Mu Heavy Chain Research Articles

Expression of the (recombinant) endogenous immunoglobulin heavy-chain locus requires the intronic matrix attachment regions.

The elements which regulate gene expression have traditionally been identified by their effects on reporter genes which have been transfected into cell lines or animals. It is generally assumed that these elements have a comparable role in expression of the corresponding endogenous locus. Nevertheless, several studies of immunoglobulin heavy-chain (IgH) gene expression have reported that the requirements for expressing IgH-derived transgenes differ from the requirements for expression of the endogenous IgH locus. Thus, although expression of transgenes requires multiple elements from the J(H)-C mu intron--the E mu core enhancer, the matrix attachment regions (MARs) which flank E mu, and several switch-associated elements--B-cell lines in which expression of the endogenous heavy-chain gene is maintained at the normal level in the absence of these intronic elements have occasionally been reported. Gene targeting offers an alternative method for assessing regulatory elements, one in which the role of defined segments of endogenous genes can be evaluated in situ. We have applied this approach to the IgH locus of a hybridoma cell line, generating recombinants which bear predetermined modifications in the functional, endogenous mu heavy-chain gene. Our analysis indicates the following. (i) Ninety-eight percent of the expression of the recombinant endogenous mu gene depends on elements in the MAR-E mu-MAR segment. (ii) Expression of the recombinant mu gene depends strongly on the MARs of the J(H)-C mu intron but not on the adjoining E mu core enhancer and switch regions; because our recombinant cell lines bear only a single copy of the mu gene, our results indicate that mu expression is activated by MAR elements lying within that same mu transcription unit. (iii) The MAR segment includes at least one activating element in addition to those defined previously by the binding of presumptive activating proteins in the nuclear matrix. (iv) Close association of the MARs with the E mu enhancer is not required for MAR-stimulated expression. (v) The other MARs in the IgH locus do not in their normal context provide the requisite MAR function.

The roles of preB cell receptor in early B cell development and its signal transduction.

The preB cell receptor is expressed for a short period after mu heavy chain is produced, that is, at the large preB cell stage in B cell development. The severe impairment of B cell differentiation observed in mice deficient for the preB cell receptor clearly demonstrated the importance of the preB cell receptor in B cell development. Analyses of bone marrow precursor B cells in normal and B cell-deficient mutant mice indicated the preB cell receptor transduced signals to drive cell cycle and to induce allelic exclusion. The proliferation of the preB cell receptor-expressing cells leads to the selective expansion of cells which have succeeded in the productive rearrangement of mu heavy chain gene. This process builds up a preB cell pool large enough to generate sufficient numbers of mature B cells. The preB cell receptor appears to induce allelic exclusion by shutting off the expression of recombinase activation gene (RAG). In order to analyse the signal transduction pathway downstream of the preB cell receptor, we have developed a new system in which cross-linking of Ig beta expressed on bone marrow proB cells mimics the signalling through the preB cell receptor to induce differentiation from proB to small preB cells.

Detection of two isotypically different antibodies produced by a murine hybridoma.

A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized. This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells. The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains. Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality. Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted. PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa. A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products. These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.

A Three-protein-DNA Complex on a B Cell-specific Domain of the Immunoglobulin μ Heavy Chain Gene Enhancer

The lymphoid-specific immunoglobulin mu heavy chain gene intron enhancer (muE) contains multiple binding sites for trans-acting nuclear factors. We have used a combination of in vitro and in vivo assays to reconstruct protein-DNA interactions on a minimal B cell-specific mu enhancer that contains three motifs, muA, muB, and muE3. Using ETS-domain proteins that transactivate the minimal enhancer in non-lymphoid cells, we show that (i) PU.1 binds coordinately to both muA and muB sites in vitro and (ii) in the presence of Ets-1, this factor binds to the muA site and PU.1 to the muB site. Two factors, TFE3 and USF, bind to the muE3 element. When the ETS proteins are present together with muE3 binding proteins, a three-protein-DNA complex is generated. Furthermore, we provide evidence for protein-protein interactions between Ets-1 and PU.1 proteins that bind to muA and muB sites, and between Ets-1 and TFE3 bound to the muA and mu3 sites. We propose that this domain of the mu enhancer is assembled into a nucleoprotein complex that contains two tissue-restricted ETS domain proteins that recognize DNA from the same side of the helix and one ubiquitously expressed bHLH-leucine zipper protein that binds between them, recognizing its site from a different side of the helix.

Enhancers of hypermutation.

Hypermutation at the immunoglobulin (Ig) loci increases the mutation rate more than 10(5)-fold over the normal, spontaneous rate. We studied two kinds of cis-acting elements - 3' enhancers and promoters - in a system in which a gene encoding the mu heavy (H) chain (Igm) is transfected in vitro into a cell line with an active Ig mutator. A construct containing a rearranged Igm gene requires the 3' H enhancer for hypermutation at a rate comparable with the one at the endogenous gene segment encoding the H chain variable region (V). Without the 3' enhancer, the basal mutational activity is much lower, but still higher than the normal, spontaneous mutation rate. Replacement of the 3' H enhancer by atopic elements of similar function also supports full hypermutation. Even though these 3' elements are defined as transcriptional enhancers, they do not seem to increase hypermutation via an increase in the rate of transcription. Replacement of the endogenous promoter by the tk promoter slightly increases hypermutability of the construct; thus, no specific sequences in the Ig promoter are likely to target hypermutation.

Mutations in the mu heavy-chain gene in patients with agammaglobulinemia.

Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit production of the membrane form of the mu chain and produce an amino acid substitution in the secreted form. In addition, a patient previously thought to have X-linked agammaglobulinemia was found to have an amino acid substitution on one chromosome at an invariant cysteine that is required for the intrachain disulfide bond and, on the other chromosome, a large deletion that included the immunoglobulin locus. Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans. This implies that an intact membrane-bound mu chain is essential for B-cell development.

Degradation of Distinct Assembly Forms of Immunoglobulin M Occurs in Multiple Sites in Permeabilized B Cells

Protein degradation is essential for quality control which retains and eliminates abnormal, unfolded, or partially assembled subunits of oligomeric proteins. The localization of this nonlysosomal pre-Golgi degradation to the endoplasmic reticulum (ER) has been mostly deduced from kinetic studies and carbohydrate analyses, while direct evidence for degradation within the ER has been provided by in vitro reconstitution of this process. In this article, we took advantage of the transport incompetence of permeabilized cells to directly demonstrate that the selective degradation of secretory IgM (sIgM) in B lymphocytes is transport-dependent. We show that, upon permeabilization of the plasma membrane with either streptolysin O or digitonin, sIgM is not degraded unless transport is allowed. Nevertheless, upon complete reduction of interchain disulfide bonds with thiols, the free mu heavy chains are degraded by a transport-independent quality control mechanism within the ER. This latter degradation is nonselective to the secretory heavy chain mus, and the membrane heavy chain mum, which is normally displayed on the surface of the B cell, is also eliminated. Moreover, the degradation of free mus is no longer restricted to B lymphocytes, and it takes place also in the ER of plasma cells which normally secrete polymers of sIgM. Conversely, when assembled with the light chain, the degradation is selective to sIgM, is restricted to B lymphocytes, and is a transport-dependent post-ER event.

Duck lymphoid organs: their contribution to the ontogeny of IgM and IgY.

The occurrence of mRNAs encoding mu, nu and nu(delta Fc) immunoglobulin heavy chains and lambda light chains in organs of duck embryos from 16 days of incubation and ducklings up to 74 days of age was assessed by Northern hybridization. The mu message was first detected in bursa of Fabricius and spleen at 16 days of incubation and in cervical lymph nodes at 23 days of incubation, but in other organs (bone marrow, buffy coat, Harderian gland, liver) not until 7 17 days after hatching; in general, the appearance of the lambda message paralleled that of mu. Messenger RNAs encoding one or both of the nu isoforms were first detected in cervical lymph nodes at 25 days of incubation, in spleen and bursa in 1-day-old ducklings, in Harderian gland, bone marrow and liver from 10 to 17 days post-hatching and in buffy coat from 46 days. In most organs, the nu(delta Fc) message was detected prior to the nu message and predominated during the experiment; Harderian gland expressed the nu(delta Fc) message exclusively. These results indicate that bursa of Fabricius, spleen and cervical lymph nodes play early roles in the development of B cells and the ontogeny of duck immunoglobulins while other lymphoid organs support the later differentiation of plasma cells, and that IgY and IgY(delta Fc) are probably not simultaneous products of the same plasma cells.

Context dependent transactivation domains activate the immunoglobulin mu heavy chain gene enhancer.

Enhancers and promoters nucleate the assembly of multiprotein complexes that are required for the transcriptional activation of eukaryotic genes. Although multimerized binding sites of individual transcription factors sometimes mimic the properties of an enhancer, the combinatorial use of factors is considered to be crucial for achieving biological specificity. The minimal B cell specific immunoglobulin mu heavy chain gene enhancer is activated by a combination of tissue-restricted ETS proteins and ubiquitously expressed basic helix-loop-helix transcription factors. Here we show that a domain of PU.1 that activates transcription from multimerized PU.1 binding sites is not required to activate the mu enhancer together with Ets-1. In contrast, a transactivation domain in Ets-1 is necessary to activate this enhancer synergistically with PU.1. Furthermore, the Ets-1 activation domain functions only when tethered to the muA site of the enhancer. These observations illuminate two forms of context dependence: first, all possible transcription activation domains may not be required to achieve combinatorial specificity; second, functional transcription activation domains may require appropriate positioning on DNA.

Combined Signaling Through Interleukin-7 Receptors and flt3 But Not c-kit Potently and Selectively Promotes B-Cell Commitment and Differentiation From Uncommitted Murine Bone Marrow Progenitor Cells

Multiple cytokines can synergize to stimulate the in vitro proliferation and exclusive myeloid differentiation of multipotent bone marrow progenitor cells. The ligand for c-kit (stem cell factor [SCF]) plays a key role in stimulating myeloid and erythroid cell production of primitive hematopoietic progenitors. SCF in combination with interleukin-7 (IL-7) can also stimulate the combined myeloid and B-cell differentiation of uncommitted hematopoietic progenitor cells as well as the growth of early B-cell progenitor cells, although the involvement of c-kit in early B lymphopoiesis remains controversial. In the present study, the flt3-ligand (FL), which, in combination with other cytokines, has overlapping activities with SCF on myeloid cell production from uncommitted progenitors, was investigated for its ability to induce selective stroma-independent B-cell commitment from uncommitted Lin-Sca-1+ bone marrow progenitor cells. IL-7 alone did not induce any clonal growth and FL alone gave rise to a few clusters (< 50 cells) but no colonies (> 50 cells), whereas the combined stimulation with FL and IL-7 resulted in clonal growth of 10% of Lin-Sca-1+ bone marrow cells. After 12 days of incubation of Lin-Sca-1+ cells in FL + IL-7, an almost 400-fold increase in cell production was observed. Phenotyping showed that greater than 99% of the cells produced were of the B-cell lineage, in that they expressed B220, but not cell surface markers specific for myeloid, erythroid, or T-cell lineages. Furthermore, the cells did not express cytoplasmic mu-heavy chain (cmu) or surface IgM, but were positive for CD24 (heat stable antigen [HSA]) and CD43 (leukosialin), suggesting that the cells produced were blocked at a late pro-B-cell stage. Interestingly, although all FL + IL-7-responsive Lin-Sca-1+ progenitor cells and the resulting pro-B cells expressed c-kit, FL + IL-7 was much more potent (62-fold) than SCF + IL-7 in stimulating production of cells of the B-cell lineage. In addition, whereas FL + IL-7 selectively stimulated the production of pro-B cells, SCF + IL-7 predominantly stimulated the production of mature granulocytes. Replating studies showed that FL + IL-7-responsive Lin-Sca-1+ progenitors were not committed to the B-cell lineage, because after 2 days of incubation in FL + IL-7, 80% of the progenitors retained a myeloid potential. As much as 27% of the FL + IL-7-responsive progenitors remained uncommitted after 7 days of incubation, but all had committed to the B-cell lineage after 10 days of incubation in FL + IL-7. These results show that FL much more potently and selectively than SCF synergizes with IL-7 to enhance B-cell commitment and development from uncommitted progenitor cells.

A Negative Cofactor Containing Dr1/p19 Modulates Transcription with TFIIA in a Promoter-specific Fashion

An activity that modulated the relative levels of transcription from the adenovirus major late promoter (MLP), and the immunoglobulin heavy chain mu promoter (mu) was purified as a 90-kDa factor. This factor is suggested to be a heterotetramer of two subunits: a 20-kDa polypeptide identical to the previously described Dr1/p19 and a novel 30-kDa polypeptide. The Dr1/p19 protein has been characterized as a repressor of transcription, and the 30-kDa protein is related to a recently identified yeast gene proposed to encode a repressor of transcription. The 90-kDa factor forms a complex with TATA-binding protein on DNA and at high concentrations of both factors protects over a 150-base pair region around the promoter from DNase I cleavage. The conformation of this complex as assayed by footprinting analysis is altered by the transcription factor TFIIA on the MLP but not on the mu promoter. Similarly, TFIIA reverses the repression of transcription by the 90-kDa factor on the MLP but not on the mu promoter. Thus, the interactions of TATA-binding protein, TFIIA, and the 90-kDa factor are promoter-specific.

Precise Alignment of Sites Required for μ Enhancer Activation in B Cells

The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1.

Chimaeric monoclonal antibodies encoded by the human VH26 gene from naïve transgenic mice display a wide range of antigen-binding specificities.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.

Surrogate light chain-dependent selection of Ig heavy chain V regions.

Bone marrow B cell precursors frequently rearrange the Ig heavy chain variable (VH) gene segment VH81X. It is puzzling, therefore, that mature B cells in adult mice rarely express mu-heavy chains bearing this VH gene segment. We show in this work in transformed pre-B cell lines that two VH81X/mu-chains that differ in their VH-D-JH joining sequences are not assembled covalently with the B cell precursor-specific surrogate light (SL) chain and are not expressed on the cell surface. From these findings, we propose that a B cell precursor clonally expands and proceeds to the next developmental stage only if it expresses a mu-chain with a VH domain that, together with the SL chain, directs the formation of a signal-transducing mu/SL chain membrane complex. Therefore, a checkpoint exists early in B cell development, at which SL chain not only screens B cell precursors for the presence of a full-length mu-chain, but also for a VH domain that promotes the assembly of a mu/SL chain complex.

Malignant lymphoma of mucosa associated lymphoid tissue: a new etiology of amyloidosis

Non Hodgkin Malignant lymphomas (NHML) of mucosa associated lymphoid tissue (MALT) are known to have multiple involvement of the digestive tract. We report one case, presenting with an infiltrative process of the jejuno-ileum, associating lymphoplasmacytoid proliferating cells and amyloidosis. The plasmacytoid cells expressed Alpha and scarce Mu heavy chains, and lambda light chain. Lympho-epithelial lesions were more obvious at the second site of involvement, in the gastric mucosa. The amyloid substance was negative with the Amyloid A component antibody and gave a background noise with Alpha, Mu and lambda chains. No similar report of amyloidosis associated with MALT NHML has been found in the literature.

Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphatase activities.

Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.

Identification and localization of a developmental stage-specific promoter activity from the murine lambda 5 gene.

The lambda 5 protein is expressed in pre-B cells in association with VpreB and mu-heavy chains, and is critical for differentiation to the B cell stage. Pre-B cell-specific expression of the lambda 5 and VpreB genes is regulated at the level of transcription initiation. In this report, we have identified several DNase l-hypersensitive sites 2.5- to 6.0-kb downstream of the lambda 5 gene, which are present in the pre-B cell line 70Z/3, but not in the myeloma cell line j558L. These sites, however, were shown to have no transcriptional enhancer activity as measured by transient transfection. Enhancer activity was identified within a 361-bp fragment (-296 to +65, where +1 is the major 5' transcription initiation site) upstream of the mouse lambda 5 gene. This activity is orientation and position independent, and is also tissue and differentiation stage specific (active in pre-B but not B and T cells). Deletion constructs indicate that three adjacent areas (-210 to -169, -153 to -64, and -64 to -22) are all necessary for enhancer activity. Pre-B cell-specific promoter activity was shown to reside within the -219 to +109 fragment. Basal promoter activity resides within the -64 to +109 fragment, but is not tissue specific or stage specific. A negative element within the -101 to -64 region is active in all lymphoid cell lines tested and therefore cannot by itself be responsible for the tissue and stage specificity. The data indicate that the elements responsible for the enhancer activity (-210 to -22) are part of the lambda 5 gene promoter and likely confer the tissue and stage specificity via positive elements within the -210 to -22 region.

Ig mu heavy chains with VH81X variable regions do not associate with lambda 5.

Annals of the New York Academy of SciencesVolume 764, Issue 1 p. 39-42 Ig μ Heavy Chains with VH81X Variable Regions Do Not Associate with λ5a ULRIKE KEYNA, ULRIKE KEYNA Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorSTEVEN E. APPLEQUIST, STEVEN E. APPLEQUIST Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorJAN JONGSTRA, JAN JONGSTRA Toronto Western Hospital Toronto, Ontario, Canada M5T 2S8Search for more papers by this authorGABRIELE B. BECK-ENGESER, GABRIELE B. BECK-ENGESER Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorHANS-MARTIN JÄCK, HANS-MARTIN JÄCK Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this author ULRIKE KEYNA, ULRIKE KEYNA Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorSTEVEN E. APPLEQUIST, STEVEN E. APPLEQUIST Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorJAN JONGSTRA, JAN JONGSTRA Toronto Western Hospital Toronto, Ontario, Canada M5T 2S8Search for more papers by this authorGABRIELE B. BECK-ENGESER, GABRIELE B. BECK-ENGESER Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this authorHANS-MARTIN JÄCK, HANS-MARTIN JÄCK Department of Microbiology and Immunology Loyola University Chicago Stritch School of Medicine Maywood, Illinois 60153Search for more papers by this author First published: September 1995 https://doi.org/10.1111/j.1749-6632.1995.tb55803.xCitations: 10 a This work was supported by grants from the American Cancer Society of Illinois, the National Cancer Institute, and the Schweppe Foundation of Chicago to H.-M.J., U.K. is supported by a fellowship from the Deutsche Forschungsgemeinschaft, and S.E.A. by a Schmitt Fellowship. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume764, Issue1Immunoglobulin Gene Expression in Development and DiseaseSeptember 1995Pages 39-42 RelatedInformation

Signal transduction by the B-cell antigen receptor.

The antigen receptor of B lymphocytes (BCR) plays important roles in recognition of foreign antigens and self-components to allow the immune system to make appropriate antibody responses. The BCR is a complex between membrane immunoglobulin and the Ig-alpha and Ig-beta heterodimer. Site-directed mutagenesis experiments have shown that the mu heavy chain transmembrane domain plays a key role in the association of mIgM with Ig-alpha/Ig-beta. In the absence of complex formation, mIgM is retained in the endoplasmic reticulum, and this function is also specified by the mu chain transmembrane domain. The ability of various mutant mIgM molecules to associate with Ig-alpha/Ig-beta correlates well with their ability to induce signal transduction reactions such as protein tyrosine phosphorylation and phosphoinositide breakdown. Thus, the signaling ability of the BCR appears to reside in the Ig-alpha/Ig-beta heterodimer. The cytoplasmic domains of Ig-alpha and Ig-beta each contain an ITAM sequence, which is defined by its limited homology with subunits of the T-cell antigen receptor and of Fc receptors. Moreover, chimeric proteins containing these ITAMs and surrounding sequences from the cytoplasmic domains of Ig-alpha or Ig-beta exhibit signaling function characteristics of the intact BCR. The Ig-alpha and Ig-beta chimeras are each capable of inducing all of the BCR signaling events tested and thus represent redundant functions. Cross-linking these chimeras leads to their phosphorylation and to binding of the intracellular tyrosine kinases Lyn and Syk. The BCR expressed in the nonlymphoid AtT20 cells, which express the Src-family tyrosine kinase Fyn but not Syk, was not able to trigger vigorous signaling reactions. Introduction of the active form of Syk into these cells restored some signaling events. These results are consistent with a model in which the ITAMs act to initiate the BCR signaling reactions by binding and activating tyrosine kinases.

Roles of heavy and light chains in IgM polymerization.

IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.