Antibodies to citrullinated alpha-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase

OBJECTIVE: To map the antibody response to human citrullinated alpha-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine cross-reactivity with bacterial enolase. METHODS: Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated alpha-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting. RESULTS: An immunodominant peptide, citrullinated alpha-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37-62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated alpha-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r2=0.803, P<0.0001). Affinity-purified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase. CONCLUSION: We have identified an immunodominant epitope in citrullinated alpha-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.