Background/Purpose:

Anti-nuclear antibodies (ANA) are present in approximately 90% of sera from systemic sclerosis (SSc) patients and play an important role in the diagnosis and prognosis of that disease. Besides the classical autoantibodies that are part of the SSc classification criteria, several other antibodies can be found, albeit with lower prevalence, some of which clearly associate with disease phenotypes. Anti-U11/U12 Ribonucleoprotein (RNP) antibodies have been reported in a small portion of patients with pulmonary fibrosis. RNPC-3, also known as U11/U12 Small Nuclear Ribonucleoprotein 65 kDa Protein has been reported as an autoantibody target with strong association with malignancy in SSc patients. The aim of this project was to analyze the B-cell epitopes of anti-RNPC-3 antibodies and to study the prevalence of anti-RNPC-3 antibodies in SSc and controls.

Methods:

Two SSc sera with anti-RNPC-3 antibodies, assayed by an addressable laser bead-based immunoassay (ALBIA) with recombinant RNPC-3, were used for epitope discovery with peptide arrays covering the full-length amino acid sequence of human RNPC-3. As controls, three samples with anti-centromere antibodies were included as well as peptides derived from CENP-A and BICD2. The identified candidate epitopes were subsequently utilized to synthesize synthetic, biotinylated, soluble peptides for testing using a novel particle-based multi-analyte technology (PMAT). To analyze the marker in SSc, sera from 88 SSc patients were included where 32 cases had documented cancer. Sera from 64 systemic lupus erythematosus (SLE) patients, 65 idiopathic inflammatory myopathies (IIM), and 20 health individuals (HI) were included as a comparator group. Co-localization by indirect immunofluorescence (IIF) of human anti-RNPC3 with rabbit anti-RNPC3 (rabbit polyclonal) was performed.

Results:

Epitope mapping revealed an immunodominant epitope in the C-terminus of the protein (see Figure). The reactivity to recombinant RNPC-3 (rRNPC-3) and to the RNPC-3 derived peptide (pRNPC-3) were highly correlated (Spearman’s rho=0.64, 95% Confidence interval 0.58-0.69; p<0.0001). Autoantibodies to both rRNPC-3 and pRNPC-3 tended to be higher in SSc and SLE compared to IIM and HI. When the cohort of patients with known history of cancer was compared with an unselected SSc cohort, no significant difference was found. Experiments performed by IIF confirmed the speckled pattern shown in previous studies.

Conclusion:

Our study is the first to report a linear epitope on RNPC-3 as a target of autoantibodies in patients with SSc. Further studies are needed to validate the association of anti RNPC-3 antibodies with cancer as well as the utility of the novel RNPC-3 derived peptide.

Figure 1   Epitope mapping of RNPC-3 using solid phase peptide arrays (a) and correlation of peptide reactivity with RNPC-3 protein along with co-localized IIF pattern (b).