Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.1c00796

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2021.11.105

researchmap

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

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

PubMed

researchmap

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

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

PubMed

researchmap

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

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

PubMed

researchmap

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

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

PubMed

researchmap

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

BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

DOI： 10.1016/j.bbagen.2018.12.012

PubMed

researchmap

Authorship：Last author,　Corresponding author  

DOI： 10.1074/jbc.RA118.006226

PubMed

researchmap

DOI： 10.1007/978-1-4939-9030-6_23

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)/5-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKK that exhibits higher basal activity (autonomous activity), activation of the CaMKK/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKK appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKK activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKK at multiple residues by CaMKK-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKK activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKK indicated that Thr(144) phosphorylation by activated AMPK converts CaMKK into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKK mutant. CaMKK mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr(144) phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr(144) antibody revealed phosphorylation of Thr(144) in CaMKK in transfected COS-7 cells that was further enhanced by exogenous expression of AMPK. These results indicate that AMPK-mediated feedback phosphorylation of CaMKK regulates the CaMKK/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKK.

DOI： 10.1074/jbc.M117.805085

Web of Science

PubMed

researchmap

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

Web of Science

PubMed

researchmap

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

Web of Science

PubMed

researchmap

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

Web of Science

PubMed

researchmap

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

Web of Science

PubMed

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background:Rev7 encodes a subunit of Pol for translesion DNA synthesis (TLS). Results: We found a Rev7 mutation in mice that causes developmental defects and increases susceptibility for genotoxicity. Conclusion:Rev7 is essential for mouse development through its function in cell proliferation. Significance: These findings demonstrate a unique function of Pol in development that is absent in other TLS polymerases. Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase (Pol), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Pol. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive H2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Pol preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

DOI： 10.1074/jbc.M113.514752

Web of Science

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

Language：English   Publisher：(NPO)日本免疫学会  

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2010&ichushi_jid=J02769&link_issn=&doc_id=20101112230046&doc_link_id=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D212741&url=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D212741&type=%8D%91%97%A7%8F%EE%95%F1%8Aw%8C%A4%8B%86%8F%8A%81FNii%83v%83%8D%83W%83F%83N%83g%83%8A%83%7C%83W%83g%83%8A&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F80182_3.gif

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

Web of Science

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2007&ichushi_jid=J02769&link_issn=&doc_id=20080304300129&doc_link_id=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D210759&url=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D210759&type=%8D%91%97%A7%8F%EE%95%F1%8Aw%8C%A4%8B%86%8F%8A%81FNii%83v%83%8D%83W%83F%83N%83g%83%8A%83%7C%83W%83g%83%8A&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F80182_3.gif

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2007&ichushi_jid=J02769&link_issn=&doc_id=20080304300127&doc_link_id=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D210757&url=https%3A%2F%2Fprojects.repo.nii.ac.jp%2F%3Faction%3Drepository_uri%26item_id%3D210757&type=%8D%91%97%A7%8F%EE%95%F1%8Aw%8C%A4%8B%86%8F%8A%81FNii%83v%83%8D%83W%83F%83N%83g%83%8A%83%7C%83W%83g%83%8A&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F80182_3.gif

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

Language：English   Publisher：(NPO)日本免疫学会  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, Fc gamma RIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

Web of Science

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(NPO)日本免疫学会  

researchmap

Language：Japanese   Publisher：(公社)日本生物工学会  

researchmap

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

Web of Science

researchmap

Language：Japanese   Publisher：(NPO)日本免疫学会  

researchmap

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

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

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

Web of Science

researchmap

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

researchmap

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

Web of Science

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

Web of Science

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2016231963

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2016231962

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2014229322

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2014229319

J-GLOBAL

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2016046032

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

Web of Science

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2014247581

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2014247582

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2012371265

J-GLOBAL

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2013125690

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2013125691

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2011331258

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2012048528

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2012048674

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/j.jbiotec.2010.09.628

Web of Science

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：PHARMACEUTICAL SOC JAPAN  

Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. The generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and sometimes mAbs for molecules conserved between species are difficult to obtain because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been attempting to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during Culture, thereby generating a useful Ab library for screening mAbs. First, we generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Ab generation system using DT40-SW is useful in the following ways: (1) mAbs for various model antigens including antoantigens can be obtained from the DT40-SW Ab library that is free from immunological tolerance; (2) the switching device of the mutation machinery enables fixing desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Ab production. The Ab generation system will be applicable for obtaining valuable Abs such as antitumor Abs.

DOI： 10.1248/yakushi.129.11

Web of Science

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本薬学会  

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2009066344

J-GLOBAL

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2006268139

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2004156456

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2003123261

Language：Japanese  

CiNii Article

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2001220188

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.30 - 2022.12.2

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.9 - 2022.11.11

Language：Japanese   Presentation type：Poster presentation  

researchmap

Event date： 2022.11.9 - 2022.11.11

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.11.9 - 2022.11.11

Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.5.28 - 2022.5.29

Language：Japanese   Presentation type：Oral presentation (general)  

researchmap

Event date： 2021.12.1 - 2021.12.3

Presentation type：Poster presentation  

researchmap

Event date： 2021.12.1 - 2021.12.3

Presentation type：Poster presentation  

researchmap

Event date： 2021.12.1 - 2021.12.3

Presentation type：Poster presentation  

researchmap

Event date： 2021.12.1 - 2021.12.3

Presentation type：Poster presentation  

researchmap

Event date： 2021.12.1 - 2021.12.3

Presentation type：Poster presentation  

researchmap

Event date： 2021.12.1 - 2021.12.3

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

Event date： 2021.12.1 - 2021.12.3

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

Event date： 2021.12.1 - 2021.12.3

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

Event date： 2020.12.2 - 2020.12.4

Language：Japanese   Presentation type：Poster presentation  

Event date： 2020.12.2 - 2020.12.4

Language：Japanese   Presentation type：Poster presentation  

Event date： 2020.11.14

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.5.23 - 2020.5.24

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2019

researchmap

Event date： 2018.8

Language：English  

researchmap

Event date： 2018

researchmap

Event date： 2018

researchmap

Event date： 2015.12

Language：Japanese  

researchmap

Event date： 2015

Language：Japanese  

researchmap

Event date： 2014.10

Language：Japanese  

researchmap

Event date： 2011.11

Language：English  

researchmap

Event date： 2011.9

Language：Japanese  

researchmap

Event date： 2008.7

Language：Japanese  

researchmap

Event date： 2008.7

Language：Japanese  

researchmap

Event date： 2007.8

Language：Japanese  

researchmap

Event date： 2007.8

Language：Japanese  

researchmap

Event date： 2007.8

Language：Japanese  

researchmap

Event date： 2005.11

Language：Japanese  

researchmap

Event date： 2005.9

Language：Japanese  

researchmap

Event date： 2004.11

Language：Japanese  

researchmap

Event date： 2002.10

Language：Japanese  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

Country：Japan

researchmap

Country：Japan

researchmap