Directed Mutagenesis in Region 713-720 of Human Thyroperoxidase Assigns 713KFPED717 Residues as Being Involved in the B Domain of the Discontinuous Immunodominant Region Recognized by Human Autoantibodies

Abstract

Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys 713 and Tyr 772 , have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1. Thyroid peroxidase (TPO) 1 is an essential membrane-bound enzyme involved in the biosynthesis of iodinated thyroid hormones. As well as thyroglobulin and the thyrotropin receptor, TPO is a major thyroid autoantigen (aAg) targeted by the immune system during autoimmune thyroid diseases (AITD). Human TPO (hTPO) is a 933 amino acid molecule and possesses a highly convoluted three-dimensional structure formed by three distinct modules, respectively, homologous with the myeloperoxidase (MPO), the complement control protein (CCP), and the epidermal growth factor (EGF) (1). This architectural complexity is a strong obstacle for producing high resolution crystals (2, 3), explaining why the only available three-dimensional model has been generated by computer approaches (1). Autoantibodies (aAbs) against hTPO, strongly expressed in patients' sera, are sensitive and specific diagnostic markers for AITD and may play a role in the process leading to thyroid cellular dysfunction and destruction (4-8). More importantly, anti-TPO aAbs have been found to be involved in antigen presentation to autoaggressive T cells (9) and may consequently enhance AITD as previously shown for antithyroglobulin aAbs (10). Therefore, a precise delineation of the amino acids constituting the epitopes recognized by human aAbs on TPO would be very helpful for understanding how this aAg is recognized during the onset of AITD, as well as for developing specific immune interventions to prevent or block such pathologies.A high proportion of anti-TPO aAbs from patients' sera recognizes conformational and discontinuous epitopes, restricted to an immunodominant region (IDR) composed of two overlapping domains called A and B (11-13). Several strategies have been used to localize these domains and, in particular, the critical amino acids involve...

Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

“…These regulatory proteins are expressed on NPA cell membrane and probably partially protect them from CDC. Combining the various human recombinants anti-TPO aAbs we selected by phage display and characterised in term of epitopes (Bresson et al, 2003(Bresson et al, , 2004Chapal et al, 2000;Rebuffat et al, 2006), could make it possible to improve complement activation.…”

“…Indeed, Garcia et al (1998) investigated TPO expression in a series of thyroid tumours by immunostaining using the anti-TPO mAb47 . We have previously shown that lysine 713 has a critical function for TPO epitope recognition by mAb47 Bresson et al, 2004), which is probably strongly impaired by cross-linking of the amino acid on formaldehyde slide fixation. Czarnocka et al (2001) used a TPO capture method that has the advantage to preserve integrity of the antigen structure and thereby allows immunological detection.…”

“…These human recombinant anti-TPO aAbs mimic human anti-TPO aAbs present in the sera of patients suffering from AITD and recognise the human TPO (hTPO) immunodominant region (Bresson et al, 2003, 2004, 2005, Rebuffat et al, 2006. To evaluate if these anti-TPO aAbs may be useful tools in the treatment of thyroid carcinomas, we selected a human recombinant anti-TPO aAb and expressed it in baculovirus (B4) and in CHO cells (B4 0 ).…”

An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes

“…Alternative approaches are to identify and remove B-cell or T-cell epitopes (13)(14)(15)(16)(17). We have recently used a mouse model to identify the major B-cell epitopes in the PE38 portion of RITs made by our group (18).…”

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

“…These regions were firstly defined in competition experiments with a panel of murine monoclonal antibodies and patient autoantibodies (5), and subsequent studies with recombinant human anti-TPO Fab fragments confirmed these findings (6,7). Numerous attempts have been made to identify the amino acids in the autoantibodies' epitopes (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18); however, to clearly interpret these data and arbitrate between some conflicting results, the determination of the three-dimensional structure of TPO in complex with various autoantibodies is needed.…”