Anti-filaggrin antibodies (AFA), directed against the 37-40 kD epidermal protein filaggrin are one of the most specific markers of rheumatoid arthritis (RA) and include anti-keratin antibodies (AKA) and anti-perinuclear factor (APF). Although the antigen triggering autoimmune response to filaggrin related proteins has not been identified, recent studies confirmed that citrulline is essential constituent of protein related antigenic determinats recognised by RA specific autoantibody population.
The aim of our study was to identify epitopes of filaggrin derived-peptides targeted by RA specific antibodies to provide further information about the nature of the initial autoantigenic substance.
Citrullin containing peptides of human profilaggrin region (amio acid residues 306-324) derived from known cDNA and on the basis of the data published by Shellekens were synthetised by the multipin peptide synthesis on solid support and were reacted in situ by patient sera. Two 19-mer peptides were prepared with single cit-rullin substitution at position 312 or 321 respectively and four additional ones with simultanious replacements of two Arg by Cit. Shortened versions of the 306SHQESTCitGRSGRSGRSGR324 peptidewere also produced by removal of 1-6 amino acid residues from its N-terminal and the 14 -mer truncated one was further shortened from its C-terminal as well. The reactivities of these peptides with RA sera and healthy controls were determined. The results showed that substitution of arginine 312 by citrulline plays major role in the anigenicity of filaggrin-derived sequences. Peptides not containing Cit in position 312 almost lost their ability to bind antibodies from RA sera. Replacement of one additional Arg by Cit in different positions did not improved the antigenicity. When the peptide with Cit in position 312 were shortened from its N-terminal, the 14-mer one showed the highest reactivity. Further shortening of this sequence from its C-terminal showed that TXGRS motif seems to be essential to comprise the autoanigenic epitope.
In conclusion our results provide further evidence that not simply the presence of citrullin but also the nature of its surronding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies.