PPAD is not targeted as a citrullinated protein in rheumatoid arthritis, but remains a candidate for inducing autoimmunity

We thank Dr Quirke and colleagues for their interest in our study. We have some concerns, however, that the authors’ attempt to reconcile our work on Porphyromonas gingivalis peptidylarginine deiminase (PPAD) autocitrullination with their model of periodontal disease in rheumatoid arthritis (RA) misrepresents our data. 2 The discovery that citrullination is the primary antigenic determinant recognised by autoantibodies in RA has generated considerable interest in the family of enzymes, peptidylarginine deiminases (PADs), that catalyse this post-translational protein modification. The identification of a bacterial PAD from P gingivalis led to the initial hypothesis by Rosenstein et al that infection with this periodontal pathogen may be aetiologically linked to RA. Two distinct mechanisms have since been hypothesised by which PPAD may trigger loss of tolerance to citrullinated proteins and autoimmunity in RA—first, by acting as a citrullinating enzyme on bacterial and host proteins, 5 and second, through the process of enzyme autocitrullination. In the absence of experimental evidence to support PPAD autocitrullination in P gingivalis or in vivo, Quirke et al posited a third mechanism in which PPAD acts as a substrate for human PADs, thereby becoming the target of a citrulline-specific antibody response. The first two hypotheses support P gingivalis as a unique periodontal bacterium relevant to RA pathogenesis. The uniqueness of P gingivalis for RA is lost, however, when considering the third hypothesis of bacterial proteins as putative substrates for human PADs. Under this premise (not supported by experimental data), human PADs may generate citrullinated antigens from any conceivable pathogen, making P gingivalis expendable as a model organism. To strengthen the credibility of PPAD for inducing autoimmunity in RA, Quirke et al variably use these three hypotheses. Importantly, our study only addressed the question of PPAD autocitrullination as a possible link of periodontal disease and RA. This mechanism, in which tolerance to peptidyl-citrulline is presumably lost due to an immune response to autocitrullinated bacterial PPAD, was supported by two findings in a recent study by Quirke et al, in which they reported that (1) full-length PPAD is autocitrullinated when artificially expressed in Escherichia coli, and (2) antibodies directed against glutathione S-transferase (GST)-tagged PPAD appeared to show preferential reactivity towards their autocitrullinated protein in 38% of patients with RA. While compelling as a model, their study did not address the nature of PPAD in P gingivalis. By studying PPAD directly within cultured P gingivalis, we found that PPAD was not autocitrullinated in bacterial cells (nor as a secreted enzyme in cell supernatants). PPAD was expressed as a truncated protein when analysed in its native bacterial species, raising questions about the biological relevance of the ‘full-length’ PPAD proenzyme. To reconcile the absence of PPAD autocitrullination in P gingivalis with the previous report of Quirke et al, we expressed the mature protein and the full-length proenzyme in E coli, and demonstrated that the truncated enzyme present in P gingivalis cannot autocitrullinate. If autocitrullination is indeed absent in vivo, a citrulline-specific antibody response to PPAD in patients with RA cannot be expected (unless the result of anti-citrullinated protein antibody (ACPA) cross-reactivity). Testing this hypothesis, we demonstrated that antibodies in patients with RA cannot distinguish autocitrullinated and native PPAD by two independent techniques, ELISA and immunoprecipitation. Moreover, differential reactivity to PPAD by individual patient