Nobuo Sakaguchi

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser502 that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.

Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating C→U during transcription of Ig-variable (V) and Ig-switch (S) region DNA, which is essential to produce high-affinity antibodies. Here we report the crystal structure of a soluble human AID variant at 2.8Å resolution that favors targeting WRC motifs (W=A/T, R=A/G) in vitro, and executes Ig V SHM in Ramos B-cells. A specificity loop extending away from the active site to accommodate two purine bases next to C, differs significantly in sequence, length, and conformation from APOBEC proteins Apo3A and Apo3G, which strongly favor pyrimidines at -1 and -2 positions. Individual amino acid contributions to specificity and processivity were measured in relation to a proposed ssDNA binding cleft. This study provides a structural basis for residue contributions to DNA scanning properties unique to AID, and for disease mutations in human HIGM-2 syndrome.

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines.

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A‐MuLV)‐transformed cell lines, which were able to differentiate from the mu‐ to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A‐MuLV‐transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM‐bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.

Activation of cyclic AMP-dependent protein kinase activity during LPS stimulation of macrophage tumor cell line, J774.1.

Abstract Stimulation of J774.1 macrophage cell line with lipopolysaccharide (LPS)- or 8Br-cyclic AMP induced phosphorylation of nonhistone nuclear proteins (NHP) particularly of size range 35,000 daltons. In vitro phosphorylation of nuclei isolated from LPS- or 8Br-cyclic AMP-stimulated cells also showed an increased incorporation of 32 P into NHP indicating an activation of a NHP-specific protein kinase in nuclei. Incubation of normal nuclei with cytoplasmic extracts from LPS- or 8Br-cyclic AMP-stimulated cells increased NHP-specific phosphorylation in nuclei. Incubation of normal nuclei with normal cytoplasm in the presence of 1 × 10 −8 M cyclic AMP also induced an increased phosphorylation of NHP, suggesting that the LPS-induced increase of intracellular cyclic AMP activates a cyclic-AMP-dependent NHP-specific protein kinase in the cytoplasm, which moves into the nucleus to phosphorylate NHP. The increased phosphorylation of NHP proceeded in advance of the secretion of T-cell activating factor(s) and the optimum concentrations of LPS- or 8Br-cyclic AMP for the maximal induction of NHP-specific protein kinase activity were similar to those required for the induction of T-cell activating factor(s) in J774.1. These experiments suggest the intimate involvement of NHP phosphorylation in the induction processes of macrophage factor production.

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse‐free survival, hazard ratio = 2.37, 95% confidence interval, 1.27–4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42–5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap‐cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland‐specific GANP‐deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP‐heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.

Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation.

Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells.

Induction of 19S IgM secretion in a murine pre-B cell line, 70z/3, by cell hybridization with non-secreting myeloma cells

Somatic cell hybrids were prepared between the TK-deficient variant of murine pre-B cell line, 70Z/3, cells and the HGPRT-deficient variant of non-secreting myeloma cells. Several hybrid clones which secreted IgM but did not express surface IgM were isolated. LPS stimulation did not induce the expression of surface IgM. SDS-PAGE analysis indicated that the IgM secreted by one of the hybrid clones was a 19S pentamer and that the size of its mu-chains was the same as that of mu-chains from MOPC 104E myeloma IgM. Two-dimensional gel electrophoresis of biosynthetically labeled immunoglobulin showed that the same pattern was obtained with kappa-chains from two hybrid clones and from the LPS-induced 70Z/3 cells. The result showed that cell hybridization could induce L-chain synthesis in the pre-B cell line. Anti-idiotypic antibody against the secreted IgM was prepared and it was shown that the surface IgM expressed on all LPS-stimulated 70Z/3 cells bore the same idiotype. Those results indicated that the specificity commitment has already occurred in 70Z/3 cells.

Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

Lymphoid lineage commitment is an important process in haematopoiesis, which forms the immune system to protect the host from pathogen invasion. However, how multipotent progenitors (MPP) switch into common lymphoid progenitors (CLP) or common myeloid progenitors (CMP) during this process remains elusive. Here we show that PCI domain-containing protein 2 (Pcid2) is highly expressed in MPPs. Pcid2 deletion in the haematopoietic system causes skewed lymphoid lineage specification. In MPPs, Pcid2 interacts with the Zinc finger HIT-type containing 1 (ZNHIT1) to block Snf2-related CREBBP activator protein (SRCAP) activity and prevents the deposition of histone variant H2A.Z and transcription factor PU.1 to key lymphoid fate regulator genes. Furthermore, Znhit1 deletion also abrogates H2A/H2A.Z exchange in MPPs. Thus Pcid2 controls lymphoid lineage commitment through the regulation of SRCAP remodelling activity.Haematopoiesis and the generation of lymphoid cell subsets are controlled by delicate genetic programs enforced via epigenetic regulation. Here the authors show that Pcid2 interacts with ZNHIT1, a component of the SRCAP chromatin remodelling complex, to critically modulate the differentiation of multipotent progenitors.

Immunodominant SARS coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates

Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471–503, S604–625, and S1164–1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597–603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597–603 and S604–625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597–603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

GANP maintains antibody structural integrity during SHM