Acetylation is mediated by acetyltransferases and leads to a conversion of lysine-residues into acetyllysines. Carbamylation is a non-enzymatic process, resulting from the modification of a lysine-residue into homocitrulline. Anti-carbamylated protein antibodies (ACarPA) and anti-acetylated protein antibodies (AAPA) are present in approximately 50% and 40% of RA patients, respectively. More recently, autoantibodies towards other post-translational modifications (PTMs) have been described in RA. ACPA recognize a wide spectrum of antigens and are found in different isotypes. About 50–70% of RA patients harbour anti-citrullinated protein antibodies (ACPA), directed against citrullinated proteins generated by the conversion of arginine to citrulline. The presence of autoantibodies is an immunological hallmark of rheumatoid arthritis (RA). Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation. We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA).
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