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• W2000469539 abstract "Marginal zone (MZ) B cells of the spleen and B1 cells, termed innate-like B cells, differ from follicular B cells by their attenuated Ca2+ mobilization, fast antibody secretion, and increased cell adhesion. We identified and characterized Mzb1 as an endoplasmic reticulum-localized and B cell-specific protein that was most abundantly expressed in MZ B and B1 cells. Knockdown of Mzb1 in MZ B cells increased Ca2+ mobilization and nuclear NFAT transcription factor localization, but reduced lipopolysaccharide-induced antibody secretion and integrin-mediated cell adhesion. Conversely, ectopic expression of an Lck-Mzb1 transgene in peripheral T cells resulted in attenuated Ca2+ mobilization and augmented integrin-mediated cell adhesion. In addition to its interaction with the substrate-specific chaperone Grp94, Mzb1 augmented the function of the oxidoreductase ERp57 in favoring the expression of integrins in their activated conformation. Thus, Mzb1 helps to diversify peripheral B cell functions by regulating Ca2+ stores, antibody secretion, and integrin activation. Marginal zone (MZ) B cells of the spleen and B1 cells, termed innate-like B cells, differ from follicular B cells by their attenuated Ca2+ mobilization, fast antibody secretion, and increased cell adhesion. We identified and characterized Mzb1 as an endoplasmic reticulum-localized and B cell-specific protein that was most abundantly expressed in MZ B and B1 cells. Knockdown of Mzb1 in MZ B cells increased Ca2+ mobilization and nuclear NFAT transcription factor localization, but reduced lipopolysaccharide-induced antibody secretion and integrin-mediated cell adhesion. Conversely, ectopic expression of an Lck-Mzb1 transgene in peripheral T cells resulted in attenuated Ca2+ mobilization and augmented integrin-mediated cell adhesion. In addition to its interaction with the substrate-specific chaperone Grp94, Mzb1 augmented the function of the oxidoreductase ERp57 in favoring the expression of integrins in their activated conformation. Thus, Mzb1 helps to diversify peripheral B cell functions by regulating Ca2+ stores, antibody secretion, and integrin activation. Mzb1 regulates cell proliferation and ER calcium stores in B cells Mzb1 augments integrin-mediated cell adhesion Expression of Mzb1 in T cells attenuates calcium fluxes and enhances cell adhesion Mzb1 associates with ERp57 and Grp94 Peripheral B lymphocytes consist of multiple cell populations that differ in their phenotype, functional properties, and anatomic locations (Allman and Pillai, 2008Allman D. Pillai S. Peripheral B cell subsets.Curr. Opin. Immunol. 2008; 20: 149-157Crossref PubMed Scopus (327) Google Scholar, Hardy et al., 2007Hardy R.R. Kincade P.W. Dorshkind K. The protean nature of cells in the B lymphocyte lineage.Immunity. 2007; 26: 703-714Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, Martin and Kearney, 2000Martin F. Kearney J.F. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a “natural immune memory”.Immunol. Rev. 2000; 175: 70-79Crossref PubMed Google Scholar). In addition to the vast majority of conventional B cells, also termed follicular B (FoB) cells, which reside in lymph nodes and in the follicles of the spleen, marginal zone (MZ) B cells occupy the marginal sinus of the spleen, and B1 cells are predominantly found in the peritoneal and pleural cavities. MZ B and B1 cells have been termed “innate-like B cells,” given that these cells can be stimulated by ligands of Toll-like receptors (TLRs) to quickly differentiate into antibody-secreting cells that produce “natural,” polyreactive antibodies (Martin and Kearney, 2000Martin F. Kearney J.F. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a “natural immune memory”.Immunol. Rev. 2000; 175: 70-79Crossref PubMed Google Scholar, Rubtsov et al., 2008Rubtsov A.V. Swanson C.L. Troy S. Strauch P. Pelanda R. Torres R.M. TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses.J. Immunol. 2008; 180: 3882-3888Crossref PubMed Scopus (68) Google Scholar). Follicular B cells, in contrast, produce specific antibodies with much slower kinetics and require stimulation by both B cell receptor (BCR) and TLR for differentiation into antibody-secreting cells (Richards et al., 2008Richards S. Watanabe C. Santos L. Craxton A. Clark E.A. Regulation of B-cell entry into the cell cycle.Immunol. Rev. 2008; 224: 183-200Crossref PubMed Scopus (42) Google Scholar). Murine marginal zone B cells have been shown to require weak BCR signals, whereas strong BCR signaling has been associated with follicular B cell development (Casola et al., 2004Casola S. Otipoby K.L. Alimzhanov M. Humme S. Uyttersprot N. Kutok J.L. Carroll M.C. Rajewsky K. B cell receptor signal strength determines B cell fate.Nat. Immunol. 2004; 5: 317-327Crossref PubMed Scopus (434) Google Scholar, Pillai and Cariappa, 2009Pillai S. Cariappa A. The follicular versus marginal zone B lymphocyte cell fate decision.Nat. Rev. Immunol. 2009; 9: 767-777Crossref PubMed Scopus (332) Google Scholar). B1 cells also differ from follicular B cells in altered Ca2+ signaling and cell proliferation (Chumley et al., 2002Chumley M.J. Dal Porto J.M. Cambier J.C. The unique antigen receptor signaling phenotype of B-1 cells is influenced by locale but induced by antigen.J. Immunol. 2002; 169: 1735-1743Crossref PubMed Scopus (57) Google Scholar). In particular, B1 cells display a reduced Ca2+ mobilization and exist in an “anergic” state in which they are less responsive to BCR stimulation (Dal Porto et al., 2004Dal Porto J.M. Burke K. Cambier J.C. Regulation of BCR signal transduction in B-1 cells requires the expression of the Src family kinase Lck.Immunity. 2004; 21: 443-453Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Wong et al., 2002Wong S.C. Chew W.K. Tan J.E. Melendez A.J. Francis F. Lam K.P. Peritoneal CD5+ B-1 cells have signaling properties similar to tolerant B cells.J. Biol. Chem. 2002; 277: 30707-30715Crossref PubMed Scopus (76) Google Scholar). Another distinguishing hallmark of MZ B cells is the abundant expression of integrins, which helps the anchoring of these cells to the marginal zone of the spleen (Lu and Cyster, 2002Lu T.T. Cyster J.G. Integrin-mediated long-term B cell retention in the splenic marginal zone.Science. 2002; 297: 409-412Crossref PubMed Scopus (306) Google Scholar). Integrins are cell-surface receptors composed of alpha- and beta-chain heterodimers that regulate cell adhesion and cell migration through bidirectional signaling (Hynes, 2002Hynes R.O. Integrins: Bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6369) Google Scholar, Luo et al., 2007Luo B.H. Carman C.V. Springer T.A. Structural basis of integrin regulation and signaling.Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1145) Google Scholar, Moser et al., 2009Moser M. Legate K.R. Zent R. Fassler R. The tail of integrins, talin, and kindlins.Science. 2009; 324: 895-899Crossref PubMed Scopus (527) Google Scholar). Regulation of integrin-mediated cell adhesion depends on conformational changes of integrins, whereby three distinct states have been defined (Hynes, 2002Hynes R.O. Integrins: Bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6369) Google Scholar, Luo et al., 2007Luo B.H. Carman C.V. Springer T.A. Structural basis of integrin regulation and signaling.Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1145) Google Scholar). A bent conformation represents a low-affinity state, in which integrins have 1/500 and 1/10,000 the affinity for ligand binding compared to the extended-closed and extended-open conformations, respectively (Luo et al., 2007Luo B.H. Carman C.V. Springer T.A. Structural basis of integrin regulation and signaling.Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1145) Google Scholar). Integrin conformations have been proposed to depend on long-range disulfide bonds in the β subunits, and mutations that introduce disulfide bonds to lock the conformation in the extended or bent states have been found to alter ligand binding affinity (Lu and Cyster, 2002Lu T.T. Cyster J.G. Integrin-mediated long-term B cell retention in the splenic marginal zone.Science. 2002; 297: 409-412Crossref PubMed Scopus (306) Google Scholar, Luo et al., 2007Luo B.H. Carman C.V. Springer T.A. Structural basis of integrin regulation and signaling.Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1145) Google Scholar). MZ B cells and B1 cells also differ from FoB cells by their fast kinetics of antibody secretion in response to lipopolysaccharide (LPS) stimulation, and B1 cells have been reported to secrete antibodies spontaneously (Rubtsov et al., 2008Rubtsov A.V. Swanson C.L. Troy S. Strauch P. Pelanda R. Torres R.M. TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses.J. Immunol. 2008; 180: 3882-3888Crossref PubMed Scopus (68) Google Scholar). All these processes depend on functions of the endoplasmic reticulum (ER), which acts as a major intracellular calcium store and provides a strongly oxidizing environment that facilitates disulfide bond formation and folding of complex glycoproteins, such as immunoglobulins and integrins (Cahalan, 2009Cahalan M.D. STIMulating store-operated Ca(2+) entry.Nat. Cell Biol. 2009; 11: 669-677Crossref PubMed Scopus (357) Google Scholar, Todd et al., 2008Todd D.J. Lee A.H. Glimcher L.H. The endoplasmic reticulum stress response in immunity and autoimmunity.Nat. Rev. Immunol. 2008; 8: 663-674Crossref PubMed Scopus (457) Google Scholar, Tu and Weissman, 2004Tu B.P. Weissman J.S. Oxidative protein folding in eukaryotes: Mechanisms and consequences.J. Cell Biol. 2004; 164: 341-346Crossref PubMed Scopus (760) Google Scholar). A family of ER oxidoreductases, including protein disulfide isomerase (PDI) and ERp57 (PDIA3), allows for the efficient folding of glycoproteins by catalyzing the formation and isomerization of intra- and intermolecular disulfide bonds (Ellgaard and Ruddock, 2005Ellgaard L. Ruddock L.W. The human protein disulphide isomerase family: Substrate interactions and functional properties.EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (586) Google Scholar). Oxidative protein folding of complex proteins in the ER also depends on chaperones. In particular, ERp57 forms a complex with calnexin and calreticulin, whereby these lectin chaperones mediate substrate recognition. The abundant chaperone BiP (Grp78) is found in a complex with other ER chaperones, such as Grp94 (gp96, Hsp90b1) and PDI (Ni and Lee, 2007Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). Grp94 has a limited substrate specificity, which includes integrins, TLRs and immunoglobulins, and conditional inactivation of Grp94 in B lymphocytes diminished surface expression of TLR and attenuated secretion of antibodies upon TLR stimulation (Liu and Li, 2008Liu B. Li Z. Endoplasmic reticulum HSP90b1 (gp96, grp94) optimizes B-cell function via chaperoning integrin and TLR but not immunoglobulin.Blood. 2008; 112: 1223-1230Crossref PubMed Scopus (91) Google Scholar, Melnick et al., 1994Melnick J. Dul J.L. Argon Y. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum.Nature. 1994; 370: 373-375Crossref PubMed Scopus (358) Google Scholar, Randow and Seed, 2001Randow F. Seed B. Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability.Nat. Cell Biol. 2001; 3: 891-896Crossref PubMed Scopus (275) Google Scholar). Given the multiple phenotypic differences between peripheral B cell subsets, the question arises as to which genes regulate the characteristic properties of innate-like B cells. Here, we show that Mzb1, an ER protein that is abundantly expressed in marginal zone B and B1 cells, regulates calcium signaling, integrin-mediated adhesion, and antibody secretion by interacting with ERp57 and forming a substrate-specific complex with the BiP and Grp94 chaperones. Thus, Mzb1 may help to diversify the functions of peripheral B cell subsets. To identify genes that are differentially expressed in peripheral B cell subsets, we screened cDNA clones of a pre-B cell minus erythroid subtractive cDNA library (Travis et al., 1991Travis A. Amsterdam A. Belanger C. Grosschedl R. LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function.Genes Dev. 1991; 5: 880-894Crossref PubMed Scopus (484) Google Scholar) for their expression in FoB cells, MZ B cells, and B1 cells. One clone, termed Mzb1, was found to be expressed in MZ B and B1 cells at a substantially higher amount than in FoB cells. We confirmed the differential expression of Mzb1 by both immunoblot analysis with an Mzb1 monoclonal antibody and by quantitative RT-PCR (Figure 1A and Figure S1A available online). By RNA blot analysis, abundant Mzb1 transcripts could be detected in all transformed B cell lines examined, regardless of their stage of differentiation (Figure S1B). Analysis of the tissue distribution of Mzb1 transcripts indicated that the Mzb1 gene is expressed predominantly in the spleen and lymph nodes (Figure S1C). Mzb1 transcripts found in the thymus and lung could be accounted for by contaminating B cells, given that immunoglobulin κ transcripts were also detected. Mzb1 cDNA contains an open reading frame of 188 amino acids (aa) with marked conservation from human to the chordate Ciona (Figure S1D). Mzb1 protein includes a CXXC thioredoxin motif at position 49, a potential amino-terminal signal sequence and a carboxy-terminal ER-retrieval sequence. We examined the processing of Mzb1 protein by mass-spectometric analysis and found protein cleavages after amino acids 18 and 20 (Figure S1D and Table S1). Two recent reports independently described the cloning of Mzb1 as an ER protein, termed pERp1, which associates with IgM in plasmacytoma cells (Shimizu et al., 2009Shimizu Y. Meunier L. Hendershot L.M. pERp1 is significantly up-regulated during plasma cell differentiation and contributes to the oxidative folding of immunoglobulin.Proc. Natl. Acad. Sci. USA. 2009; 106: 17013-17018Crossref PubMed Scopus (42) Google Scholar, van Anken et al., 2009van Anken E. Pena F. Hafkemeijer N. Christis C. Romijn E.P. Grauschopf U. Oorschot V.M. Pertel T. Engels S. Ora A. et al.Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1.Proc. Natl. Acad. Sci. USA. 2009; 106: 17019-17024Crossref PubMed Scopus (49) Google Scholar). However, no activity was ascribed to pERp1, other than an observed 50% decrease in antibody secretion after knockdown in plasmacytoma cells. We determined the subcellular localization of Mzb1 by fractionation of K46 B cells and immunohistochemical analysis of MZ B cells and NIH 3T3 cells stably transfected with an Mzb1-GFP gene construct (Figure 1B and Figures S1E and S1F). In transfected NIH 3T3 cells, the Mzb1-GFP fusion protein, but not a fusion protein lacking the N-terminal signal sequence, colocalized with the ER marker Bap31. Moreover, in the microsomal fraction of K46 B cells, the endogenous Mzb1 showed a similar resistance to proteinase-K treatment as the luminal ER protein BiP, and both proteins were efficiently digested by proteinase-K in the presence of the detergent Triton X-100 (Figure 1B). Thus, Mzb1 appears to be localized in the ER lumen. To gain insight into the function of Mzb1, we generated stably transfected K46 B cell clones in which the expression of Mzb1 was markedly downregulated by an Mzb1-specific siRNA. As controls for the specificity of the siRNA-mediated knockdown, we used both cells carrying a nonspecific siRNA construct and Mzb1-knockdown cells retransfected with an siRNA-resistant (Mzb1∗) expression construct. Immunoblot analysis with Mzb1 antibody revealed a marked reduction of protein expression in three independent Mzb1-knockdown clones and an efficient rescue of Mzb1 protein expression in knockdown cells retransfected with the siRNA-resistant Mzb1∗ construct (Figure 1C). The Mzb1-knockdown cells grew more quickly than the control-knockdown and Mzb1∗-rescue cells (Figure 1E). By determining [H3]-thymidine incorporation, we found that the unstimulated Mzb1-knockdown clones incorporated 4- to 6-fold more thymidine than control-knockdown cells (Figure 1D). No marked differences in the apoptosis of untreated or tunicamycin-treated Mzb1- and control-knockdown K46 cells were observed (Figure S1G). BCR stimulation resulted in similar thymidine incorporation in Mzb1- and control-knockdown cells, suggesting that Mzb1 may curb the proliferation of K46 B cells (Figure 1D). To examine whether Mzb1-knockdown results in activation of K46 cells similar to stimulation by BCR signaling, we analyzed tyrosine phosphorylation. Immunoblot analysis of lysates prepared from serum-deprived control-knockdown, Mzb1-knockdown, and Mzb1∗-rescue cells demonstrated that tyrosine phosphorylation is enhanced in unstimulated Mzb1-knockdown cells and resembles the pattern observed with BCR-stimulated control-knockdown cells (Figure 1F). In this experiment, the stimulation of the BCR further increased tyrosine phosphorylation, suggesting that Mzb1 may be involved in setting a threshold for BCR-mediated signaling. We also examined the effect of Mzb1 downregulation and Mzb1 overexpression on the proliferation of sorted FoB and MZ B cells by transduction of primary cells with bicistronic retroviruses expressing GFP and Mzb1-specific siRNA or GFP and Mzb1. To allow for retroviral transduction of FoB cells, we used anti-CD23, which stimulates them by inducing Ca2+ mobilization (Kolb et al., 1990Kolb J.P. Renard D. Dugas B. Genot E. Petit-Koskas E. Sarfati M. Delespesse G. Poggioli J. Monoclonal anti-CD23 antibodies induce a rise in [Ca2+]i and polyphosphoinositide hydrolysis in human activated B cells. Involvement of a Gp protein.J. Immunol. 1990; 145: 429-437PubMed Google Scholar). Consistent with anti-CD23-induced Ca2+ mobilization, NFAT2 was detected in the nucleus and cytosol of these cells (Figure S1H). In contrast, FoB cells purified by a different scheme, involving depletion of other B cell types with anti-CD9 (Won and Kearney, 2002Won W.J. Kearney J.F. CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice.J. Immunol. 2002; 168: 5605-5611Crossref PubMed Scopus (122) Google Scholar), showed a predominant cytosolic localization of NFAT2 (Figure S1I). Anti-CD23-stimulated and transduced GFP-positive cells were sorted and analyzed for Mzb1 expression and cell proliferation (Figures 1G and 1H and Figure S1J). The transduced lymphocytes kept their surface phenotype (data not shown). In both MZ B and FoB cells, the downregulation of Mzb1 increased cell proliferation, whereas Mzb1 overexpression decreased proliferation. Consistent with the more abundant Mzb1 expression in MZ B versus FoB cells, the effects of Mzb1 downregulation were more pronounced in MZ B cells and those of Mzb1 overexpression were stronger in FoB cells (Figure 1H). Thus, the downregulation of Mzb1 increased cell proliferation and overall tyrosine phosphorylation, whereas Mzb1 overexpression decreased the proliferation rate. Ca2+ is a second messenger that regulates multiple processes in immune cells (Vig and Kinet, 2009Vig M. Kinet J.P. Calcium signaling in immune cells.Nat. Immunol. 2009; 10: 21-27Crossref PubMed Scopus (287) Google Scholar). In unstimulated cells, a low cytosolic Ca2+ concentration is maintained by sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA)-mediated pumping of Ca2+ into the ER and by the inactive states of Ca2+ channels. In stimulated cells, Ca2+ release from the ER stores results in Ca2+ influx and nuclear translocation of the Ca2+-responsive transcription factor NFAT (Cahalan, 2009Cahalan M.D. STIMulating store-operated Ca(2+) entry.Nat. Cell Biol. 2009; 11: 669-677Crossref PubMed Scopus (357) Google Scholar, Crabtree and Olson, 2002Crabtree G.R. Olson E.N. NFAT signaling: Choreographing the social lives of cells.Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar). To examine Ca2+ signaling in response to BCR stimulation, we measured the changes in free intracellular Ca2+ by flow cytometry. We observed an increase in stimulated Ca2+ concentrations relative to control-knockdown and rescue cells in all of the three analyzed clones of Mzb1-knockdown K46 B cells (Figures 2A and S2A). Complementation of Mzb1-knockdown cells with gene constructs expressing siRNA-resistant Mzb1∗ or Mzb1∗CXXA with a mutated CXXA motif resulted in similar attenuation of Ca2+ signaling (Figures 1C and 2A), suggesting that the function of Mzb1 is independent of its thioredoxin motif. We confirmed the enhancement of Ca2+ signaling in Mzb1-knockdown cells by determining the real-time changes in free Ca2+ of individual cells (Figure 2B). The altered Ca2+ flux in BCR-stimulated Mzb1-knockdown cells could be a consequence of changes in the ER Ca2+ store content, an impaired Ca2+ influx from the cellular environment, or a defect in the BCR signaling pathway. Because Mzb1 is an ER resident, we measured potential changes in BCR-independent store-operated Ca2+ entry by treating cells with thapsigargin, an inhibitor of the SERCA pump. Thapsigargin treatment of Mzb1-knockdown and control K46 cells followed by the addition of extracellular Ca2+ showed that Mzb1 downregulation augmented ER Ca2+ depletion and increased Ca2+ influx (Figure 2C). Treatment of cells with the ionophore ionomycin in the absence of extracellular Ca2+ resulted in an increased Ca2+ flux in Mzb1-knockdown cells relative to control and rescue cells, suggesting that Mzb1 regulates the ER Ca2+ store, rather than store-operated Ca2+ entry (Figure S2B). Chemokine signaling also induces Ca2+ fluxes (Muller and Lipp, 2001Muller G. Lipp M. Signal transduction by the chemokine receptor CXCR5: Structural requirements for G protein activation analyzed by chimeric CXCR1/CXCR5 molecules.Biol. Chem. 2001; 382: 1387-1397Crossref PubMed Scopus (22) Google Scholar); however, the effect of the Mzb1-knockdown on Ca2+ mobilization was unaffected by exposure to the chemokine CXCL13 prior to triggering of Ca2+ fluxes (Figures S2C and S2E). Thus, the effect of Mzb1 on Ca2+ mobilization appears to be independent of CXCR5 signaling. As additional readout of altered Ca2+ fluxes, we analyzed the effects of Mzb1 downregulation or overexpression on the nuclear localization and/or activity of the Ca2+-regulated NFATc transcription factors (Crabtree and Olson, 2002Crabtree G.R. Olson E.N. NFAT signaling: Choreographing the social lives of cells.Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar). In K46 B cells, which normally have low NFAT activation, Mzb1 downregulation resulted in enhanced DNA binding by NFAT in electrophoretic mobility shift assays (Figure S2F) and activation of a luciferase reporter construct containing multimerized NFAT-binding sites (Figure S2G). In primary MZ B and B1 cells, Mzb1 downregulation enhanced nuclear localization of NFAT2 (NFATc1) relative to control siRNA-transduced cells (Figures 2D and 2E and Figure S2H). Conversely, overexpression of Mzb1 in FoB cells increased cytosolic localization of NFAT2 (Figures 2E and 2F), consistent with the reduction of BCR-mediated Ca2+ mobilization in Mzb1-overexpressing FoB cells (Figure 2G). Similarly, a reduced Ca2+ store content, released by ionomycin treatment, was observed in individual NIH 3T3 fibroblastic cells stably expressing Mzb1 (Figure 2H). Finally, we directly measured the Ca2+ store content in individual Mzb1-expressing and control NIH 3T3 cells by measuring the fluorescence lifetime of a transiently transfected ER-localized cameleon Ca2+ indicator (Figures 2I and 2J; Luik et al., 2008Luik R.M. Wang B. Prakriya M. Wu M.M. Lewis R.S. Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation.Nature. 2008; 454: 538-542Crossref PubMed Scopus (406) Google Scholar). Mzb1-expressing NIH 3T3 cells showed a significant decrease in the Ca2+ store content relative to mock-transfected NIH 3T3 cells. Thus, the down- and upregulation of Mzb1 in primary MZ B and FoB cells result in opposite effects on experimentally induced Ca2+ fluxes and activation of the Ca2+-responsive NFAT transcription factors. Taken together, these data indicate that Mzb1 regulates Ca2+ signaling via modulating the ER Ca2+ store content. To identify potential Mzb1 interaction partners, we performed a gel filtration experiment with the microsomal fraction of K46 B cells. Immunoblot analysis of individual fractions indicated that the majority of Mzb1 is found in fractions corresponding to 25 kDa, whereas a small amount of Mzb1 was detected in higher molecular weight fractions of ∼220 kDa (Figure 3A ). The higher molecular weight complex of Mzb1 was sensitive to changes in Ca2+ concentration, given that its abundance was decreased by the addition of 2.5 mM Ca2+. We crosslinked proteins in vivo to stabilize interactions prior to cell lysis and purified the Mzb1-associated proteins by precipitation with anti-Mzb1. The immunoprecipitates were separated by SDS-PAGE, and gel regions corresponding to Mzb1-specific bands were isolated from the anti-Mzb1 and the anti-EBNA isotype control lanes (Figure 3B). Mass spectrometry of the eluted proteins allowed for the identification of the chaperones Grp94 and BiP, as well as the protein disulfide isomerases ERp57 and PDIA6 (CaBP1) as Mzb1-associated proteins (Figure 3B and Table S2). In the anti-Mzb1 lane, we also detected a lower abundance of peptides derived from SERCA2b, a client protein of ERp57 (Li and Camacho, 2004Li Y. Camacho P. Ca2+-dependent redox modulation of SERCA 2b by ERp57.J. Cell Biol. 2004; 164: 35-46Crossref PubMed Scopus (187) Google Scholar), and integrin β1, a client protein of ERp57 and Grp94 (Yang and Li, 2005Yang Y. Li Z. Roles of heat shock protein gp96 in the ER quality control: Redundant or unique function?.Mol. Cells. 2005; 20: 173-182Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To confirm the associations of Mzb1 with the identified protein partners, we coimmunoprecipitated Mzb1 protein complexes from K46 B cell extracts in the absence and presence of Ca2+. Both Grp94, a protein with high-affinity binding sites for Ca2+, and ERp57 interact with their client proteins at high Ca2+ concentrations (Biswas et al., 2007Biswas C. Ostrovsky O. Makarewich C.A. Wanderling S. Gidalevitz T. Argon Y. The peptide-binding activity of GRP94 is regulated by calcium.Biochem. J. 2007; 405: 233-241Crossref PubMed Scopus (44) Google Scholar, Li and Camacho, 2004Li Y. Camacho P. Ca2+-dependent redox modulation of SERCA 2b by ERp57.J. Cell Biol. 2004; 164: 35-46Crossref PubMed Scopus (187) Google Scholar). However, Grp94, ERp57, and BiP were efficiently coimmunoprecipitated with anti-Mzb1 in the absence of Ca2+ and less efficiently in the presence of 2.5 mM Ca2+, suggesting that these proteins associate with Mzb1 in a calcium-dependent manner (Figures 3C and 3D). These interactions were also detected by coimunoprecipitations with antibodies against ERp57 and Grp94 (Figures S3A and S3B). Moreover, reprobing the immunoblot of the gel filtration experiments described above revealed an overlap of the ∼220 kDa Mzb1 complex with ERp57 at low but not high Ca2+ concentrations (Figure 3A). In GST pull-down assays, recombinant Mzb1 interacted with both GST-BiP and GST-ERp57, but not with GST alone (Figure 3E). Addition of 10 mM DTT to a K46 cell lysate impaired coimmunoprecipitation of ERp57 and Mzb1, but not that of Grp94 and Mzb1, suggesting that the association of Mzb1 and ERp57 involves disulfide bonds (Figure 3F). Finally, we analyzed whether the association of Mzb1 with ERp57 competes for the interaction of ERp57 with calnexin and calreticulin. In coimmunoprecipitations with anti-ERp57, we detected abundant amounts of calnexin and calreticulin in lysates from siMzb1-knockdown cells, but not in lysates from control-knockdown and Mzb1∗-rescue cells. Conversely, we observed coimmunoprecipitation of both Mzb1 and Grp94 in lysates from control-knockdown and Mzb1∗-rescue cells (Figure 3G). These data suggest that the interaction of Mzb1 with ERp57 results in an exchange of ERp57-associated chaperones. MZ B cells differ from FoB cells by their fast kinetics of antibody secretion, and therefore we examined whether Mzb1 downregulation or Mzb1 overexpression in sorted MZ B and FoB cells alters LPS-induced antibody secretion. ELISpot analysis of Mzb1 siRNA-expressing MZ B cells showed ∼1/7 the number of ASCs relative to control siRNA-expressing MZ B cells (Figure 4A ). Conversely, retroviral Mzb1 overexpression in FoB cells stimulated with anti-CD23 and suboptimal concentrations of LPS resulted in a 5-fold increase in the number of ASCs relative to mock-transduced cells. Moreover, overexpression of Mzb1 in MZ B cells increased the number of ASCs, whereas the downregulation of Mzb1 in FoB cells decreased the frequency of ASCs (Figure 4B). In ELISA assays, we detected an ∼50% decrease of IgM secretion in Mzb1-knockdown MZ B cells and a two-fold increase in Mzb1-overexpressing FoB cells, relative to control cells (Figure S4). Flow cytometry analysis to detect CD138, a marker of ASCs (Tumang et al., 2005Tumang J.R. Frances R. Yeo S.G. Rothstein T.L. Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expres" @default.
• W2000469539 created "2016-06-24" @default.
• W2000469539 creator A5010195864 @default.
• W2000469539 creator A5016354098 @default.
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• W2000469539 date "2010-11-01" @default.
• W2000469539 modified "2023-10-12" @default.
• W2000469539 title "Mzb1 Protein Regulates Calcium Homeostasis, Antibody Secretion, and Integrin Activation in Innate-like B Cells" @default.
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