Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

The B cell antigen receptor of class IgD induces a stronger and more prolonged protein tyrosine phosphorylation than that of class IgM.

Most mature B lymphocytes coexpress two classes of antigen receptor, immunoglobulin (Ig)M and IgD. The differences in the signal transduction from the two receptors are still a matter of controversy. We have analyzed B cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen, or class-specific antibodies, results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetic and the intensity of phosphorylation, however, was quite different between the two receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum after 1 minute and diminished after 60 minutes whereas, in the membrane IgD-expressing cells, the substrate phosphorylation increased further over time, reached its maximum at 60 minutes, and persisted longer than 240 minutes after exposure to antigen. As a result, the intensity of protein tyrosine phosphorylation induced by cross-linking of membrane IgD was stronger than that induced by membrane IgM. Studies of chimeric receptors demonstrate that only the membrane-proximal C domain and/or the transmembrane part of membrane-bound IgD molecule is required for the long-lasting substrate phosphorylation. Together, these data suggest that the signal emission from the two receptors is controlled differently.

Ordered activation of the Ig lambda locus in Abelson B cell lines.

Derivatives of the mu-producing Abelson line P8 have been analyzed for L chain gene rearrangements. Two of seven clones studied assembled their V lambda genes while growing in culture. V lambda gene rearrangements occurred only in those Abelson subclones that either were rearranging or had rearranged their recombining sequence (RS) element on both Ig kappa alleles. Our data suggest that (a) RS rearrangements are preferentially initiated in kappa- pre-B cells; and (b) the deletion or inactivation of sequences lying between J kappa and RS is a requirement for the activation of the Ig lambda locus.

The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface.

We constructed expression vectors coding for the two pre-B-specific genes, VpreB and lambda 5, and transfected them together with a mu vector (mu tm) into Ig- myeloma cells. In a transfectant expressing all three introduced genes, the mu tm chain is transported on the cell surface. A biochemical analysis demonstrated that, in these cells, the mu tm chain is associated noncovalently with an 18-kD protein and covalently with a 22-kD protein, which are most likely the products of VpreB and lambda 5, respectively. Our results, thus, strongly suggest that the products of lambda 5 and VpreB bind to mu chains and have the same capacity as conventional Ig L chains to allow surface expression of mu chains.

Transfected plasmacytoma cells do not transport the membrane form of IgM to the cell surface.

Expression vectors coding for membrane-bound IgM antibodies were introduced into myeloma and B lymphoma cells. Only the lymphoma but not the myeloma cells were able to express the antibodies on the cell surface, although in both cases, complete antibodies were assembled intracellularly. In myeloma cells, the Ig molecules did not reach the Golgi compartment. Thus, the intracellular transport of membrane-bound antibodies is controlled in the B cell lineages in a developmentally ordered fashion.

SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.

Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31.

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.

Endosomal targeting by the cytoplasmic tail of membrane immunoglobulin.

Membrane-bound immunoglobulin (mIg) of the IgG, IgA, and IgE classes have conserved cytoplasmic tails. To investigate the function of these tails, a B cell line was transfected with truncated or mutated gamma2a heavy chains. Transport to the endosomal compartment of antigen bound by the B cell antigen receptor did not occur in the absence of the cytoplasmic tail; and one or two mutations, respectively, in the Tyr-X-X-Met motif of the tail partially or completely interrupted the process. Experiments with chimeric antigen receptors confirmed these findings. Thus, a role for the cytoplasmic tail of mIg heavy chains in endosomal targeting of antigen is revealed.

Aberrant B cell development and immune response in mice with a compromised BCR complex.

The immunoglobulin alpha (Ig-alpha)-Ig-beta heterodimer is the signaling component of the antigen receptor complex on B cells (BCR) and B cell progenitors (pre-BCR). A mouse mutant that lacks most of the Ig-alpha cytoplasmic tail exhibits only a small impairment in early B cell development but a severe block in the generation of the peripheral B cell pool, revealing a checkpoint in B cell maturation that ensures the expression of a functional BCR on mature B cells. B cells that do develop demonstrate a differential dependence on Ig-alpha signaling in antibody responses such that a signaling-competent Ig-alpha appears to be critical for the response to T-independent, but not T-dependent, antigens.

Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein.

The BRAF(V600E) mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf(wt)), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf(V600E), although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf(V600E) was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf(wt) and B-Raf(V600E) and suggests that B-Raf(V600E) cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.

Immunodeficiency. Trapping the nude mouse gene.

Hairless nude mice are immunodeficient because they lack a thymus. The nude gene has now been identified; it encodes a winged-helix transcription factor that is expressed specifically in skin and thymus.

Molecular mimicry of the antigen receptor signalling motif by transmembrane proteins of the Epstein-Barr virus and the bovine leukaemia virus.

BACKGROUND: Many transmembrane proteins of eukaryotic cells have only a short cytoplasmic tail of 10 - 100 amino acids, which has no obvious catalytic function. These tails are thought to be involved either in signal transduction or in the association of transmembrane proteins with the cytoskeleton. We have previously identified, in the cytoplasmic tails of components of B and T lymphocyte antigen receptors, an amino-acid motif that is required for signalling. The same motif is also found in the cytoplasmic tails of two viral proteins: the latent membrane protein, LMP2A, of Epstein Barr virus and the envelope protein, gp30, of bovine leukaemia virus. Interestingly, both viruses can activate infected B lymphocytes to proliferate, as does signalling by the B-cell receptor. RESULTS: In this study, we show that the cytoplasmic tails of the two viral proteins, and the cytoplasmic tail of the B-cell receptor immunoglobulin-alpha chain, when linked to CD8 in chimeric transmembrane proteins, can transduce signals in B cells. Cross-linking of these chimeric receptors activates B-cell protein tyrosine kinases and results in calcium mobilization. Furthermore, these cytoplasmic sequences are also protein tyrosine kinase substrates and may interact with cytosolic proteins carrying SH2 protein-protein interaction domains. CONCLUSION: Our findings suggest that viral transmembrane proteins can mimic the antigen-induced stimulation of the B-cell antigen receptor and thus can influence the activation and/or survival of infected B lymphocytes.

B cell antigen receptors.

Antigen receptors have a dual role on the surface of B lymphocytes, namely the binding and internalization of antigens and the activation of the antigen-recognizing B cell. The structural requirements for internalization are still a matter of controversy; but as far as signalling is concerned it is becoming increasingly apparent that the antigen receptor is functionally divided into the ligand-binding immunoglobulin (Ig) molecule and the signal-transducing Ig-alpha/Ig-beta heterodimer. This knowledge has been exploited to design variant antigen receptors which combine both functions in a single molecule.

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

Stability of the B cell antigen receptor complex.

The B cell antigen receptor (BCR) comprises the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer. By comparing the stability of the IgD-BCR and IgM-BCR in different detergent lysates, we find that the IgD-BCR is more stable than the IgM-BCR. Analysis of chimeric mIgD molecules suggests that the deltam transmembrane region is responsible for the more stable association of mIgD with the Ig-alpha/Ig-beta heterodimer. Further, the differential glycosylation of Ig-alpha molecules, in the two different BCR complexes, is determined solely by the ectodomains of the mIg molecules. The implications of these findings for the intracellular transport and the signalling capacity of the BCRs are discussed.

Fc-dependent effector functions of idiotype-anti-idiotype immune complexes.

Some effector functions of antigen-antibody and antibody-antibody (idiotype-anti-idiotype) complexes were analyzed. As a model system a monoclonal IgM antibody specific for the hapten NP (antibody B1-8) was reacted either with hapten and hapten-carrier conjugates or with monoclonal anti-idiotope antibodies with specificity for B1-8 idiotopes. The precipitating, C1q-binding, complement-activating and Fc receptor binding properties of these complexes were compared. Binding of both hapten-carrier conjugates and anti-idiotope antibodies to B1-8 results in formation of complexes which depending on the B1-8:ligand ratio precipitate, activate complement, bind C1q and exhibit increased avidity for Fc mu and Fc gamma receptors of mouse spleen cells. In both types of complexes cross-linking of IgM molecules is essential for triggering these Fc-dependent functions, and a functional heterogeneity if idiotype-anti-idiotope complexes based on different idiotype-anti-idiotope ratios could also be observed. The functional similarity of B1-8-hapten-carrier and B1-8-anti-idiotope complexes suggests that regulatory functions so far assigned to antigen-antibody complexes could be carried out also by idiotype-anti-idiotope complexes.

The ribose 5-phosphate isomerase-encoding gene is located immediately downstream from that encoding murine immunoglobulin kappa.

The immunoglobulin kappa locus (Ig kappa) is active only in the B-lymphocyte cell lineage. By exon-trapping we found a gene situated downstream from the murine Ig kappa locus. This gene encodes a protein with 53% sequence identity to the ribose 5-phosphate isomerase A (RPI-A) of Escherichia coli and is therefore likely to be the murine homologue (mRPI) of this enzyme. We confirmed this assumption by showing that a glutathione S-transferase (GST)::mRPI fusion protein has enzymatic activity and that an anti-mRPI antibody detects a protein of the predicted mass of RPI (33 kDa). Cloning and sequencing of the human counterpart show that the RPI gene is evolutionarily conserved. The expression of mRPI is not influenced by the rearrangement status of the Ig kappa locus in B cells and mRPI is expressed in all tissues. We thus show that two genes with very different expression patterns, a housekeeping gene and a gene expressed in a tissue-specific manner, can be located on a chromosome in close proximity to each other.

Function of B-cell antigen receptor of different classes.

Most mature B lymphocytes co-express two classes of antigen receptor, IgM and IgD. The differences in the signal transduction from the 2 receptors are still a matter of controversy. We have analysed B-cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen or class-specific antibodies results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetics and intensity of phosphorylation, however, were quite different between the 2 receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum already after 1 min and diminished after 60 min whereas in the membrane IgD-expressing cells, the substrate phosphorylation increases further over time, reached its maximum at 60 min and persisted longer than 240 min after exposure to antigen. Recently prolonged signaling has been found to be responsible for signaling differences between tyrosine kinase receptors using otherwise similar signaling routes. Thus, the duration of a signal may be an important biological feature of signal transducing cascades.

Characterization of the B cell-specific adaptor SLP-65 and other protein tyrosine kinase substrates by two-dimensional gel electrophoresis.

The identification of substrates for protein tyrosine kinases in B cells is a critical step to a better understanding of the molecular mechanism(s) of lymphocyte activation through the antigen receptor. The substrate proteins were immunopurified from stimulated B cells and separated by two-dimensional gel electrophoresis techniques using either the isoelectric focussing (IEF)/SDS-PAGE or the non-equilibrium PH gradient electrophoresis (NEPHGE)/SDS-PAGE method. The biochemical characteristics of the proteins (isoelectric point and relative molecular mass) obtained and the subsequent use of antibodies that are specific for different cellular proteins confirmed the participation of HS1, Vav, Ig-alpha, Lyn and Btk in antigen receptor-mediated signal transduction. The heat shock cognate protein HSC70 was identified as a novel substrate protein in activated B cells. An important signaling function has previously been suggested for a 65-kDa protein (p65), whose phosphorylation can be detected before that of other substrate proteins. The analysis identified p65 as a so far unknown protein. Based on p65 peptide sequences, the full length cDNA was isolated and found to encode a B cell-specific adaptor protein, called SLP-65.

Signaling difference between class IgM and IgD antigen receptors.

Most mature B lymphocytes coexpress two classes of antigen receptor, IgM and IgD. The differences in the signal transduction from the two receptors are still a matter of controversy. We have analyzed B-cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen or class-specific antibodies results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetic and intensity of phosphorylation, however, was quite different between the two receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum after one minute and diminished after 60 minutes, whereas in the membrane IgD-expressing cells, the substrate phosphorylation increased further over time, reaching its maximum at 60 minutes and persisting longer than 240 minutes after exposure to antigen. Recently prolonged signaling has been found to be responsible for signaling differences between tyrosine kinase receptors using otherwise similar signaling routes. Thus, the duration of a signal may be an important biological feature of signal-transducing cascades.