Background/Purpose: Anti-citrullinated protein antibodies (ACPA) are the main autoantibody system in rheumatoid arthritis (RA), and due to high sensitivity and specificity testing using the anti-CCP2 assay is the gold standard in RA diagnosis.  Citrullinated proteins are formed when arginine residue(s) are converted to citrulline residue(s) by peptidylarginine deiminase (PAD) enzymes.  Five known human PAD isoforms (PAD1-4 &6) catalyse citrullination constitutively in numerous healthy tissues, and the process is essential for many pathways.  It is unknown what causes the breach of tolerance to these native proteins.  Periodontitis (PD) is a known risk factor for RA, and there is increasing evidence that the link between the two diseases is due to citrullination by a unique prokaryotic PAD enzyme expressed by the keystone PD pathogen Pophyromonas gingivalis (PPAD).  PPAD and mammalian PADs have distinct substrate specificities and low sequence homology, thus making PPAD a prime candidate for inhibition. However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme.  By solving the 3D structure of PPAD we aim to characterize activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA.  

Methods: A library of PPAD clones was synthesized, expressed and purified from E.coli.  Candidates for crystallization were identified based on high solubility and activity.  A highly active clone tPPAD^WT was identified and crystallized, alongside a catalytically inactive mutant tPPAD^C351A of corresponding sequence- with the exception of a single amino acid substitution within the active site- and their 3D crystal structures solved.  Both enzymes were crystallized with bound arginine containing ligands.  Key residues identified from 3D structures were examined by mutations.  Fibrinogen and α-enolase were incubated with tPPAD^WT and P.gingivalisarginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry.   

Results: Here, we solve the crystal structure of a truncated, highly active form of PPAD, and confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351.  In addition, we identify Arg152 and Arg154 as involved in substrate binding, and using a number of mutations demonstrate they may determine the substrate specificity of PPAD for C-terminal arginines.  We show RA autoantigens are favorable substrates for PPAD and demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD^WTand RgpB.

Conclusion: PPAD displays an unequivocal specificity for C-terminal arginine residues not shown by mammalian PAD2 or PAD4.  PPAD readily citrullinates peptides from key RA autoantigens to form novel peptides, which could be involved in the breach of tolerance to native citrullinated peptides in RA.  The lack of sequence homology to mammalian PADs makes PPAD and ideal target for inhibition, and this high-resolution crystal data presented here could pave the way for this.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/crystal-structure-of-porphyromonas-gingivalis-peptidylarginine-deiminase-implications-for-autoimmunity-in-rheumatoid-arthritis/