ARTHRITIS & RHEUMATISM Vol. 62, No. 1, January 2010, pp 126–131 DOI 10.1002/art.27230 © 2010, American College of Rheumatology Rheumatoid Arthritis–Specific Autoantibodies to Peptidyl Arginine Deiminase Type 4 Inhibit Citrullination of Fibrinogen Isabelle Auger, Marielle Martin, Nathalie Balandraud, and Jean Roudier Objective. Autoantibodies to citrullinated pro- teins are specific for rheumatoid arthritis (RA) and recognize epitopes centered by citrulline, a posttransla- tionally modified form of arginine. Peptidyl arginine deiminase type 4 (PAD-4), the enzyme that converts arginine into citrulline, is in itself a target for RA- specific autoantibodies. This study was undertaken to assess whether anti–PAD-4 autoantibodies interfere with citrullination in vitro in patients with RA, and to identify peptide targets of anti–PAD-4 antibodies that can activate or inhibit citrullination. Methods. To test whether autoantibodies to PAD-4 influence citrullination, an in-house citrullina- tion assay was developed using purified autoantibodies to PAD-4. To map B cell epitopes on PAD-4, 65 overlap- ping 20-mer peptides encompassing the entire PAD-4 were analyzed for their reactivity in RA sera. Results. Autoantibodies to PAD-4 inhibited PAD- 4–mediated citrullination. Three linear peptides on PAD-4 were recognized almost uniquely by PAD-4 auto- antibodies in the sera of patients with RA. One peptide was located in the N-terminal, calcium-binding domain of PAD-4, while 2 other peptides were located in the C-terminal, substrate-binding domain of PAD-4. Conclusion. Autoantibodies to PAD-4 inhibit in vitro citrullination of fibrinogen by PAD-4. Most anti– PAD-4–positive sera recognize peptides located both in the N-terminal domain (211–290) and the C-terminal domain (601–650) of PAD-4. Rheumatoid arthritis (RA) is a chronic inflam- matory joint disease that leads to articular cartilage and bone destruction (1). Its worldwide prevalence is 0.5%, but its cause is unknown. RA is an autoantibody- mediated disease, and the most critical autoantibodies in RA are directed at citrulline residues on different pro- teins, such as fibrin, filaggrin, and vimentin (2–5). Cit- rulline is generated by posttranslational conversion of arginine residues. This process is catalyzed by a group of enzymes called calcium-dependent peptidyl arginine de- iminases (PADs) (6). Polymorphisms in PADI4, the gene encoding PAD-4, are associated with the development of RA in Asian populations (7–10). PAD-4 not only is involved in the generation of citrullinated epitopes, but also is in itself a target for RA-specific antibodies. Autoantibodies against PAD-4 have already been described in RA (11–14). In a 2008 study using protein array and enzyme-linked immu- nosorbent assay (ELISA) analyses, we identified auto- antibodies to PAD-4 in the sera of patients with RA (15). We observed that 29% of the 116 RA patients had anti–PAD-4 antibodies, compared with none of the 33 patients with ankylosing spondylitis (AS) and 3% of the 60 healthy control subjects (P  10 -7 by chi-square test). In the present study, we developed an in-house citrullination assay to test how autoantibodies directed at PAD-4 may interfere with its activity. To map B cell epitopes on PAD-4, we tested RA sera against 65 overlapping 20-mer peptides encompassing the entire PAD-4. PATIENTS AND METHODS RA patients and controls. RA patients were chosen from the Rheumatology Ward at Hospital La Conception in Supported by grants from INSERM, the Association pour la Recherche Contre la Polyarthrite, and the Socie ´te ´ Franc ¸aise de Rhumatologie. Isabelle Auger, PhD, Marielle Martin, Nathalie Balandraud, MD, PhD, Jean Roudier, MD, PhD: INSERM Unite ´ 639, Marseilles, France. Address correspondence and reprint requests to Isabelle Auger, PhD, INSERM Unite ´ 639, Parc Scientifique de Luminy, 163 Avenue de Luminy, Case 939, 13009 Marseilles, France. E-mail: isabelle.auger@inserm.fr. Submitted for publication April 24, 2009; accepted in revised form September 16, 2009. 126