Introduction Up to 40% of patients with primary biliary cirrhosis (PBC) show reactivity against Ro-52 (Hepatology 2009), a Sjögren's syndrome (SS)-associated autoantibody. Previous studies have shown that anti-Ro-52 in SS targets the central, coiled-coil sequence of the 475-aa of the antigen. No study has dissected anti-Ro-52 reactivity in PBC.
Aim To investigate anti-Ro-52 B-cell responses in patients with PBC and compare them with those obtained in patients with SS.
Method Human recombinant full-length Ro-52 and 3 constructs spanning the whole protein were expressed in E. coli and tested for reactivity by a line immunoassay. Construct 1 (C1: aa1–129) contained the RING finger and the B-Box domains, construct 2 (C2: aa125–268) contained the coiled-coil domain and construct 3 (C3:aa 268–475) contained the B30.2/SPRY domain. Twenty-three 18-mer peptides overlapping by 12aa were synthesised and tested by an in house ELISA to better define the core epitopes within the highly immunogenic C2 region. Overall, 122 serum samples (68 from Ro-52 positive PBC patients, 39 from Ro-52 positive SS patients without liver disease and 15 from Ro-52 negative healthy blood donors) were tested.
Results Reactivity to the C2 construct of Ro-52 was present in all Ro-52 positive sera from PBC and SS patients and in none of the controls (p<0.001). Reactivity to the C3 construct was virtually absent in PBC (3%) and SS (0%) while reactivity to C1 was equally present in PBC (15%) and SS (10%). Within the immunodominant C2 sequence, 2 novel epitopic regions were identified using peptide mapping: the first sequence (aa 175–192: 1 peptide) was recognised by the great majority of patients with PBC (86%) and SS (69%), the second sequence (aa235–258: two overlapping peptides) was recognised by 35% PBC and 54% SS patients (p=NS, for both).
Conclusion This is the first systematic B-cell mapping approach of anti-Ro-52 responses in PBC patients showing that the antigenicity of this protein lies within its coiled coil region. Two new epitopes, one of which dominant, have being discovered, both in PBC and SS. The fact that the fine epitope specificity of anti-Ro-52 is virtually identical in PBC and SS suggests a common mechanism of tolerance breakdown to this autoantigen in the two conditions.
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