Zinc finger domain of Ro60kD autoantigen is essential for binding of Ro52kD and autoantibodies

The Ro60kD polypeptide is associated with both RNA and the Ro52kD protein. The specific protein–RNA and protein–protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements located on the 92–161 and 301–327 regions of Ro60kD protein, respectively. The zinc finger domain of Ro60kD is a good candidate to hold a conformational epitope, because both the binding of zinc and the redox conditions can induce specific conformational changes. In this study, we investigated the presence of antibodies against synthetic peptides corresponding to the zinc finger domain of Ro60kD protein (Zif-1b), to a truncated form possessing zinc-binding regions but lacking the intermediate loop (Zif-2b) and to the intermediate loop (310–319) of the zinc finger domain (Zif-3b). We found that the peptide Zif-1b, corresponding to the native sequence (301–327 aa) of Ro60kD, is recognized by antibodies from the majority (83%) of anti-Ro/La-positive patients with primary Sjögren's syndrome (pSS), in the absence of zinc ions. The same sera failed to react with Zif-1b in the presence of Zn²⁺ (2.5%). Its truncated form (Zif-2b) did not react against the same sera, while the peptide corresponding to loop 310–319 (Zif-3b) exhibited high reactivity (85%). The presence of zinc ions was necessary for binding of Zif-1b to recombinant Ro52kD, indicating that discrete conformational states of the Ro60kD zinc finger domain are employed in interaction with Ro52kD protein and autoantibodies. The two different states of the zinc finger domain of Ro60kD may reflect the existence of the Ro60kD autoantigen in different redox environments (e.g. in the interior of the cell and cell membrane), where the Cys residues have different capacities to coordinate zinc ions.