研究者詳細 - 曲　正樹

論文 - 曲　正樹

分割表示 >> ／全件表示 62 件中 1 - 62 件目

Development of a novel AAK1 inhibitor via Kinobeads-based screening. 査読国際誌

Akari Yoshida, Satomi Ohtsuka, Fumiya Matsumoto, Tomoyuki Miyagawa, Rei Okino, Yumeya Ikeda, Natsume Tada, Akira Gotoh, Masaki Magari, Naoya Hatano, Ryo Morishita, Ayano Satoh, Yukinari Sunatsuki, Ulf J Nilsson, Teruhiko Ishikawa, Hiroshi Tokumitsu

Scientific reports 14 ( 1 ) 6723 - 6723 2024年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and β/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.

DOI： 10.1038/s41598-024-57051-9

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids 査読

Satomi Ohtsuka, Yumi Miyai, Hiroyuki Mima, Masaki Magari, Yoichi Chiba, Futoshi Suizu, Hiroyuki Sakagami, Masaki Ueno, Hiroshi Tokumitsu

Cell Calcium 102820 - 102820 2024年1月

Rapid detection of calmodulin/target interaction via the proximity biotinylation method. 査読国際誌

Kento Nlandu Nakamura, Haruki Yamauchi, Hiroyuki Mima, Yerun Chen, Satomi Ohtsuka, Masaki Magari, Ryo Morishita, Hiroshi Tokumitsu

Biochemical and biophysical research communications 659 29 - 33 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.

DOI： 10.1016/j.bbrc.2023.03.072

The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability. 査読国際誌

Masaki Magari, Miku Nishioka, Tomomi Hari, Sayaka Ogawa, Kaho Takahashi, Naoya Hatano, Naoki Kanayama, Junichiro Futami, Hiroshi Tokumitsu

FEBS letters 2022年8月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin β receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

DOI： 10.1002/1873-3468.14468

Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases 査読

Riku Kaneshige, Satomi Ohtsuka, Yuhei Harada, Issei Kawamata, Masaki Magari, Naoki Kanayama, Naoya Hatano, Hiroyuki Sakagami, Hiroshi Tokumitsu

The FEBS Journal 2022年5月

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063 査読

Satomi Ohtsuka, Taisei Okumura, Yuna Τabuchi, Tomoyuki Miyagawa, Naoki Kanayama, Masaki Magari, Naoya Hatano, Hiroyuki Sakagami, Futoshi Suizu, Teruhiko Ishikawa, Hiroshi Tokumitsu

Biochemistry 2022年3月

Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase 査読

Yusei Fukumoto, Yuhei Harada, Satomi Ohtsuka, Naoki Kanayama, Masaki Magari, Naoya Hatano, Hiroyuki Sakagami, Hiroshi Tokumitsu

Biochemical and Biophysical Research Communications 587 160 - 165 2022年1月

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins. 査読国際誌

Seita Doi, Naoki Fujioka, Satomi Ohtsuka, Rina Kondo, Maho Yamamoto, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Takafumi Hasegawa, Hiroshi Tokumitsu

Cell calcium 96 102404 - 102404 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

DOI： 10.1016/j.ceca.2021.102404

Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target. 査読国際誌

Maho Yamamoto, Rina Kondo, Haruka Hozumi, Seita Doi, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Hiroshi Tokumitsu

Biomolecules 11 ( 4 ) 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

DOI： 10.3390/biom11040510

Development and characterization of novel molecular probes for Ca2+/calmodulin-dependent protein kinase kinase, derived from STO-609. 査読国際誌

Satomi Ohtsuka, Yui Ozeki, Moeno Fujiwara, Tomoyuki Miyagawa, Naoki Kanayama, Masaki Magari, Naoya Hatano, Futoshi Suizu, Teruhiko Ishikawa, Hiroshi Tokumitsu

Biochemistry 59 ( 17 ) 1701 - 1710 2020年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes including neuronal, metabolic, and pathophysiological pathways. Herein we developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analog (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and β) with a similar potency (Ki = 0.35 µM for CaMKKα and Ki = 0.2 µM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV and endogenous AMPKα in HeLa cells with an IC50 of ~0.3 µM, and it suppressed the CaMKK isoforms-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

DOI： 10.1021/acs.biochem.0c00149

Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells. 査読国際誌

Shota Takabatake, Yusei Fukumoto, Satomi Ohtsuka, Naoki Kanayama, Masaki Magari, Hiroyuki Sakagami, Naoya Hatano, Hiroshi Tokumitsu

Biochemical and biophysical research communications 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

DOI： 10.1016/j.bbrc.2020.02.056

Regulation of Ca²⁺/calmodulin-dependent protein kinase kinase β by cAMP signaling. 査読国際誌

Takabatake S, Ohtsuka S, Sugawara T, Hatano N, Kanayama N, Magari M, Sakagami H, Tokumitsu H

Biochimica et biophysica acta. General subjects 1863 ( 4 ) 672 - 680 2019年4月

Interleukin 34 (IL-34) cell-surface localization regulated by the molecular chaperone 78-kDa glucose-regulated protein facilitates the differentiation of monocytic cells. 査読

Ogawa S, Matsuoka Y, Takada M, Matsui K, Yamane F, Kubota E, Yasuhara S, Hieda K, Kanayama N, Hatano N, Tokumitsu H, Magari M

The Journal of biological chemistry 294 ( 7 ) 2386 - 2396 2019年2月

Interaction of S100A6 with Target Proteins In Vitro and in Living Cells. 査読

Sakane K, Yamaguchi F, Tsuchiya M, Kondo R, Kanayama N, Magari M, Hatano N, Kobayashi R, Tokumitsu H

Methods in molecular biology (Clifton, N.J.) 1929 367 - 377 2019年

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca²⁺/calmodulin (CaM) dependence of Ca²⁺/CaM-dependent protein kinase kinase β. 査読

Nakanishi A, Hatano N, Fujiwara Y, Bin Shari A, Takabatake S, Akano H, Kanayama N, Magari M, Nozaki N, Tokumitsu H

The Journal of Biological Chemistry 292 ( 48 ) 19804 - 19813 2017年12月

Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target 査読

Kyohei Sakane, Miyu Nishiguchi, Miwako Denda, Fuminori Yamagchi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 491 ( 4 ) 980 - 985 2017年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, All, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.07.161

Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help 査読

Ting-ting Zhang, David G. Gonzalez, Christine M. Cote, Steven M. Kerfoot, Shaoli Deng, Yuqing Cheng, Masaki Magari, Ann M. Haberman

ELIFE 6 255 - 267 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELIFE SCIENCES PUBLICATIONS LTD

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6(hi) follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4(+) T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6(hi) follicular state is promoted by a regional and transient diminution of T cell help.

DOI： 10.7554/eLife.19552

SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus 査読

Yuka Kawaguchi, Hiroaki Nariki, Naoko Kawamoto, Yuichi Kanehiro, Satoshi Miyazaki, Mari Suzuki, Masaki Magari, Hiroshi Tokumitsu, Naoki Kanayama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 485 ( 2 ) 261 - 266 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSFI-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSFI-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, over expression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSFI-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C terminus-dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.02.097

Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen 査読

Yusui Furuya, Miwako Denda, Kyohei Sakane, Tomoko Ogusu, Sumio Takahashi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

CELL CALCIUM 60 ( 1 ) 32 - 40 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CHURCHILL LIVINGSTONE

To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ceca.2016.04.004

Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms 査読

Yuya Fujiwara, Yoshinori Kawaguchi, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

JOURNAL OF BIOLOGICAL CHEMISTRY 291 ( 26 ) 13802 - 13808 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK phosphorylates Thr(172) in the AMPK subunit more efficiently than CaMKK, with a lower K-m (approximate to 2 m) for AMPK, whereas the CaMKI phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK/CaMKK chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK/Ile(322) in CaMKK confer, at least in part, a distinct recognition of AMPK but not of CaMKI.

DOI： 10.1074/jbc.M116.727867

Ca2+/Calmodulin結合型転写因子の網羅的同定

太尾田泰成, 大西和貴, 古谷雄穂, 傳田美和子, 金山直樹, 曲正樹, 森下了, 徳光浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3P0185] - [3P0185] 2015年12月

インターラクトーム解析法を用いたヒトS100A6標的分子の探索

坂根恭平, 西口みゆ, 古谷雄穂, 傳田美和子, 山口文徳, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1P0204] - [1P0204] 2015年12月

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells 査読

Yuya Fujiwara, Yuri Hiraoka, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

BIOCHEMISTRY 54 ( 25 ) 3969 - 3977 2015年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

To assess the isoform specificity of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKK alpha mutant (Ala292Thr/Leu233Phe) and a CaMKK beta mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wildtype or the STO-609-resistant mutant of CaMKK alpha was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKK beta mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKK beta is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK. isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

DOI： 10.1021/acs.biochem.5b00149

Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators 査読

Takafumi Miyamoto, Elmer Rho, Vedangi Sample, Hiroki Akano, Masaki Magari, Tasuku Ueno, Kirill Gorshkov, Melinda Chen, Hiroshi Tokumitsu, Jin Zhang, Takanari Inoue

CELL REPORTS 11 ( 4 ) 657 - 670 2015年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CELL PRESS

AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell health, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPK alpha 1, not AMPK alpha 2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.

DOI： 10.1016/j.celrep.2015.03.057

ヒトCalmodulin標的分子の網羅的同定

古谷雄穂, 西口みゆ, 傳田美和子, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会プログラム・講演要旨集 87回 [4T14a - 01] 2014年10月

A Missense Mutation in Rev7 Disrupts Formation of Polζ, Impairing Mouse Development and Repair of Genotoxic Agent-induced DNA Lesions 査読

Maryam Khalaj, Abdolrahim Abbasi, Hiroshi Yamanishi, Kouyou Akiyama, Shuso Wakitani, Sotaro Kikuchi, Michiko Hirose, Misako Yuzuriha, Masaki Magari, Heba A. Degheidy, Kuniya Abe, Atsuo Ogura, Hiroshi Hashimoto, Tetsuo Kunieda

The Journal of Biological Chemistry 289 ( 6 ) 3811 - 3824 2014年2月

CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34 査読

Fumihiro Yamane, Yumiko Nishikawa, Kazue Matsui, Miki Asakura, Eriko Iwasaki, Koji Watanabe, Hikaru Tanimoto, Hiroki Sano, Yuki Fujiwara, E. Richard Stanley, Naoki Kanayama, Neil A. Mabbott, Masaki Magari, Hitoshi Ohmori

JOURNAL OF LEUKOCYTE BIOLOGY 95 ( 1 ) 19 - 31 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FEDERATION AMER SOC EXP BIOL

Occurrence of a CSF-1 receptor-dependent monocyte differentiation process driven by IL-34, but not CSF-1. With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b(+) monocytic cells (F4/80(+), Gr-1(-), Ly6C(-), I-A/E-/lo, CD11c(-), CD115(+), CXCR4(+), CCR2(+), CX(3)CR1(-)) when cultured with a Lin(-)c-kit(+) population from mouse spleen cells. The developed CD11b(+) cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

DOI： 10.1189/jlb.0613311

In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5 '-triphosphate as a phosphate donor 査読

Saki Yurimoto, Tomohito Fujimoto, Masaki Magari, Naoki Kanayama, Ryoji Kobayashi, Hiroshi Tokumitsu

BMC BIOCHEMISTRY 13 ( 27 ) 2012年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOMED CENTRAL LTD

Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases -including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
Results: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKI alpha (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K-m values of CaMKK isoforms for GTP (400-500 mu M) were significantly higher than those for ATP (similar to 15 mu M), and a 2- to 4-fold decrease in V-max was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of similar to 45 kDa and similar to 35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
Conclusions: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.

DOI： 10.1186/1471-2091-13-27

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1 査読

Yuichi Kanehiro, Kagefumi Todo, Misaki Negishi, Junji Fukuoka, Wenjian Gan, Takuya Hikasa, Yoshiaki Kaga, Masayuki Takemoto, Masaki Magari, Xialu Li, James L. Manley, Hitoshi Ohmori, Naoki Kanayama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 ( 4 ) 1216 - 1221 2012年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

DOI： 10.1073/pnas.1120368109

B細胞免疫記憶 AIDに依存したIgV変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である(AID-dependent IgV hypermutation requires a splice isoform of the SR protein SRSF1)

金山直樹, 金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, Li Xialu, Manley James L., 大森斉

日本免疫学会総会・学術集会記録 40 172 - 172 2011年11月

IL-21-Dependent B Cell Death Driven by Prostaglandin E-2, a Product Secreted from Follicular Dendritic Cells 査読

Masaki Magari, Yumiko Nishikawa, Yasumasa Fujii, Yumi Nishio, Koji Watanabe, Michiya Fujiwara, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF IMMUNOLOGY 187 ( 8 ) 4210 - 4218 2011年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC IMMUNOLOGISTS

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21-induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE(2). Treatment of B cells with IL-21 plus PGE(2), but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE(2) receptor isoform, EP4, was responsible for IL-21/PGE(2)-induced B cell death. Thus, PGE(2) is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE(2)-induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3-positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE(2)-induced B cell death in GC B cell selection. The Journal of Immunology, 2011, 187: 4210-4218.

DOI： 10.4049/jimmunol.1100934

AIDに依存した体細胞突然変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である

金山直樹, 金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, Li Xialu, Manley James L., 大森斉

日本生化学会大会プログラム・講演要旨集 84回 3T15a - 15 2011年9月

変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

鴨下佳代子, 小島聡史, 藤井忍, 北村幸一, 井上和恵, 岡山展久, 松田修一, 小嶋宏侑, 池田美香, 金広優一, 藤堂景史, 曲正樹, 大森齊, 金山直樹

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2T6 - 10 2010年12月

抗体遺伝子への体細胞突然変異におけるsplicing factor ASF/SF2の寄与

金広優一, 藤堂景史, 根岸美咲, 福岡純司, 曲正樹, 大森齊, 金山直樹

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2P - 0478 2010年12月

Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation 査読

Masamichi Kajita, Takahiro Okazawa, Mika Ikeda, Kagefumi Todo, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 110 ( 3 ) 351 - 358 2010年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with similar to 600-fold lower K(D)) than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.03.006

変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

金山直樹, 小島聡史, 藤井忍, 北村幸一, 井上知恵, 岡山展久, 松田修一, 鴨下佳代子, 藤堂景史, 池田美香, 曲正樹, 大森齊

日本生物工学会大会講演要旨集平成22年度 81 - 81 2010年9月

Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire 査読

Masaki Magari, Yuichi Kanehiro, Kagefumi Todo, Mika Ikeda, Naoki Kanayama, Hitoshi Ohmori

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 396 ( 2 ) 353 - 358 2010年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SW Delta C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW Delta C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW Delta C cells, although the protein might be highly susceptible to degradation. In DT40-SW Delta C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW Delta C cells may be useful for constructing AID libraries for efficient screening of mAbs in vitro. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.04.096

Conditional transformation of immunoglobulin mutation pattern from gene conversion into point mutation by controlling XRCC3 expression in the DT40 B cell line 査読

Masamichi Kajita, Masaki Magari, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 109 ( 4 ) 407 - 410 2010年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

A hypermutating B cell line DT40 is useful for screening antibodies and improving affinity of the selected antibodies in vitro. To perform affinity maturation efficiently, we generated an engineered DT40 line whose immunoglobulin mutation pattern can be transformed from gene conversion into point mutation by conditional suppression of XRCC3 expression. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.09.050

Enhancement of antibody production from a chicken B cell line DT40 by reducing Pax5 expression 査読

Masaki Magari, Takahiro Aya, Mika Ikeda, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 107 ( 2 ) 206 - 209 2009年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

We developed a novel in vitro antibody (Ab) generation system using a hypermutating chicken B cell line (DT40-SW). We suppressed the expression of the Pax5 transcription factor by targeted disruption of the gene to increase Ab production in isolated clones and produce the desired Abs. This single genetic manipulation resulted in a significant enhancement of Ab production without significantly affecting maximum cell density. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2008.09.014

変異導入能力を有する培養B細胞株を用いたin vitro抗体作製システムの高機能化

金山直樹, 曲正樹, 梶田真道, 金広優一, 藤堂景史, 大森斉

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 1T9 - 7 2008年11月

ニワトリB細胞株DT40-SWを用いたin vitro抗体作製システムの高機能化抗体遺伝子への変異導入効率の増強

金広優一, 曲正樹, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成20年度 109 - 109 2008年7月

ニワトリB細胞株を用いたin vitro抗体作製システムの高機能化変異様式の転換の高性能抗体作製への応用

梶田真道, 藤堂景史, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成20年度 109 - 109 2008年7月

Analysis of B cell selection in the germinal center reaction during a T-dependent antibody response at a single cell level 査読

Takahiro Okazawa, Masaki Magari, Takafumi Kimoto, Emi Kouyama, Hitoshi Ohmori, Naoki Kanayama

IMMUNOLOGY LETTERS 117 ( 1 ) 96 - 105 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V-H-encoded H chain (VHT) paired with the lambda 1 or lambda 2 L chain. It has been reported that both two VHT/lambda 1 and VHT/lambda 2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only VHT/lambda 2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the VHT. The VHT/lambda 2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than VHT/lambda 1 B cells. Here, to uncover how VHT/lambda 2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of VHT+ GC B cells at a single cell level. VHT/lambda 2 B cells bearing the unmutated VHT gene were found in the GCs more frequently than VHT/lambda 1 and mutated VHT/lambda 2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of VHT/lambda 2 B cells that mutated their VHT genes preferentially expanded, and finally VHT/lambda 2 B cells bearing the T313A mutation occupied VHT+ GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2008.01.002

ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発抗原特異的クローンのセルソーターによる効率的単離

岡澤貴裕, 藤堂景史, 梶田真道, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 149 - 149 2007年8月

ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発 Pax5発現抑制による抗体産生の増強

曲正樹, 綾高宏, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 149 - 149 2007年8月

ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発変異導入の促進による迅速な抗体ライブラリーの構築

金広優一, 曲正樹, 藤堂景史, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成19年度 148 - 148 2007年8月

Analysis of antigen-stimulated B cell migration into germinal centers during the early stage of a T-dependent immune response 査読

Emi Kouyama, Yumiko Nishikawa, Takahiro Okazawa, Masaki Magari, Hitoshi Ohmori, Naoki Kanayama

IMMUNOLOGY LETTERS 109 ( 1 ) 28 - 35 2007年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (VHT) encoded H chain and the lambda 1 or lambda 2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although VHT/lambda 1 and VHT/lambda 2 IgM were equally produced, VHT/lambda 2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of VHT/lambda 2 B cells for pNP was approximately 50-100-fold higher than that of VHT/lambda 1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of VHT/lambda 2 B cells for affinity maturation. VHT/lambda 2 B cells were more responsive to pNP than VHT/lambda 1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the VHT/lambda 2 B cell population was more potent in migration into GCs than the VHT/lambda 1 counterpart. Thus, it is suggested that the higher BCR affinity of VHT/lambda 2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2006.12.011

Novel in vitro screening system for monoclonal antibodies using hypermutating chicken B cell library 査読

Kagefumi Todo, Kenji Miyake, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 102 ( 5 ) 478 - 481 2006年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

DOI： 10.1263/jbb.102.478

B細胞免疫反応と免疫記憶超変異ニワトリB細胞株を用いたin vitro抗原生産システム(In vitro antibody generation system using a hypermutating chicken B cell line)

Kanayama Naoki, Todo Kagefumi, Okazawa Takahiro, Ikeda Mika, Fujita Risako, Magari Masaki, Ohmori Hitoshi

日本免疫学会総会・学術集会記録 36 55 - 55 2006年11月

Establishment of lymphotoxin β receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes. 査読

Yumiko Nishikawa, Masaki Hikida, Masaki Magari, Naoki Kanayama, M. Mori, Tomohiro Kurosaki, Hitoshi Ohmori

The Journal of Immunology 177 ( 8 ) 5204 - 5214 2006年10月

Suppressive effects of mometasone furoate on an antigen-specific IgE antibody response and production of IL-4 in mice 査読

Masaki Magari, Mika Ikeda, Miki Asakura, Naoki Kanayama, Masami Ogawa, Hitoshi Ohmori

IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY 28 ( 3 ) 491 - 500 2006年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：TAYLOR & FRANCIS INC

A nasal formulation of mometasone furoate (MF) is advantageous in avoiding systemic activity characteristic of glucocorticoids when it is applied topically. To confirm anti-allergic effects of this glucocorticoid formulation elaborately, we investigated whether the drug can suppress the production of IgE antibodies and related cytokines. It we showed that IgE production induced in mice immunized via intranasal route was significantly reduced when the mice were administered MF intranasally. Further, MF was effective in inhibiting production of type-2 helper T cell cytokines in vivo and in vitro. These results provide a immunopharmacological basis for clinical efficacy of this drug.

DOI： 10.1080/08923970600928155

Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line 査読

N Kanayama, K Todo, S Takahashi, M Magari, H Ohmori

NUCLEIC ACIDS RESEARCH 34 ( 2 ) e10 2006年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

DOI： 10.1093/nar/gnj013

抗体遺伝子変換機構を利用した非抗体タンパクのDNAシャッフリングによる機能改変

藤堂景史, 高橋佐都子, 片岡大輔, 曲正樹, 金山直樹, 大森斉

日本免疫学会総会・学術集会記録 35 80 - 80 2005年11月

培養B細胞株の変異機能を利用する新規なin vitro抗体作製システム

三宅健司, 藤堂景史, 曲正樹, 金山直樹, 大森斉

日本生物工学会大会講演要旨集平成17年度 118 - 118 2005年9月

Immunogenicity of autologous IgG bearing the inflammation-associated marker 3-nitrotyrosine 査読

H Ohmori, M Oka, Y Nishikawa, H Shigemitsu, M Takeuchi, M Magari, N Kanayama

IMMUNOLOGY LETTERS 96 ( 1 ) 47 - 54 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

To explore the link between inflammation and autoimmunity, we analyzed the immunogenicity of 3-nitrotyrosine(NT)-bearing self-proteins, an inflammation-associated marker that is formed by nitration of protein tyrosine residues with peroxynitrite generated during inflammation. An interesting feature of NT is its structural similarity to a synthetic hapten, 4-hydroxy-3-nitrophenylacetyl (NP), with which some anti-DNA antibodies (Abs) have been reported to show cross-reactivity. We confirmed that some of anti-DNA monoclonal Abs (mAbs) obtained from MRL/lpr mice also bound NT as well as NP. Based on these findings, we examined whether NIT-bearing autologous IgG (NT-1gG) as a model of NT-self proteins is immunogenic to induce a DNA-cross-reactive anti-NT Ab response in autologous normal mice. Anti-NT IgM and IgG Ab responses were elicited after the third immunization with NT-IgG. concomitant with an increase in anti-single stranded (ss)DNA titer. Interestingly, a part of anti-NT mAbs thus induced showed cross-reactivity with ssDNA. some of which used VH sequences that were highly homologous to those reported in anti-DNA Abs from NZB/WF1 mice. Splenic T cells primed with NT-IgG. but not with unmodified IgG. showed a proliferative response to the inducing antigen. Collectively, NT-IgG is immunogenic in autologous hosts. and can induce anti-NT Abs that are cross-reactive with ssDNA. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2004.07.004

B細胞株の遺伝子改変能を利用したタンパク分子改変システムの構築

藤堂景史, 高橋佐都子, 曲正樹, 金山直樹, 大森斉

日本免疫学会総会・学術集会記録 34 263 - 263 2004年11月

Generation of IgM and IgG 1 monoclonal antibodies with the identical variable regions: comparison of avidity

Naoki Kanayama, Kimi Yamakoshi, Masaaki Kiyomi, Masaki Magari, Hitoshi Ohmori

Memoris of the Faculty of Engineering, Okayama University 38 91 - 96 2004年

B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity 査読

N Kanayama, T Kimoto, K Todo, Y Nishikawa, M Hikida, M Magari, M Cascalho, H Ohmori

JOURNAL OF IMMUNOLOGY 169 ( 12 ) 6865 - 6874 2002年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC IMMUNOLOGISTS

The quasi-monoclonal mouse has limited B cell diversity, whose major (similar to80%) B cell Ag receptors are comprised of the knockin V-H 17.2.25 (VHT)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/lambda1 and VHT/lambda2 IgM were equally produced, VHT/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 Of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that VHT/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an,affinity maturation process.

Role for complement receptors (CD21/CD35) in the regulation of recombination activating gene expression in murine peripheral B cells 査読

H Ohmori, M Magari, Y Nakayama, N Kanayama, M Hikida

IMMUNOLOGY LETTERS 83 ( 2 ) 95 - 99 2002年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-2478(02)00083-4

Contribution of light chain rearrangement in peripheral B cells to the generation of high-affinity antibodies 査読

M Magari, T Sawatari, Y Kawano, M Cascalho, M Wabl, N Kanayama, M Hikida, H Ohmori

EUROPEAN JOURNAL OF IMMUNOLOGY 32 ( 4 ) 957 - 966 2002年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-V C H VERLAG GMBH

Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high-affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)-knockin mouse whose B cell diversity is limited due to the expression of the site-directed transgene. Immunization of the mouse with p-nitrophenylacetyl (pNP)-conjugated chicken gamma-globulin preferentially led to the production of anti-pNP IgG Ab comprised of non-VHT-encoded H chains and lambda chains. lambda(+) IgG constituted a majority of high-affinity Ab to this hapten. RAG-2 mRNA and the recombination signal sequence break of the lambda1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating X, gene rearrangement and the production of lambda(+) high-affinity anti-pNP IgG. These observations were reproduced in RAG-1-deficient mice that were reconstituted with the spleen cells of the knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.

Regulatory role for complement receptors (CD21/CD35) in the recombination activating gene expression in mouse peripheral B cells

Masaki Hikida, Masaki Magari, Yasunori Nakayama, Naoki Kanayama, Hitoshi Ohmori

Memoirs of the Faculty of Engineering Okayama Univiersity 36 51 - 60 2002年

Use of secondarily revised VH genes in IgE antibodies produced in mice infected with the nematode Nippostrongylus brasiliensis 査読

N Kanayama, C Hukue, M Magari, K Ohtani, M Hikida, M Yamada, S Matsuda, H Ohmori

IMMUNOLOGY LETTERS 77 ( 3 ) 181 - 186 2001年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Although a high level of IgE is produced after primary infection with Nippostrongylus brasiliensis (Nb), most of the IgE antibodies (Abs) are not specific to the worm. Analyses with Western blotting and enzyme-linked immunosorbent assay (ELISA) revealed that the IgE Abs from Nb-infected BALB/c mice did not show reactivity with Nb-derived excretory-secretory proteins (NES) and antigens present in the cell-free extracts of the worm. Monoclonal IEE Abs obtained from the Nb-infected mice were not reactive with these Nb antigen either. To characterize Nb-induced IgE response, we used (QM x C57BL/6)F1 (QBF1) mice that bear the knock-in 17.2.25 VHDJH segment (VHT) encoding a VH region specific to 4-hydroxy-3-nitrophenylacetyl hapten, and express VHT-encoded antigen receptors on 80-85% of their B cells. Consistent with the frequency of VHT-positive B cells, more than 80% of IgE Abs induced in QBF1 B cells that were cultured with LPS plus IL-4 were found to bear VHT-encoded H chains. In contrast, when QBF1 mice were infected with Nb, less than 10% of Nb-induced IgE Abs were found to use VHT. The QBF1-derived IgE did not react with Nb antigens either. Taken together, data suggest that Nb-induced IgE response in mice is not merely the result of polyclonal activation of B cells, but may involve a mechanism that revises Ig genes secondarily. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0165-2478(01)00216-4

　前のページ - 次のページ　

1