To investigate the B-cell autoimmune epitopes on human thyroid peroxidase (TPO), we generated proteolytic peptides by enzymatic hydrolysis of TPO in nondenaturing and nonreducing conditions. The hydrolysate was chromatographed on a reverse phase column. We eluted a material immunoreactive with both a TPO monoclonal antibody recognizing a linear epitope (mAb47, amino acid 713-721) and TPO autoantibodies (aAb) from patients. The aAb immunoreactivity, but not that of mAb47, was lost after reduction. Western blots after electrophoresis without reduction showed that the aAb and mAb47 were immunoreactive with a 66-kDa band and that aAb identified a doublet at 20 kDa. For electrophoresis under reducing conditions, the 66-kDa band resolved into two peptides of 40 and 26 kDa, whereas the doublet at 20 kDa remained unchanged. None of these reduced peptides was immunoreactive with aAb, whereas the 40-kDa peptide was immunoreactive with mAb47. The 40-kDa peptide extends from amino acid 549 to 933 of TPO, and its last 192 amino acids overlap the autoimmune 20-kDa peptide. After iodine labeling, the 20-kDa peptide lost its immunoreactivity. We conclude that the C-terminal end of the extracellular part of TPO, which includes all the tyrosine residues of the 20-kDa peptide, contains at least one conformational B-cell epitope involved in autoimmune thyroid diseases.Thyroperoxidase (TPO) 1 is a membrane-bound enzyme that faces the colloid and that acts at the apical pole of the thyrocytes. TPO catalyzes the iodination of thyroglobulin and the coupling of some iodotyrosine residues to form thyroid hormone residues. This catalysis is under thyrotropin control through its specific receptor (1, 2). Like thyroglobulin and the thyrotropin receptor, TPO is one of the main autoantigens (aAg) in autoimmune thyroid disease (AITD). However, the immune response to this sequestered aAg is not clear (for review, see Ref.3). After TPO was identified as microsomal aAg (4, 5), many studies investigated the human immune response to this enzyme. We showed there were two autoimmune domains on the surface of the molecule (6); this was confirmed by another group (7,8). Disappointingly, no difference was observed in autoantibody (aAb) response to TPO for patients with Graves' disease and Hashimoto's thyroiditis, the two well defined AITD (9).One of the major tasks in AITD is to identify the immunodominant B-cell epitopes of the main aAg. This may help explain the mechanisms causing immunopathological states and, consequently, may provide targets for diagnosis and therapeutic strategies. TPO is a valuable model for such studies, given the preponderance of its corresponding aAb in AITD. Moreover, TPO is implicated in the physiological function of the thyroid, and aAb binding to TPO might impair thyroid hormone synthesis through cytotoxic processes (10), thus leading to hypothyroidism. Some authors (11-14) but not others (5, 15) claimed that TPO aAb inhibit the catalytic activity of the enzyme at the iodine and the aromatic sites. However, aAb from patien...
