AUTOANTIBODIES TO PROTEIN-ARGININE DEIMINASE (PAD) 1 FROM THE SERA OF RHEUMATOIR ARTHRITIS (RA) PATIENTS RECOGNIZE THEIR ANTIGEN INDEPENDENTLY OF ITS AUTOCITRULLINATION STATUS
- Laura Martinez-Prat (Spain)
Abstract
Background and Aims
Protein citrullination, key in Rheumatoid Arthritis (RA), is catalyzed by the protein-arginine deiminase (PAD) enzymes, which require calcium for their activity. These proteins can autocitrullinate but the impact on antibody recognition isn’t fully understood. The objective of this study was to evaluate the effect of PAD1 autocitrullination on the recognition by anti-PAD1 antibodies.
Methods
Full-length PAD1 proteins were recombinantly generated in the absence or presence of calcium. Citrullination status of the produced antigens was evaluated by immunoblotting using the anti-Citrulline (Modified) Detection Kit (MerckMillipore), alongside a commercial PAD1 protein (Cayman). A panel for the detection of anti-PAD1 IgG for the particle-based multi-analyte technology [PMAT, research use only (RUO), Inova Diagnostics] was created by conjugating the citrullinated and non-citrullinated PAD1 versions to magnetic beads. Sera from RA patients (n=14) and healthy controls (n=8) were tested with this panel on the Aptiva instrument (RUO, Inova Diagnostics).
Results
Autocitrullination of PAD1 antigens was confirmed by anti-citrulline immunoblot only for the proteins generated in the presence of calcium (Figure 1). Partial citrullination was observed for the commercial PAD1 antigen. Strong reactivity to both the citrullinated and non-citrullinated PAD1 antigens was observed with the PMAT anti-PAD1 IgG panel, with apparent higher reactivity to the non-citrullinated versions (Figure 2).
Conclusions
Both citrullinated and non-citrullinated PAD1 were recognized by anti-PAD1 IgG antibodies in the sera of RA patients, supporting the idea that these antibodies recognize unique epitopes in the PAD1 enzyme and are distinct from ACPA.