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Biomedical subjects

Masamichi Muramatsu

Publications and source records attributed to Masamichi Muramatsu.

At least 19 recordsLinked to original sources
PubMed2006

Identification of a specific domain required for dimerization of activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA.

APOBEC-1 Deaminase↗
PubMed2006

Negative regulation of activation-induced cytidine deaminase in B cells.

Both class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes require the activity of activation-induced cytidine deaminase (AID). Expression of AID is restricted to B cells in the germinal centers of the lymphoid organs, where activated B cells undergo CSR and SHM. We previously showed that constitutive and systemic expression of AID leads to tumorigenesis in T cells and lung epithelium, but not in B cells. This finding led us to suspect that transgenic AID may be inactivated at least in part in B cells. To address this issue, we generated conditional AID-transgenic mice that constitutively express AID only in B cells. Studies on the cross between the AID-transgenic and AID-deficient mice showed that abundant AID protein accumulated by constitutive expression is inactivated in B cells, possibly providing an explanation for the absence of deregulation of CSR and SHM in AID-transgenic B cells.

Animals↗
PubMed2005

CXCR5-dependent seeding of follicular niches by B and Th cells augments antiviral B cell responses.

The chemokine receptor CXCR5 and its ligand CXCL13 define the structure of B cell follicles within secondary lymphoid organs. Here, we examined the impact of CXCR5 on antiviral B cell responses in vivo. CXCR5-/- mice showed a normal production of IgM and IgG acutely after infection with vesicular stomatitis virus (VSV) and developed VSV-specific germinal centers. However, impaired Ig class switch and Ab production were observed under conditions of limited availability of Ag (i.e., after immunization with nonreplicating viral particles or soluble Ag). Adoptive transfer of CXCR5-deficient, VSV-specific B and Th cells demonstrated that CXCR5 expression on both B and Th cells is required for an efficient Ig class switch. These experiments revealed that CXCR5 is critical for the coordinated interaction of antiviral T and B cells through its impact on initial B cell expansion and the recruitment of Ag-specific B and Th cells to germinal centers.

Adoptive Transfer↗
PubMed2005

Evolution of class switch recombination function in fish activation-induced cytidine deaminase, AID.

Following activation of mammalian B cells, class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig heavy chain (IgH) gene can improve the functions of the expressed antibodies. Activation-induced cytidine deaminase (AID) is the only known B cell-specific protein required for inducing CSR and SHM in mammals. Lower vertebrates have an AID homologue, and there is some evidence of SHM in vivo. However there is no evidence of CSR in the cartilaginous or bony fishes, and this may be due in part to a lack of cis-elements in the IgH gene that are the normal targets of AID-mediated recombination. We have tested whether bony fish (zebrafish and catfish) AID can mediate CSR and SHM in mammalian cells. As expected, ectopic expression of fish AID in mouse fibroblasts resulted in mutations in an introduced SHM reporter gene, indicating that fish AID can mediate SHM. Unexpectedly, expression of fish AID in mouse AID-/- B cells induced surface IgG expression as well as switched transcripts from Ig gene loci, clearly indicating that the fish AID protein can mediate CSR, at least in mouse cells. These results suggest that the AID protein acquired the ability to mediate CSR before the IgH locus evolved the additional exon clusters and switch regions that are the targets of recombination. We discuss how pleiotropic functions of specific domains within the AID protein may have facilitated the early evolution of CSR in lower vertebrates.

Animals↗
PubMed2005

A target selection of somatic hypermutations is regulated similarly between T and B cells upon activation-induced cytidine deaminase expression.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutations (SHM) and class switch recombination. Overexpression of AID in non-B cells can induce SHM in artificial constructs inserted in various loci in the genome. AID overexpression was thus proposed to introduce mutations in a wide variety of genes with little specificity. We previously showed that AID transgenic mice developed T cell lymphomas in which the variable region beta genes of the T cell receptor and c-myc were mutated as frequently as SHM in activated B cells. To understand the target specificity of SHM in AID-expressing T lymphomas, we sequenced six oncogenes (c-myc, pim1, p53, atm, tgfbr-2, and k-ras) and two genes (cd4 and cd5) that are actively transcribed in T lymphomas. SHM was found only in c-myc, pim1, cd4, and cd5, which share the E47 binding motif in the enhancer/promoter. The rest that are not mutated in B cells were not mutated in AID-induced T lymphomas either, although they are transcribed in T and B cells. Comparison of several features of SHM, including selection of targets and mutation distribution, suggests that the regulatory mechanism of SHM is similar between T and B cells. SHM base specificities in the CD4 and CD5 genes were biased to AT, indicating that the preference of target bases of the mutations generated by overexpression of AID is not always GC bases but variable between target genes.

Animals↗
PubMed2005

DNA cleavage in immunoglobulin somatic hypermutation depends on de novo protein synthesis but not on uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requires de novo protein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.

Animals↗
PubMed2005

AID to overcome the limitations of genomic information.

The limitations of genomic information forced our ancestors to adopt a strategy for introducing somatic DNA alterations with the risk of genome instability. Although activation-induced deaminase (AID) is involved in DNA cleavage in somatic hypermutation and class-switch recombination, its mechanism of action has been debated extensively, with the two main hypotheses being distinguished by the chief target of AID: RNA or DNA. The principle distinction between the two hypotheses is the requirement for translation of edited mRNA or uracil removal from DNA for DNA cleavage. Although a series of experiments has provided support for the 'RNA-editing' hypothesis and requires reevaluation of the 'DNA-deamination' hypothesis, definitive proof is yet to come.

DNA↗
PubMed2004

AID is required for c-myc/IgH chromosome translocations in vivo.

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.

Animals↗
PubMed2004

Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step in immunoglobulin class switch recombination (CSR). AID is proposed to deaminate cytosine to generate uracil (U) in either mRNA or DNA. In the second instance, DNA cleavage depends on uracil DNA glycosylase (UNG) for removal of U. Using phosphorylated histone gamma-H2AX focus formation as a marker of DNA cleavage, we found that the UNG inhibitor Ugi did not inhibit DNA cleavage in immunoglobulin heavy chain (IgH) locus during CSR, even though Ugi blocked UNG binding to DNA and strongly inhibited CSR. Strikingly, UNG mutants that had lost the capability of removing U rescued CSR in UNG-/- B cells. These results indicate that UNG is involved in the repair step of CSR yet by an unknown mechanism. The dispensability of U removal in the DNA cleavage step of CSR requires a reconsideration of the model of DNA deamination by AID.

Animals↗
PubMed2004

De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination.

Activation-induced cytidine deaminase is required for the DNA cleavage step of Ig class switch recombination (CSR). However, its molecular mechanism is controversial. RNA-editing hypothesis postulates that activation-induced cytidine deaminase deaminates cytosine in an unknown mRNA to generate a new mRNA encoding an endonuclease for CSR and thus predicts that DNA cleavage depends on de novo protein synthesis. On the other hand, DNA deamination hypothesis proposes that DNA cleavage is initiated by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase. By using the chromatin immunoprecipitation assay to detect gamma-H2AX focus formation as a marker for DNA cleavage, we found that cycloheximide inhibited DNA cleavage in the Ig heavy-chain locus during CSR. Requirement of protein synthesis in the DNA cleavage step of CSR strengthens the RNA-editing hypothesis.

Animals↗
PubMed2004

Separate domains of AID are required for somatic hypermutation and class-switch recombination.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM). Mutants with changes in the C-terminal region of AID retain SHM but lose CSR activity. Here we describe five mutants with alterations in the N-terminal region of AID that caused selective deficiency in SHM but retained CSR, suggesting that the CSR and SHM activities of AID may dissociate via interaction of CSR- or SHM-specific cofactors with different domains of AID. Unlike cells expressing C-terminal AID mutants, B cells expressing N-terminal AID mutants had mutations in the switch micro region, indicating that such mutations are generated by reactions involved in CSR but not SHM. Thus, we propose that separate domains of AID interact with specific cofactors to regulate these two distinct genetic events in a target-specific way.

Active Transport, Cell Nucleus↗
PubMed2004

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1.

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms.

APOBEC-1 Deaminase↗
PubMed2004

Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut.

The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID(-/-) mice. Reconstitution of lamina propria IgA production in AID(-/-) mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system.

Anaerobiosis↗
PubMed2004

AID: how does it aid antibody diversity?

Activation-induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion (GC). AID is known to be required for DNA cleavage of S regions in CSR. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We discuss available evidence for the two proposed models. Recent findings, namely requirement of protein synthesis for DNA breakage and dispensability of U removal activity of uracil DNA glycosylase, force us to reconsider DNA deamination hypothesis.

Animals↗
PubMed2004

B1b lymphocytes confer T cell-independent long-lasting immunity.

Many microbial pathogens employ antigenic variation as a strategy to evade the immune system, posing a challenge in vaccine development. To understand the requirements for immunity against such pathogens, we studied Borrelia hermsii, a relapsing fever bacterium. We found that mice deficient in T, follicular B, marginal zone B, or B1a lymphocytes resolved B. hersmii bacteremia and became resistant to reinfection. The resolution of bacteremia coincided with an expansion and persistence of B1b lymphocytes, and purified B1b lymphocytes from convalescent wild-type or TCR-betaxdelta-/- mice conferred immunity to Rag1-/- mice. The B1b lymphocytes in the reconstituted Rag1-/- mice provided long-lasting immunity by rapidly generating B. hermsii-specific IgM but not IgG upon bacterial challenge. Unmutated IgM is sufficient to eliminate B. hermsii, because AID-/- mice deficient in somatic hypermutation and class switch recombination efficiently resolved all bacteremic episodes. These data demonstrate that B1b lymphocytes can provide long-lasting T cell-independent IgM memory.

Adoptive Transfer↗
PubMed2003

RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells.

Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to induce somatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro.

3T3 Cells↗
PubMed2003

AID mutant analyses indicate requirement for class-switch-specific cofactors.

Activation-induced cytidine deaminase (AID) is the essential and sole B cell-specific factor required for class-switch recombination (CSR) and somatic hypermutation (SHM). However, it is not known how AID differentially regulates these two independent events. Involvement of several cofactors interacting with AID has been indicated by scattered distribution of loss-of-function point mutations and evolutionary conservation of the entire 198-amino-acid protein. Here, we report that human AID mutant proteins with insertions, replacements or truncations in the C-terminal region retained strong SHM activity but almost completely lost CSR activity. These results indicate that AID requires interaction with a cofactor(s) specific to CSR.

3T3 Cells↗