研究者データベース

相沢 智康(アイザワ トモヤス)
先端生命科学研究院
教授

基本情報

所属

  • 先端生命科学研究院

職名

  • 教授

学位

  • 博士(理学)(北海道大学)

ホームページURL

J-Global ID

研究キーワード

  • 抗菌ペプチド   核磁気共鳴   蛋白質工学   構造生物学   蛋白質科学   

研究分野

  • ライフサイエンス / 食品科学
  • ナノテク・材料 / 生体化学
  • ライフサイエンス / 構造生物化学
  • ライフサイエンス / 生物物理学

職歴

  • 2019年01月 - 現在 北海道大学 教授
  • 2019年01月 - 現在 北海道大学(大学院先端生命科学研究院) 教授
  • 2007年 - 2018年 北海道大学(大学院先端生命科学研究院) 助教授
  • 2007年 - 2018年 Associate Professor
  • 2006年 - 2007年 北海道大学(大学院先端生命科学研究院) 助手
  • 2006年 - 2007年 Research Associate
  • 2007年 - 北海道大学(大学院先端生命科学研究院) 准教授
  • 2001年 - 2006年 北海道大学(大学院理学研究科生物科学専攻) 助手
  • 2001年 - 2006年 Research Associate
  • 2001年 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
  • 2001年 Researcher

学歴

  •         - 2001年   北海道大学   理学研究科   生物科学専攻
  •         - 2001年   北海道大学
  •         - 1998年   北海道大学   理学研究科   生物科学専攻
  •         - 1998年   北海道大学
  •         - 1996年   北海道大学   理学部   高分子学科
  •         - 1996年   北海道大学

所属学協会

  • 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   

研究活動情報

論文

  • Poncet P, Aizawa T, Sénéchal H
    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 49 8 1163 - 1166 2019年08月 [査読有り][通常論文]
  • Iizuka A, Kajimoto K, Fujisawa T, Tsukamoto T, Aizawa T, Kamo N, Jung KH, Unno M, Demura M, Kikukawa T
    Scientific reports 9 1 10711  2019年07月 [査読有り][通常論文]
  • Hasemi T, Kikukawa T, Watanabe Y, Aizawa T, Miyauchi S, Kamo N, Demura M
    Biochimica et biophysica acta. Bioenergetics 1860 2 136 - 146 2019年02月 [査読有り][通常論文]
  • Sénéchal H, Keykhosravi S, Couderc R, Selva MA, Shahali Y, Aizawa T, Busnel JM, Arif R, Mercier I, Pham-Thi N, Charpin DA, Poncet P
    Allergy, asthma & immunology research 11 1 143 - 151 2019年01月 [査読有り][通常論文]
  • Tsukamoto T, Kikuchi C, Suzuki H, Aizawa T, Kikukawa T, Demura M
    Scientific reports 2018年09月 [査読有り][通常論文]
  • Penkhrue W, Sujarit K, Kudo T, Ohkuma M, Masaki K, Aizawa T, Pathom-Aree W, Khanongnuch C, Lumyong S
    International journal of systematic and evolutionary microbiology 2018年03月 [査読有り][通常論文]
  • 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
    JOURNAL OF EXPERIMENTAL MEDICINE 214 12 3507 - 3518 2017年12月 [査読有り][通常論文]
     
    The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as alpha-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 a-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of a-defensins. Administration of R-Spo1 or recombinant a-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年11月 [査読有り][通常論文]
     
    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. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520-1528, 2017
  • Sénéchal H, Šantrůček J, Melčová M, Svoboda P, Zídková J, Charpin D, Guilloux L, Shahali Y, Selva MA, Couderc R, Aizawa T, Poncet P
    The Journal of allergy and clinical immunology 2017年08月 [査読有り][通常論文]
  • Nakamura S, Kikukawa T, Tamogami J, Kamiya M, Aizawa T, Hahn MW, Ihara K, Kamo N, Demura M
    Biochimica et biophysica acta 1857 12 1900 - 1908 2016年12月 [査読有り][通常論文]
  • Hiroaki Ishida, Leonard T. Nguyen, Ramamourthy Gopal, Tomoyasu Aizawa, Hans J. Vogel
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 138 35 11318 - 11326 2016年09月 [査読有り][通常論文]
     
    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 FSW, lactoferrampin B, MIP3 alpha(51-70), and human beta-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年06月 [査読有り][通常論文]
     
    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 H-1 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. (C) 2016 Elsevier Inc. All rights reserved.
  • 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 22 4 214 - 221 2016年04月 [査読有り][通常論文]
     
    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 -helical structure in a solution containing LPS. For NMR experiments, we expressed N-15-labeled and C-13-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 N-15-edited and C-13-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 -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. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.
  • 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 - 125 2016年02月 [査読有り][通常論文]
     
    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 (c) two types of transient interactions between FMBP-1 and chromosomal DNA, and (d) stable binding of FMBP-1 to chromosomal DNA.
  • Tamaki, Hajime, Egawa, Ayako, Kido, Kouki, Kameda, Tomoshi, Kamiya, Masakatsu, Kikukawa, Takashi, Aizawa, Tomoyasu, Fujiwara, Toshimichi, Demura, Makoto
    JOURNAL OF BIOMOLECULAR NMR 64 1 87 - 101 2016年01月 [査読有り][通常論文]
     
    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 C alpha RMSD of 1.49 angstrom relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn2+ 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.
  • Tomisawa S, Sato Y, Kamiya M, Kumaki Y, Kikukawa T, Kawano K, Demura M, Nakamura K, Ayabe T, Aizawa T
    Protein expression and purification 112 21 - 28 2015年08月 [査読有り][通常論文]
     
    Mammalian alpha-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of alpha-defensins, large amounts of alpha-defensins are essential. Although many expression systems for the production of recombinant alpha-defensins have been developed, attempts to obtain large amounts of alpha-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 alpha-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil 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 alpha-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step. (C) 2015 Elsevier Inc. All rights reserved.
  • Kikukawa T, Kusakabe C, Kokubo A, Tsukamoto T, Kamiya M, Aizawa T, Ihara K, Kamo N, Demura M
    Biochimica et biophysica acta 1847 8 748 - 758 2015年08月 [査読有り][通常論文]
  • 相沢智康, 米北太郎, 北條江里
    Bio Ind 32 3 42 - 48 2015年03月12日 [査読無し][通常論文]
  • Taro Miyoshi, Yuhei Nagai, Tomoyasu Aizawa, Katsuki Kimura, Yoshimasa Watanabe
    WATER SCIENCE AND TECHNOLOGY 72 6 844 - 849 2015年 [査読有り][通常論文]
     
    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 beta-barrel protein. These findings strongly suggest the importance of beta-barrel proteins in developing membrane fouling in MBRs.
  • Kushibiki T, Kamiya M, Aizawa T, Kumaki Y, Kikukawa T, Mizuguchi M, Demura M, Kawabata S, Kawano K
    Biochimica et biophysica acta 1844 527 - 534 3 2014年03月 [査読有り][通常論文]
  • Kousuke Shibasaki, Hiroaki Shigemura, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOCHEMISTRY 52 51 9257 - 9268 2013年12月 [査読有り][通常論文]
     
    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, P-3, was a quasi-equilibrium complex between the N and O intermediates, where a N <-> O + Cl- equilibrium was attained. The K-d,K-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, Delta V-N -> O, were estimated from the pressure dependence of K-d,K-N <-> O. A comparison between K-d,K-N <-> O and Delta V-N -> O led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K-d,K-N <-> O. For some mutants, however, large Delta V-N -> O values were found, whereas the K-d,K-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,K-N <-> O. Mutants with a small K-d,K-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.
  • Takashi Tsukamoto, Xianglan Li, Hiromi Morita, Takashi Minowa, Tomoyasu Aizawa, Nobutaka Hanagata, Makoto Demura
    PLOS ONE 8 9 e75831  2013年09月 [査読有り][通常論文]
     
    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.
  • 相沢智康
    Bio Ind 30 7 35 - 40 2013年07月12日 [査読無し][通常論文]
  • Taro Yonekita, Ryuji Ohtsuki, Eri Hojo, Naoki Morishita, Takashi Matsumoto, Tomoyasu Aizawa, Fumiki Morimatsu
    JOURNAL OF MICROBIOLOGICAL METHODS 93 3 251 - 256 2013年06月 [査読有り][通常論文]
     
    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. a-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. (C) 2013 Elsevier B.V. All rights reserved.
  • Nakamura T, Aizawa T, Kariya R, Okada S, Demura M, Kawano K, Makabe K, Kuwajima K
    The Journal of biological chemistry 288 20 14408 - 14416 20 2013年05月 [査読有り][通常論文]
     
    Although HAMLET human alpha-lactalbumin made lethal to tumor cells), a complex formed by human alpha-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 alpha-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 degrees 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 beta(2)-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.
  • 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 45  2013年 [査読有り][通常論文]
     
    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.
  • Mineyuki Mizuguchi, Makoto Takeuchi, Shinya Ohki, Yuko Nabeshima, Takahide Kouno, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Katsuhide Yutani
    BIOCHEMISTRY 51 31 6089 - 6096 2012年08月 [査読有り][通常論文]
     
    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 degrees 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 DI state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of alpha 4- and alpha 6-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 DI state. Therefore, in the folding process from the DI state to the native state, the alpha 4- and alpha 6-helices become separated and the central beta-sheet is folded between these helices. That is, the non-native interaction between the alpha 4- and a6-helices may be responsible for the unusually slow folding of PCP-0SH.
  • 相沢 智康, 出村 誠, 河野 敬一
    蚕糸・昆虫バイオテック = Sanshi-konchu biotec 81 2 115 - 123 2012年08月01日 [査読無し][通常論文]
  • Takashi Tsukamoto, Takanori Sasaki, Kazuhiro J. Fujimoto, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOPHYSICAL JOURNAL 102 12 2906 - 2915 2012年06月 [査読有り][通常論文]
     
    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.
  • 原田 尚樹, 柏崎 晴彦, 赤澤 敏之, 村田 勝, 相沢 智康, 出村 誠, 田中 順三, 飯塚 正, 井上 農夫男
    北海道歯学雑誌 32 2 166 - 176 北海道歯学会 2012年03月15日 [査読無し][通常論文]
     
    【背景・目的】ハイドロキシアパタイト(HAp)は生体親和性と骨伝導性に優れた生体材料であるが,その硬さと脆性のため,望む形状に成形することが困難である.それゆえ,HApの欠点である成形性を改善するHAp/高分子の新規複合材料の開発に多くの関心が集まっている.キトサンは甲殻類の外殻などに含まれる天然高分子で,その生体吸収性や高い熱安定性などの性質から生体材料として注目されている.我々はこれまでに,多孔性キトサン/HAp複合体を作製し,歯槽骨再生材料に適した柔軟性のある物性を持つことを報告してきた.本研究では,この複合体の骨形成蛋白質(rhBMP-2)担体としての有用性を評価する目的で,ラット頭頂骨骨膜下埋入実験を行い,骨形成過程と担体複合体の吸収変化を組織形態学的に検討した.【方法】共沈澱法とポローゲンリーチング法により多孔性キトサン/HAp複合体を作製し,5μg のrhBMP-2を添加した.rhBMP-2無添加の複合体を対照として,10週齢のSDラット頭頂骨骨膜下に埋入し,4,8週後に屠殺し,組織学的観察および形態計測を行った.【結果】rhBMP-2添加多孔性キトサン/HAp複合体では,埋入4,8週後に複合体の周辺部から中央部にかけて多数の細胞侵入および骨形成が認められた.対照群では骨形成はみられなかった.形態計測した結果,埋入4,8週後における複合体の占有率はrhBMP-2...
  • Yamashita Y, Kikukawa T, Tsukamoto T, Kamiya M, Aizawa T, Kawano K, Miyauchi S, Kamo N, Demura M
    Biochimica et biophysica acta 1808 2905 - 2912 12 2011年12月 [査読有り][通常論文]
  • 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 - 569 2011年01月 [査読有り][通常論文]
     
    Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a beta-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the beta-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down beta-sheets. This up-and-down beta-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 beta-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 beta-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. (C) 2010 Elsevier Ltd. All rights reserved.
  • Wei Wang, Soichiro Itoh, Tomoyasu Aizawa, Atsushi Okawa, Katsuyoshi Sakai, Tsuneo Ohkuma, Makoto Demura
    BIOMEDICAL MATERIALS 5 6 065009  2010年12月 [査読有り][通常論文]
     
    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-alpha) assay by ELISA. The inflammatory cell infiltration and release of TNF-alpha 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 - 8375 2010年09月 [査読有り][通常論文]
     
    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 a-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 a-helix structure content.
  • Takahashi M, Mizuguchi M, Shinoda H, Aizawa T, Demura M, Okazawa H, Kawano K
    Biochimica et biophysica acta 1804 1500 - 1507 7 2010年07月 [査読有り][通常論文]
  • 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 16 5 242 - 248 2010年05月 [査読有り][通常論文]
     
    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. Copyright (C) 2010 European Peptide Society and John Wiley & Sons, Ltd.
  • Takanori Sasaki, Tomoyasu Aizawa, Masakatsu Kamiya, Takashi Kikukawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    BIOCHEMISTRY 48 51 12089 - 12095 2009年12月 [査読有り][通常論文]
     
    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. Oil 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 wits determined via size-exclusion chromatography, depended oil 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 similar to 2 at file 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
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 43 29625 - 29634 2009年10月 [査読有り][通常論文]
     
    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.
  • Shigeharu Takiya, Shin Saito, Takuya Yokoyama, Daisuke Matsumoto, Tomoyasu Aizawa, Masakatsu Kamiya, Makoto Demura, Keiichi Kawano
    JOURNAL OF BIOCHEMISTRY 146 1 103 - 111 2009年07月 [査読有り][通常論文]
     
    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.
  • Takahashi M, Mizuguchi M, Shinoda H, Aizawa T, Demura M, Okazawa H, Kawano K
    Biochimica et biophysica acta 1794 936 - 943 6 2009年06月 [査読有り][通常論文]
  • 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 19 9 779 - 785 2009年05月 [査読有り][通常論文]
     
    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
    FEBS JOURNAL 276 8 2192 - 2200 2009年04月 [査読有り][通常論文]
     
    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.
  • Megumi Kubo, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 2 547 - 555 2009年03月 [査読有り][通常論文]
     
    Halorhodopsin (HR) acts as a light-driven chloride pump which transports a chloride ion from the extracellular (EC) to the cytoplasmic space during a photocycle reaction that includes some photointermediates initiated by illumination. To understand the chloride uptake mechanisms, we focused on a basic residue Arg123 of HR from Natronomonas pharaonis (NpHR), which is the only basic residue located in the EC half ion channel. By the measurements of the visible absorption spectra in the dark and the light-induced inward current through the membrane, it was shown that the chloride binding and transport ability of NpHR completely disappeared by the change of arginine to glutamine. From flashphotolysis analysis, the photocycle of R123Q differed from that of wildtype NpHR completely. The response of the R123H mutant depended on pH. These facts imply that the positive charge at position 123 is essential for chloride binding in the ground state and for the chloride uptake under illumination. On the basis of the molecular structures of HR and the anion-transportable mutants of bacteriorhodopsin, the effects of the positive charge and the conformational change of the Arg123 side chain as well as the chloride-pumping mechanism are discussed.
  • Takanori Sasaki, Megumi Kubo, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
    PHOTOCHEMISTRY AND PHOTOBIOLOGY 85 1 130 - 136 2009年01月 [査読有り][通常論文]
     
    Halorhodopsin (HR) is a transmembrane seven-helix retinal protein, and acts as an inward light-driven Cl(-) pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 mm) to 1% (20 mm) dodecyl-beta-d-maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 mm). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton-coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR-DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross-linked NpHR by glutaraldehyde gave the SDS-PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR (Escherichia coli and Halobacterium salinarum), CD spectra showed exciton-coupling, which suggests strongly the trimer structure in the cell membrane.
  • 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 - 214 2008年11月 [査読有り][通常論文]
     
    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. (C) 2008 Elsevier Inc. All rights reserved.
  • Takahide Kouno, Mineyuki Mizuguchi, Masakiyo Sakaguchi, Eiichi Makino, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demur, Nam-Ho Huh, Keiichi Kawano
    JOURNAL OF PEPTIDE SCIENCE 14 10 1129 - 1138 2008年10月 [査読有り][通常論文]
     
    S100A11 protein is a member of the S100 family containing two EF-hand motifs. It undergoes phophorylation on residue T10 after cell stimulation such as an increase in Ca2+ concentration. Phosphorylated S100A11 I 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. Copyright (C) 2008 European Peptide Society and John Wiley & Sons, Ltd.
  • 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-STRUCTURE FUNCTION AND BIOINFORMATICS 72 1 414 - 426 2008年07月 [査読有り][通常論文]
     
    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 wit 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 alamne 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 -i-helix-inducing and DNA-binding abilities. From these results, we predicted the DNA-binding mode of the STPR domain.
  • Yasuhiro Nonaka, Tomoyasu Aizawa, Daisuke Akieda, Masanori Yasui, Masahiro Watanabe, Nobuhisa Watanabe, Isao Tanaka, Masakatsu Kamiya, Mineyuki Mizuguchi, Makoto Demura, Keiichi Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72 1 313 - 322 2008年07月 [査読有り][通常論文]
     
    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 85A 4 919 - 928 2008年06月 [査読有り][通常論文]
     
    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. (C) 2007 Wiley Periodicals, Inc.
  • Yu Kitago, Shuichi Karita, Nobuhisa Watanabe, Masakatsu Kamiya, Tomoyasu Aizawa, Kazuo Sakka, Isao Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 282 49 35703 - 35711 2007年12月 [査読有り][通常論文]
     
    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 angstrom. 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 - 13741 2007年12月 [査読有り][通常論文]
     
    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 H-1 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 antifungai 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.
  • Hiroyasu Nakatani, Kosuke Maki, Kimiko Saeki, Tomoyasu Aizawa, Makoto Demura, Keiichi Kawano, Shuji Tomoda, Kunihiro Kuwajima
    BIOCHEMISTRY 46 17 5238 - 5251 2007年05月 [査読有り][通常論文]
     
    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 13 4 269 - 279 2007年04月 [査読有り][通常論文]
     
    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 angstrom, 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. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.
  • Makoto Takeuchi, Mineyuki Mizuguchi, Takahide Kouno, Yoshinori Shinohara, Tomoyasu Aizawa, Makoto Demura, Yoshihiro Mori, Hiroyuki Shinoda, Keiichi Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 66 3 716 - 725 2007年02月 [査読有り][通常論文]
     
    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. Proteins 2007;66:716-725. (c) 2006 Wiley-Liss, Inc.
  • 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 - 1713 2007年02月 [査読有り][通常論文]
     
    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.
  • Masanori Yasui, Taku Miyahara, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta
    PROTEIN JOURNAL 25 7-8 475 - 482 2006年12月 [査読有り][通常論文]
     
    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
    AMERICAN JOURNAL OF PATHOLOGY 169 2 416 - 423 2006年08月 [査読有り][通常論文]
     
    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 H-1 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.
  • 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 - 822 2006年 [査読有り][通常論文]
     
    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 H-1 NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core P-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 Asn(2). 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.
  • 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 - 34 2006年 [査読有り][通常論文]
     
    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.
  • M Mizuguchi, A Matsuura, Y Nabeshima, K Masaki, M Watanabe, T Aizawa, M Demura, K Nitta, Y Mori, H Shinoda, K Kawano
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 61 2 356 - 365 2005年11月 [査読有り][通常論文]
     
    The N-terminal half of the a-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of a-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.
  • M Kubo, M Sato, T Aizawa, C Kojima, N Kamo, M Mizuguchi, K Kawano, M Demura
    BIOCHEMISTRY 44 39 12923 - 12931 2005年10月 [査読有り][通常論文]
     
    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.
  • T Nakamura, H Takasugi, T Aizawa, M Yoshida, M Mizuguchi, Y Mori, H Shinoda, Y Hayakawa, K Kawano
    JOURNAL OF BIOTECHNOLOGY 116 3 211 - 219 2005年03月 [査読有り][通常論文]
     
    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. (C) 2004 Elsevier B.V. All rights reserved.
  • M Demura, T Yoshida, T Hirokawa, Y Kumaki, T Aizawa, K Nitta, Bitter, I, K Toth
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 15 5 1367 - 1370 2005年03月 [査読有り][通常論文]
     
    The molecular recognition of neurotransmitters, dopamine and acetylcholine with an amphiphilic resorcinarene receptor was investigated in an aqueous sodium dodecylsulfate (SDS) micelle system by H-1 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. (c) 2005 Elsevier Ltd. All rights reserved.
  • M Sato, M Kubo, T Aizawa, N Kamo, T Kikukawa, K Nitta, M Demura
    BIOCHEMISTRY 44 12 4775 - 4784 2005年03月 [査読有り][通常論文]
     
    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 Ar123. 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.
  • M Yoshida, T Aizawa, T Nakamura, K Shitara, Y Hayakawa, K Matsubara, K Miura, T Kouno, KD Clark, MR Strand, M Mizuguchi, M Demura, K Nitta, K Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 49 51331 - 51337 2004年12月 [査読有り][通常論文]
     
    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 Phe(3)) 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 Gly(5) or Gly(6) 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 Phe(3) is a binding determinant of the N-terminal domain.
  • Watanabe M, Aizawa T, Demura M, Nitta K
    Biochimica et biophysica acta 1702 129 - 136 2 2004年11月 [査読有り][通常論文]
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta
    PROTEIN AND PEPTIDE LETTERS 11 4 325 - 330 2004年08月 [査読有り][通常論文]
     
    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 60degreesC. but 110 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.
  • M Watanabe, Y Kobashigawa, T Aizawa, M Demura, K Nitta
    PROTEIN JOURNAL 23 5 335 - 342 2004年07月 [査読有り][通常論文]
     
    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.
  • K Sato, T Nakamura, M Mizuguchi, K Miura, M Tada, T Aizawa, T Gomi, K Miyamoto, K Kawano
    FEBS LETTERS 553 3 232 - 238 2003年10月 [査読有り][通常論文]
     
    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. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • M Tada, T Aizawa, Y Shinohara, K Matsubara, K Miura, M Yoshida, K Shitara, T Kouno, M Mizuguchi, K Nitta, Y Hayakawa, K Kawano
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 12 10778 - 10783 2003年03月 [査読有り][通常論文]
     
    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 N-15-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.
  • N Fujitani, M Kanagawa, T Aizawa, T Ohkubo, S Kaya, M Demura, K Kawano, S Nishimura, K Taniguchi, K Nitta
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 1 223 - 229 2003年01月 [査読有り][通常論文]
     
    It has been well established that phosphotylation 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. (C) 2002 Elsevier Science (USA). All rights reserved.
  • K Miura, M Kamimura, T Aizawa, M Kiuchi, Y Hayakawa, M Mizuguchi, K Kawano
    PEPTIDES 23 12 2111 - 2116 2002年12月 [査読有り][通常論文]
     
    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. (C) 2002 Elsevier Science Inc. All rights reserved.
  • A Matsuura, M Yao, T Aizawa, N Koganesawa, K Masaki, M Miyazawa, M Demura, Tanaka, I, K Kawano, K Nitta
    BIOCHEMISTRY 41 40 12086 - 12092 2002年10月 [査読有り][通常論文]
     
    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.
  • Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein
    T Suetake, T Aizawa, N Koganesawa, T Osaki, Y Kobashigawa, M Demura, S Kawabata, K Kawano, S Tsuda, K Nitta
    PROTEIN ENGINEERING 15 9 763 - 769 2002年09月 [査読有り][通常論文]
     
    Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The H-1-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, N-15-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 N-15 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.
  • N Koganesawa, T Aizawa, H Shimojo, K Miura, A Ohnishi, M Demura, Y Hayakawa, K Nitta, K Kawano
    PROTEIN EXPRESSION AND PURIFICATION 25 3 416 - 425 2002年08月 [査読有り][通常論文]
     
    A small multifunctional cytokine, growth-blocking peptide (GBP), front 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 48 h 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 50 mg GBP was recovered pet, 1 L of yeast culture supernatant. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Structure and activity of insect cytokine GBP which stimulates the EGF receptor
    T Aizawa, Y Hayakawa, K Nitta, K Kawano
    MOLECULES AND CELLS 14 1 1 - 8 2002年08月 [査読有り][通常論文]
     
    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.
  • K Miura, H Doura, T Aizawa, H Tada, M Seno, H Yamada, K Kawano
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 294 5 1040 - 1046 2002年06月 [査読有り][通常論文]
     
    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. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Y Kato, T Aizawa, H Hoshino, K Kawano, K Nitta, H Zhang
    BIOCHEMICAL JOURNAL 361 2 221 - 230 2002年01月 [査読有り][通常論文]
     
    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 Grain-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.
  • Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system
    N Koganesawa, T Aizawa, K Masaki, A Matsuura, T Nimori, H Bando, K Kawano, K Nitta
    PROTEIN ENGINEERING 14 9 705 - 710 2001年09月 [査読有り][通常論文]
     
    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.
  • Aizawa T, Hayakawa Y, Ohnishi A, Fujitani N, Clark KD, Strand MR, Miura K, Koganesawa N, Kumaki Y, Demura M, Nitta K, Kawano K
    The Journal of biological chemistry 276 34 31813 - 31818 34 2001年08月 [査読有り][通常論文]
  • K Masaki, T Aizawa, N Koganesawa, T Nimori, H Bando, K Kawano, K Nitta
    JOURNAL OF PROTEIN CHEMISTRY 20 2 107 - 113 2001年02月 [査読有り][通常論文]
     
    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 degreesC 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.
  • H Zhang, S Yoshida, T Aizawa, R Murakami, M Suzuki, N Koganezawa, A Matsuura, M Miyazawa, K Kawano, K Nitta, Y Kato
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 44 10 2701 - 2705 2000年10月 [査読有り][通常論文]
     
    ASABF is a CS alpha beta-type antimicrobial peptide that contains four intramolecular disulfide bridges (Y. Kato and S. Komatsu, J. Biol. Chem. 271:30493-30498, 1996). In the present study, a recombinant ASABF was produced hy using a yeast expression system, and its antimicrobial activity was characterized in detail. The recombinant ASABF was active against all gram-positive bacteria tested (7 of 7; minimum bactericidal concentration [MBC], 0.03 to 1 mu g/ml) except Leuconostoc mesenteroides, some gram-negative bacteria (8 of 14; MBC, >0.5 mu g/ml), and some yeasts (3 of 9; MBC >3 mu g/ml). Slight hemolytic activity (4.2% at 100 mu g/ml) against human erythrocytes was observed only under low-ionic-strength conditions. Less than 1 min of contact was enough to kill Staphylococcus aureus ATCC 6538P. The bactericidal activity against S. aureus was inhibited by salts.
  • 河野 敬一, 相沢 智康
    生物物理 39 2 109 - 112 日本生物物理学会 1999年03月25日 [査読無し][通常論文]
  • T Aizawa, N Fujitani, Y Hayakawa, A Ohnishi, T Ohkubo, Y Kumaki, K Kawano, K Hikichi, K Nitta
    JOURNAL OF BIOLOGICAL CHEMISTRY 274 4 1887 - 1890 1999年01月 [査読有り][通常論文]
     
    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 H-1 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 - 178 1998年01月 [査読有り][通常論文]
     
    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-l 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. (C) 1998 Federation of European Biochemical Societies.

その他活動・業績

特許

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 相沢 智康
  • 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))
    研究期間 : 2011年 -2012年 
    代表者 : 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。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2007年 -2008年 
    代表者 : 相沢 智康
     
    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。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2004年 -2005年 
    代表者 : 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。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2003年 -2003年 
    代表者 : 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))
    研究期間 : 2002年 -2003年 
    代表者 : 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.

教育活動情報

主要な担当授業

  • ソフトマター科学特別講義
    開講年度 : 2018年
    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
  • ソフトマター科学特別講義
    開講年度 : 2018年
    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, high pressure NMR, protein, three-dimensional structure, molecular interaction, pressure, structural calculation
  • 生命科学特別講義Ⅲ
    開講年度 : 2018年
    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
  • 生命科学特別講義Ⅲ
    開講年度 : 2018年
    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, high pressure NMR, protein, three-dimensional structure, molecular interaction, pressure, structural calculation
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2018年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
  • 生命情報分子科学特論
    開講年度 : 2018年
    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
  • ソフトマター解析学特論
    開講年度 : 2018年
    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
  • 国際研究集会企画プログラム
    開講年度 : 2018年
    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
  • 高分子機能学特別講義
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : high-resolution NMR, MRI, protein, peptide, three-dimensional structure, metabolomics, drug screening
  • 高分子機能学特別講義
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : NMR, solution NMR, solid state NMR, high pressure NMR, protein, three-dimensional structure, molecular interaction, pressure, structural calculation
  • 生物系の分析化学
    開講年度 : 2018年
    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
  • 分子遺伝科学Ⅱ
    開講年度 : 2018年
    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
  • 生体高分子学実験Ⅰ
    開講年度 : 2018年
    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
  • 高分子機能学基礎実験
    開講年度 : 2018年
    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

大学運営

委員歴

  • 2019年04月 - 2020年03月   日本核磁気共鳴学会   評議員
  • 2017年 - 2018年   日本生物物理学会   代議員
  • 2010年 - 2012年   日本生物物理学会   ホームページ編集委員長   日本生物物理学会
  • 2009年 - 2011年   日本生物物理学会   学会委員   日本生物物理学会
  • 2009年 - 2011年   日本生物物理学会   運営委員   日本生物物理学会
  • 2007年 - 2010年   日本生物物理学会   ホームページ編集委員   日本生物物理学会
  • 2007年 - 2009年   日本生物物理学会   運営委員   日本生物物理学会
  • 2007年 - 2009年   日本生物物理学会   学会委員   日本生物物理学会
  • 2005年 - 2006年   日本生物物理学会   生物物理編集地区委員   日本生物物理学会


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