Although the primary structure of human thyroid peroxidase (hTPO) has been recently deciphered, little is known about its spatial conformation. Such information is of crucial importance in any attempt to relate the structure with the function of hTPO. To probe the antigenic surface of hTPO and to correlate its immunological structure to its biochemical properties, we used 13 monoclonal antibodies (mAb) displaying various affinity for hTPO. Criss-cross experiments showed 7 clusters of reactivity which were interpreted as reflecting 7 epitopes on the surface of the hTPO molecule. Extending our analysis to partial and nonsymmetrical cross-reactivities, these epitopes were shown to be localized in 4 antigenic domains of the hTPO. We further investigated the nature of these 7 hTPO epitopes by testing mAb binding to peroxidases from various origins and chemically modified hTPO; 3 epitopes were shown to be evolutionary conserved, and 5 resistant to reduction and denaturation. We also analyzed the role of the hTPO epitopes in the enzymatic activity and autoimmune targeting of the molecule. Nine epitopes were shown to be localized at the vicinity of both catalytic sites as the binding of their respective mAb modulated the enzyme activity. Autoantibodies from patients presenting with autoimmune thyroid disorders were essentially directed to epitopes similar or adjacent to those recognized by 8 of the 13 mAb and present on only 2 antigenic domains of hTPO. Taken together these data allowed us to propose a tentative map of the surface of the hTPO molecule which associates its epitopic structure with its biochemical functions.

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Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffrnity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A,,, to A,,, ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen. Microsomal antigen Thyroidperoxidase AutoantibodyMonoclonal antibody Autoimmunity