Relationship between Immunological Structure and Biochemical Properties of Human Thyroid Peroxidase

Abstract

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.

Is there loss or qualitative changes in the expression of thyroid peroxidase protein in thyroid epithelial cancer?

“…These and other data reported by this group suggest that although the TPO molecule is present in thyroid cancers at low levels, the epitope recognized by mAb#47 is not (DeMicco et al, 1991(DeMicco et al, , 1994Faroux et al, 1997;Garcia et al, 1998). To detect any modifications or abnormalities of the TPO molecule expressed in thyroid carcinomas, we chose a structural approach of mapping the antigenic surface with a panel of 13 well characterized mAbs directed against native human TPO, and a panel of human anti-TPO IgGκ Fab fragments (Ruf et al, 1989;McIntosh et al, 1997). TPO purified from Graves' tissues was previously used to map the interaction of a panel of 13 mouse mAbs to 4 antigenic domains of TPO (Ruf et al, 1989).…”

“…To detect any modifications or abnormalities of the TPO molecule expressed in thyroid carcinomas, we chose a structural approach of mapping the antigenic surface with a panel of 13 well characterized mAbs directed against native human TPO, and a panel of human anti-TPO IgGκ Fab fragments (Ruf et al, 1989;McIntosh et al, 1997). TPO purified from Graves' tissues was previously used to map the interaction of a panel of 13 mouse mAbs to 4 antigenic domains of TPO (Ruf et al, 1989). Human monoclonal autoantibodies expressed as IgGκ Fab fragments recognized exclusively epitopes located in only 2 of the 4 domains, A and B (Czarnocka et al, 1997).…”

“…A panel of 12 murine anti-TPO monoclonal antibodies (mAb), previously produced and characterized, directed to epitopes from 3 antigenic domains of TPO was used (Ruf et al, 1989). Antibodies mAb#2, #9, #47, #60 -recognize domain A; mAb#15, #18, #59, #64 -domain B, and mAb#1, #30, #40, #53 -domain D. Domains A and B, defining the immunodominant region of TPO, are adjacent whereas domain D is topologically distant.…”

“…MAb #47 is one of a panel of well characterized murine monoclonal antibodies produced against native human TPO. The immunochemical properties of these mAbs have been published elsewhere (Ruf et al, 1989).…”

Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens

“…47 and no. 53) generated using soluble TPO and adjuvant [23] were all shown to be of a single subclass (IgG1; data not shown).…”