミン(HHHおよびBBB)のドメインを入れかえたキメラ体(HBB,BHB,BBH)との比較から、アポトーシス誘導に関与しているドメインについて考察した。熱処理法で得られたサンプルではMG状態での安定性とアポトーシス活性の強い相関があった。(3)αラクトアルブミンの天然構造の示す水溶性と基質選択性補因子機能は、Molten Globule様構造を酸性処理という簡便な方法で脂肪酸トラップすることができ、細胞死誘導活性という新規の生物機能に変換することができた。今後、αラクトアルブミンと脂肪酸による複合体のNMRキャラクタリゼーションをさらに詳細に検討することで、構造中間体のトラップとそのダイナミクス情報が得られると期待できる。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2005 -2006 
    Author : 河野 敬一, 水口 峰之, 出村 誠, 相沢 智康
     
    カブトガニ血球蛋白質のLPS認識の分子機構を明らかにするために、tachyplesinIを取り上げ、まずDPCミセル中の立体構造を検討した。DPCミセル中のtachyplesinIの構造については議論中であるが、より詳細な解析を可能とするために遺伝子工学を用いた組み換え体の作成に着手し、NMR解析のための同位体ラベル試料の調製に成功した。 また、DPC存在下のtachyplesinIのHSQCスペクトルからDPCミセルとの相互作用によりtachyplesinIの構造が変化することが明らかになった。CD14、MD2については、札医大黒木教授の協力を得て精製蛋白質を得ることができた。 精製量の少ない段階で、とりあえずCDによる相互作用解析を行い、相互作用によるスペクトル変化を観測することができた。この変化の一部は結合によるロイシンリッチリピートの円弧の大きさが変化したことによると思われる。 カブトガニ血球中に見出された抗菌タンパク質tachystatinBの水溶液中における立体構造を解析し、この蛋白質が機能時に2量体を形成することを提案した(研究発表4)。 他に膜インタフェース関連の研究としては、細胞の構成タンパク質を観察するための新規の方法をGFPトランスジェニックマウスを用いた系で開発した(研究発表1)。 細胞間の情報伝達に関わっている膜タンパク質コネキシン(CX26)のN末端部分の変異(N14Y)が角膜炎や難聴の患者に多く見られることを明らかにした。また、その変異がN末端部分の運動性を変化させることがNMRを用いた解析により明らかになり、病気のメカニズムの一端を明らかにした(研究発表2)。 昆虫のサイトカインであるGBPと膜上のレセプターとの相互作用におけるGBPのN末端部分の役割を網羅的な変異体実験により明らかにした(研究発表3)。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2006 
    Author : DEMURA Makoto, AIZAWA Tomoyasu, KAWANO Keiichi, NITTA Katsutoshi
     
    α-lactalbumin is a calcium binding protein found in mammalian milk. It is known that α-lactalbumin is one of the components of lactose synthase, which catalyzes the final step in lactose biosynthesis in the lactating mammary gland, and the other component of this system is galactosyltransferase (GT). Most of α-lactalbumin, including human, guinea pig, bovine, goat, camel, equine and rabbit proteins, consist of 123 amino acid residues. Native a-lactalbumin has two structural domains: a large α-helical domain and a small β-sheet domain. The structure is stabilized by four disulfide bridges such as lysozyme. α-lactalbumin forms molten globule state (folding intermediates) at low pH or calcium free state. In this study, development and structural characteristics of the apoptosis-inducing α-lactalbumin as a "non-native" and novel biological function were investigated in detail. A simple and useful method for preparation of non-native structure just like the MG-state which have induction activity of tumor-cell death were developed from not only human but also bovine α-lactalbumin.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2006 
    Author : KAWANO Keiichi, HAYAKAWA Yoichi, KAWABATA Shun-ichiro, MIZUGUCHI Mineyuki, AIZAWA Tomoyasu, DEMURA Makoto
     
    Growth-blocking peptide (GBP) is a small insect cytokine with various activities: larval growth regulation, cell proliferation, and stimulation of a class of lepidopteran insect blood cells (plasmatocytes). GBP was initially identified as a 25 amino acid peptide hormone (1-25 GBP), which suppress the growth of host armyworm, found in the hemolymph of the parasitized armyworm. However, in non-parasitized armyworm larvae, 23 amino acid GBP is expressed in insect plasma. Subsequent studies revealed that the parasitization by wasp induced an elongation of the C-terminal amino acid sequence of GBP (1-28 GBP). In this study, we characterized the GBP analogs, which consist of various length of C-terminal region, by comparison of their biological activities and three-dimensional structures. The results indicate that 1-28 GBP exhibits the strongest activity to suppress the larval growth. NMR analysis shows that these peptides have the same tertiary structures basically, ^-which consist of a disordered N-terminal region, a structured core stabilized by a disulfide bond and an antiparallel β-sheet in aqueous solution. However, the C-terminal region of 1-28 GBP undergoes conformational transition from a random-coiled state to an a-helical state in the presence of dodecylphosphocholine (DPC) micelles. It suggests that the C-terminal region bound to membrane would have an affects on the larval growth activity. We isolated, identified, and determined the solution structure of a novel peptide from lepidopteran, Pseudaletia separata that induces rapid hemocyte aggregation. The isolated peptide from crude integument extract, designated hemocyte-aggregation factor (AGF), was revealed to contain 32 amino acid residues (H_2N-SVQILRCPDGMQMLRSGQCVATTEPPFDPDSY-COOH) by N-terminal amino acid sequencing and 3'-RACE. NMR spectra of ^<15>N-labelled AGF were recorded at 283-313 K and pH 5.0. The full sequential assignment for all non-proline residues was achieved using ^<15>N-edited TOCSY and NOESY spectra. The solution structure was calculated by CYANA and discussed in relevance to the functional feature of AGF.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2004 -2005 
    Author : 出村 誠, 新田 勝利, 新田 勝利, 相沢 智康, 河野 敬一, 出村 誠
     
    α-ラクトアルブミン(α-LA)はミルクに含まれる123残基からなるカルシウム結合蛋白質で、β-1,4-ガラクトース転移酵素の基質選択性の補因子機能をもつ。α-LAの構造は天然状態において、N末端側1-34残基とC末端側86-123残基にα-helical構造を形成し、中間の35-85残基はβ-sheet構造を持つ。低pHやカルシウムフリーの条件下では折りたたみ中間体(MG状態)を形成する。一方、ヒトミルクから単離されたオレイン酸-α-LA複合体が癌細胞死を誘導する新たな機能が報告された。 しかし、生物種ごとのα-LAのMG状態形成との関係や、α-LAの活性化部位、オレイン酸結合部位などの詳細は不明である。本年度は昨年度に引き続き、本研究ではホモロジーの高いヒト型α-LA(HLA)、ウシ型α-LA(BLA)およびこれらの遺伝子組み換え体を用い、各種脂肪酸-α-LA複合体の調整方法の考案と細胞死誘導活性を評価した。調整方法として従来よりもより簡便な熱処理法を考案した。この方法は、従来法より多くの利点をもつので、特許出願した。 この方法により、牛乳からアポトーシス誘導型調整試料を作製できた。
  • 文部科学省：科学研究費補助金(特定領域研究)
    Date (from‐to) : 2004 -2005 
    Author : 相沢 智康, 河野 敬一, 出村 誠
     
    本年度は得られた遺伝子組換え蛋白質試料を用いて、精密な立体構造を決定し、活性発現と立体構造の関係を解明するため、相互作用解析等を進めた。変異体を用いた解析の結果、ASABFについては、現在まで知られるCSαβモチーフ抗菌ペプチドには保存されていないC末端のフレキシブル領域が存在し、抗菌活性の維持に重要な働きを有することを明らかにした。CSαβモチーフを有する抗菌ペプチドは、昆虫をはじめとする節足動物を中心に、軟体動物、植物まで有することが知られており、ごく最近、真菌からも発見されている。しかし、このいずれにもC末端のフレキシブル領域は存在しておらず、このファミリーの進化を知る上でも、また抗菌活性の発現メカニズムを解明する上でも興味深い。さらに、ミセルとの相互作用解析の結果などをあわせて考察すると、このフレキシブル領域は、膜との初期のインタラクションに重要な役割を果たしていると考えられる。また、NMR法を用いた緩和測定による分子の内部の残基の運動性に関する解析の結果から、N末端に存在するループ領域が極めて特徴的な運動性を示すことが明らかになった。昆虫由来のCSαβモチーフを有するペプチドでは、この領域の電荷の存在が、膜相互作用時に複合体を形成しポアを形成するために重要であることが示唆されており、興味深い結果といえる。サイトカインGBPについては、このGBPを持つアワヨトウ幼虫が寄生蜂であるカリヤコマユバチに寄生された際におこる成長抑制作用と免疫系の抑制に、寄生の際に感染するポリドナウイルスによるGBPのC末端の伸張が関係している可能性を分子レベルで明らかにすることに成功し、論文として投稿中である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2005 
    Author : HAYAKAWA Yoichi, SHIMADA Kimio, AIZAWA Tomoyasu
     
    Growth-blocking peptide (GBP) is a member of an insect cytokine family. GBP is a small peptide consists of 25 amino acid residues, but it exerts diverse functions including larval growth retardation, cell proliferation, paralysis induction, cardioacceleration, immune cell stimulation, and morphogenesis. In this study, deletion analogs of GBP were synthesized to investigate the relationship between the structure of GBP and the biological activities such as mitogenic and plasmatocyte (a type of hemocytes) spreading activities. The results indicated that the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP, indicating the possibility that multifunctional properties of this peptide may be mediated by different forms of GBP receptors. In order to demonstrate this speculation, we analyzed GBP receptors in different types of cells by a competitive receptor binding assay with ^<125>I-GBP. The results showed that High Five cells contain two types of GBP receptors : high affinity (K_d=0.26) and low affinity (K_d=13.7), while hemocytes contain a single class of GBP receptor with a medium affinity (K_d=1.26). These results demonstrated a heterogeneity of GBP receptors at least in insect culture cells and hemocyte cells. Further, we identified two different types of GBP receptor candidates in hemocytes, approximately 60 kDa and 77 kDa proteins. The former protein does not contain any distinct domain structures other than one transmembrane domain. However, the latter protein contains one transmembrane domain, and ITAM (immunoreceptor tyrosine-based activation motif) and IgG like domains in the outer cell region and inner cell region, respectively. We further found that tyrosine residues in the cytoplasmic region of this molecule are phosphorylated by GBP. We are now trying to demonstrate whether GBP specifically binds to these cell membrane proteins.
  • 文部科学省：科学研究費補助金(特定領域研究)
    Date (from‐to) : 2003 -2003 
    Author : 相沢 智康
     
    線虫由来の抗菌ペプチドASABF及びABFについて、そのクローニング及びメタノール代謝酵母を宿主として用いた大量発現系の構築を行い、活性、立体構造に関する解析を進めた。その結果、モデル生物として重要性の高いC.elegansにおいて世界で初めて抗菌ペプチドの同定に成功し、NMR法による立体構造解析を進め、インセクトデフェンシンファミリーと類似性の高いものであることを明らかにした。またカブトガニ由来の抗菌ペプチドであるタキサイチンについて、大腸菌を宿主として用い、インクルージョンボディーからのリフォールディングを行うことで、大量発現系の構築に成功し、安定同位体ラベル試料を調整することで、立体構造と活性の関係について詳細な検討を進めた。その結果、タキサイチンのキチン結合能を有する抗真菌活性発現と電荷が重要である推定される抗グラム陰性、抗グラム陽性活性発現では分子内の異なる部位が重要な役割を果たすことを明らかにした。この他、メタノール代謝酵母を用いた大量発現系により生産した、カイコガ由来リゾチームについてその立体構造解析、安定性解析などをすすめ、ニワトリや哺乳類などの一般的なリゾチームには見られない低温活性などの活性を有すること、またその活性と構造の関連性に関して知見を得たほか、鱗翅目昆虫由来の異物排除に関連する血球活性化因子GBPについて変異体を用いて立体構造と活性に関する研究を展開した。
  • 文部科学省：科学研究費補助金(若手研究(B))
    Date (from‐to) : 2002 -2003 
    Author : 相沢 智康
     
    多機能サイトカインGBPに対して結合能を有する調節蛋白質GBPBPの大量発現構築に、大腸菌を利用した発現系を用いて成功したのでこれを利用し、GBPBPと、GBP間の相互作用について表面プラズモン共鳴センサーを用いて解析を進めた。また、このGBPBPをN末端、C末端ドメインに分割した発現系を構築し、その結合能の解析を行なった。その結果、GBPとGBPBPの相互作用は、各ドメインのみでは観察されず、両ドメインが必要なこと、また生体内の環境と近い生理食塩水中では、GBPBPの立体構造がかなりの速さで失われ結合能も失っていくことが明らかになった。また、GBP分子のN末端の分子の自由度を高めている残基についての重要性を検討するために、これらの領域に対する変異を導入した変異体の作成を重点的に行い、機能と立体構造相関に関する知見を蓄積した。自由度を高めている要因と考えられるGly残基を自由度の低い残基に置換した変異体をプローブとすることで、受容体のリガンド結合部位に関する知見を得た。また、この知見を基にアンタゴニストとして働くペプチドの作成に成功した。さらに、GBPの立体構造形成に重要と考えられるTyr11をAlaに置換した変異体では、溶液中での立体構造が失われるにもかかわらず、活性を保持するという極めて興味深い現象を発見した。さらに詳細な解析から、受容体結合時に本来の分子の立体構造が誘導形成され、活性を発現しているという可能性を示唆する研究結果を得た。
  • NMR法と遺伝子工学を用いたタンパク質の立体構造と機能の関係の解明
  • Studies for elucidating the relationship between tertiary structure of proteins and its functions by using NMR and protein engineering.

Educational Activities

Teaching Experience

  • Soft Matter Analysis
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : high-resolution NMR, MRI, protein, peptide, three-dimensional structure, metabolomics, drug screening
  • Special Lecture in Soft Matter Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : NMR, solution NMR, solid state NMR, rheo-NMR, protein, three-dimensional structure, molecular interaction, rheology, structural calculation
  • Soft Matter Analysis
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 分光法、NMR、細胞膜、タンパク質科学、バイオマテリアル、機能性ポリマー、プレゼンテーション
  • Bioinformation and Molecular Sciences
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 分光法、NMR、細胞膜、タンパク質科学、バイオマテリアル、機能性ポリマー、プレゼンテーション
  • Soft Matter Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • Transdisciplinary Life Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • International workshop management training
    開講年度 : 2021
    課程区分 : 博士後期課程
    開講学部 : 生命科学院
    キーワード : 研究集会、国際交流
  • Molecular Genetic Science II
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 遺伝情報、ゲノム、DNA、RNA、タンパク質、転写、翻訳、遺伝子発現、転写調節因子、遺伝的変動、遺伝子重複、系統樹、相同遺伝子、点変異、トランスポゾン、ウイルス、ヒトゲノム
  • Analytical Chemistry for Life Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 分析化学、機器分析、ＮＭＲ
  • Laboratory Work on Bio-macromolecular Science II
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験，細胞生物学実験, 生物物理学実験
  • Experimental Biological Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 高分子機能学，実験技術，生命科学，物質科学，情報科学，融合科学
  • Laboratory Work on Bio-macromolecular Science I
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験，細胞生物学実験, 生物物理学実験
  • Fundamental Laboratory Work on Bio-macromolecular Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 基礎実験手法，物理学実験，化学分析実験，有機合成実験，生化学実験，安全教育，法令

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