Immunoglobulin Gκ Antithyroid Peroxidase Antibodies in Hashimoto’s Thyroiditis: Epitope-Mapping Analysis1

Abstract

Patients with autoimmune thyroid disease frequently have high affinity antibodies to thyroid peroxidase (TPO), although the role they play in disease pathogenesis is not known. We have previously prepared 37 monoclonal anti-TPO IgG kappa Fab fragments from two patients with Hashimoto's thyroiditis and demonstrated the similarity of these Fab sequences to those published previously, mainly derived from patients with Graves' disease. In this paper, we described epitope mapping of these Fabs using a previously characterized panel of murine monoclonal antibody (mAb) and show that the Fabs bind to two neighboring epitopes on native TPO. Although the epitope-mapping method differs from that used to characterize previously published TPO-reactive Fab sequences, it indicates a similarly restricted response to neighboring epitopes in both Graves' disease and Hashimoto's thyroiditis. The epitope mapping included mAb 47, which binds to a linear TPO peptide of known sequence in addition to native TPO. Although TPO-reactive Fab did not inhibit the binding of mAb 47, mAb 47 did inhibit the binding of Fab, indicating the likely site of the immunodominant region on native TPO. These results confirm the restricted nature of TPO antibody and further delineate the immunodominant region of native TPO as defined by the mAb.

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

“…This was clearly seen when TPO was bound via mAbs directed to epitopes located on antigenic domain B, indicating that both mAbs and Fabs recognize overlapping part of TPO. No cross-inhibition of Fab binding to TPO was observed when TPO was captured by mAbs reactive with antigenic domain D. This TPO region is not recognized by autoantibodies, and none of the binding of the recombinant human IgGκ Fabs used was influenced by the occupancy of those mAb epitopes on the TPO molecule (Czarnocka et al, 1997;McIntosh et al, 1997). This result therefore indicates that, when expressed, TPO in differentiated thyroid cancer tissues does not differ from TPO in normal thyroid tissue or Graves' tissue.…”

“…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). Moreover, there was no overlap between the epitopes recognized by the panel of TPOspecific IgGκ Fab and the epitopes of mAbs reacting with antigenic domains C and D (Czarnocka et al, 1997).…”

“…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). Moreover, there was no overlap between the epitopes recognized by the panel of TPOspecific IgGκ Fab and the epitopes of mAbs reacting with antigenic domains C and D (Czarnocka et al, 1997). We observed that the TPO protein present in cancer tissues did not show any differences from normal in its antigenic activity, as TPO was captured by all murine anti-TPO monoclonal antibodies used.…”

“…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. Characterization of the human Fabs has been described elsewhere (Czarnocka et al, 1997;McIntosh et al, 1997). In this study, Fabs representative for domain A (126 TP1,6,7,8,10 and 126 TO 10), and domain B reactivity (126 TP5, 9, 13, 14, 15 and 126 TO1) were used.…”

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

“…Controversial results have assigned the mAb 47/C21 epitope (713-721) sometimes inside the IDR (25,32,56) and sometimes outside this region (23,35,57). McLachlan and Rapoport's group (32) deduced from their studies using recombinant antibody TR1.9 that TPO residue Lys 713 , which is within the human aAb IDR, lies at the fringe of the mouse mAb 47/C21 epitope (32), as is also suggested for the 713-720 region identified by our investigation.…”