Epitope recognition of recoverin in cancer associated retinopathy: Evidence for calcium-dependent conformational epitopes

Recoverin, a photoreceptor-specific calcium-binding protein, is a target for antibodies in human cancer-associated retinopathy. We have studied the binding properties of antirecoverin human autoantibodies and rat monoclonal antibodies (MAbs). The majority of antibodies to recoverin, both in human disease and in animals immunized with recoverin, are directed against the same major immunodominant region, the sequence within the residue 64-70 in proximity to the calcium-binding domain EF-hand 2. The immunodominant epitopes consist of linear and conformational components. Our data demonstrate that the reactivity of autoantibodies was dependent on conformational changes induced by the binding of calcium to recoverin. Some patients' antibodies could be detected by immunocytochemical methods and enzyme linked immunosorbent assay (ELISA); however, their binding was abolished on Western blots; their epitopes were sensitive to SDS treatment. Immunization of Lewis rat with purified recoverin induces antibodies with properties strikingly similar to human autoantibodies. Comparison between the monoclonal antibodies (MAbs) and native autoantibodies showed that the calcium-induced changes in conformation were critical for specificities of the antibody generated. The recoverin region within the sequence 61-82 is a key region for antibody binding; it also contains a major T-cell epitope, and it is highly uveitogenic in Lewis rats.