AUTO-ANTIBODIES TO THE AMINO TERMINAL SEGMENT (AMINOACID 1-17) OF CENP-A DISPLAY A UNIQUE SPECIFICITY IN SYSTEMIC SCLEROSIS AND ARE INDEPENDENTLY EXPRESSED FROM THOSE AGAINST CENP-B

Background and objective: Systemic sclerosis is a highly invalidating connective tissue disease, whereby an interplay between vasculopathies, fibroblast activation and autoimmunity leads to widespread collagen deposition and tissue fibrosis. Autoimmunity is demonstrated by the presence of anti-nuclear antibodies (ANA) which include different subsets of auto-antibodies (Abs), such as anti-CENP-A Abs. Because CENP-A does not appear to have any pathogenetic (or protective) role to explain why the corresponding auto-Abs are more frequently found in the limited rather than the diffuse form of systemic sclerosis, and to get a deeper insight into the mechanism(s) by which these Abs are generated, we investigated their fine specificity, i.e., the amino acids (motif amino acids) which are recognized by this Ab population. We focused our study on the amino terminal portion of CENP-A spanning amino acid (AA) 1 to 17 (Ap1-17). This region was selected because it represents, along with Ap17-30, an immuno-dominant epitope of CENP-A. Both regions express the motif GPxRx shared with CENP-B, which is thought to be at the basis of anti-CENP-B cross reactivity with CENP-A. Methods: A CENP-A-derived peptide spanning AA 1 to 17 (Ap1-17) was insolubilized on an AffiGel-15 column, which was used for affinity purification of anti-Ap1-17 Abs from anti-CENP-positive sera of 8 patients with systemic sclerosis. SDS-GEL was employed to assess their purity and Western blot to test the reactivity of purified anti-Ap1-17 IgG with recombinant CENP-A and CENP-B. ELISAs were used to test the reactivity of sera or purified anti-peptide Abs. A phage display peptide library (PDPL) was used to identify anti-Ap1-17 Abs-specific motif AA. Web-available programs were looked up for the motif screening against protein database. Results: Anti-Ap1-17 IgG purified from these sera reacted specifically and dose-dependently with Ap1-17, whereas no reactivity was observed with Ap17-30. Panning of PDPL with anti-Ap1-17 IgG from patients pt14 and pt4 identified the 2 partially overlapping motifs <5Rx(st)xKP10.> and <9KPxxPxR15.>. Assessment of the reactivity of isolated phage clones with IgG anti-Ap1-17 divided the 8 patients into 4 groups, in function of their IgG reactivity (or lack of reactivity) with the pt14 and pt4 IgG-specific phage clones (pcs) 14 and pcs4, as follows: group #1 included pt14 IgG reacting with pcs14 only; group#2 included pt4 IgG which reacted with pcs4 only; group #3 included pt5, pt7 and pt9 IgG reacting, though to a different extent, with pc4.22, pc4.26, pc4.33 and pc4.40; group #4 included pt1, pt8 and pt15 IgG reacting neither with Pt4 nor with pt14-specific pcs. It is unlikely that pt1, pt8 and pt15 IgG reacted with the previously defined motif GPxRx expressed also by CENP-B, because of its lack of reactivity with IgG from any of the 8 patients evaluated. Conclusions: Our data show that: a) anti-Ap1-17 are independently expressed from anti-Ap17-30 and from anti-CENP-B; b) two novel motifs can be recognized by anti-CENP-A Abs; and c) IgG anti-Ap1-17 from different patients display a unique specificity, despite the recognition of the same peptide segment