Immunodetection of manganese superoxide dismutase in cultured human retroocular fibroblasts using sera directed against the thyrotropin receptor

Abstract

The identification of antigenic targets in the retroocular autoimmune response of Graves' ophthalmopathy is likely to increase our understanding of mechanisms underlying this disorder. While a number of putative autoantigens have been identified on the basis of molecular weight or cell of origin, a determination of the significance of these antigens is contingent upon an identification of the amino acid sequence. Our group has previously identified immunoreactive retroocular fibroblast (ROF) proteins recognized by thyrotropin receptor (hTSH-R) antisera (anti-p1), at molecular weights of 95, 71, 41, and 14-25 kDa. In the present study, proteins detected by anti-p1 and visualized by Ponceau staining were isolated and processed for microsequencing. Ponceau staining revealed dense bands at molecular weights of 14 and 23 kDa, and a weak band at 41 kDa. N-terminal sequencing was performed on the prominent band at approximately 23 kDa, showing it to be manganese superoxide dismutase (MnSOD), a mitochondrial enzyme responsible for protection against oxygen free radical-associated cellular damage. Sequence comparison of MnSOD to the hTSH-R peptide, p1, revealed a linear segment of amino acid homology. Preincubation of anti-p1 with p1 blocked immunodetection of the 23 kDa band corresponding to MnSOD, and immunoprecipitation of ROF protein using anti-pi yielded protein recognized by anti-MnSOD. Autoimmunity against human recombinant MnSOD was further assessed by ELISA. Patients with Graves' disease (n = 53) had significantly higher ELISA indices than normal control subjects (n = 29), while patients with Hashimoto's thyroiditis had intermediate values. These results document MnSOD autoantibodies in patients with Graves' disease and suggest that this may result from an immune cross-reactivity between MnSOD and the TSH-receptor.