Background:Rheumatoid arthritis (RA) is characterized by autoantibodies against post-translationally modified proteins (AMPA) such as citrullinated, carbamylated and acetylated proteins. Importantly, these antibodies are highly multireactive, as they often recognize more than one of these post-translational modifications. Despite extensive research, the antigens inducing the breach of tolerance remain unknown, although microbial antigens are often suspected. Various bacteria are known to be capable of acetylation, therefore, it is intriguing to know what mechanisms can underlie the breach of tolerance towards acetylated proteins and development of anti-acetylated protein antibodies (AAPA).Objectives:To investigate whether acetylated proteins of bacterial origin (1) are recognized by human derived AMPA and AMPA expressing B cells; and (2) can induce AMPA development when used to immunize mice.Methods:Acetylated E. coli proteins were acquired with two separate methods (Figure 1A): by culturing E. coli in a condition promoting auto-acetylation (intrinsically acetylated bacterial proteins, IABP), or by directly acetylating lysate-derived proteins via a chemical reaction (extrinsically acetylated BP, EABP). Acetylated ovalbumin (AcOVA) served as positive control for AAPA induction in mice, non-acetylated BP (NABP) and phosphate buffer saline (PBS) served as negative control. Mice were immunized with these proteins and the resulting antibody response was studied by ELISA. Furthermore, EABP/IABP/NABP were investigated for recognition by human-derived AAPA with ELISA and AAPA-expressing B cells with spleen tyrosine kinase (Syk) phosphorylation assay; acetylated human fibrinogen and native fibrinogen served as positive and negative control.Results:Repetitive immunization of mice with EABP resulted in an AMPA response recognizing acetylated, carbamylated and citrullinated proteins. AMPA titers in these mice exceeded the titers in the positive control mice immunized with AcOVA and were substantially higher than in the NABP-immunized mice (Figure 1B). Human-derived monoclonal AAPA recognized EABP and IABP (not shown). B cell activation (measured by Syk phosphorylation) assay indicated that AAPA expressing B cells recognized EABP and (to a lesser extent) IABP, but not NABP (Figure 1C).Conclusion:Acetylated bacterial proteins are potent antigens that can induce cross-reactive AMPA responses in mice and they are recognized by human AAPA. This suggests that acetylated bacterial proteins could possibly be involved in the breach of tolerance in RA.Acknowledgements:We thank Dr. Can Araman and Prof. Chunaram Choudhary for their advice regarding optimization of bacterial auto-acetylation.Disclosure of Interests:None declared
FLASHIda: Intelligent data acquisition for top-down proteomics that doubles proteoform level identification count
