Contribution of Peptide Backbone to Anti-Citrullinated Peptide Antibody Reactivity

Abstract

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 1–2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs), which have been found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs.

Generation and Characterization of Anti–Citrullinated Protein Antibody–Producing B Cell Clones From Rheumatoid Arthritis Patients

“…Whereas serum ACPAs are polyreactive to different citrullinated epitopes (38), our data indicate that the BCR expressed by CPreactive memory B cells were characterized by a more restricted ACPA fine-specificity pattern. This finding supports that from other studies (39) indicating that CP-reactive memory B cells recognize citrulline within a specific amino acid context beyond the citrulline residue itself. The discrepancy between our results and others may be reflective of the bifurcation of developmental pathways leading to distinct cellular fates of plasma and memory B cells.…”

Structural Basis of Cross‐Reactivity of Anti–Citrullinated Protein Antibodies

“…Several studies showed that ACPA responses are highly cross‐reactive toward citrullinated antigens, regardless of their origins . Consistent with these observations, the Luminex screening assay using a library of CCPs derived from CII and classic citrullinated peptides clearly showed that both E4 and F3 could recognize several peptides whose sequences were distinct except for the presence of a citrulline, strong preference for a glycine as the (Cit+1) residue, and the high frequency of Gly‐X‐Y repeats natively occurring in CII.…”

“…Characterization of the crystal structures of the E4 Fab –peptide complexes confirmed that the Cit‐Gly motif is essential for recognition. It was reported that only the peptides containing the Cit‐Gly motif showed significant antibody reactivity in the serum of RA patients, and that the substitution of glycine with residues harboring small side chains could reduce antibody reactivity . The preference for glycine at the (Cit+1) position is due to the steric hindrance imposed by the antibody residues at this position.…”

Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide Specific Antibodies in Rheumatoid Arthritis

“…Cross-reactivity of ACPA was reported by several groups, and the concept that Cit-Gly motif has importance in the recognition was also studied earlier [26,29,30,36,41]. ACPAs depend on a specific citrullinated epitopes, rather than a citrulline containing motif [41,44].…”

“…The suggested central role of Cit-Gly [41,44] Gly and thus Ala-Cit motif is also important in the recognition. Based on these, we designed an artificial peptide containing two Cit-Gly and two Ala-Cit motifs.…”