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

Abstract

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766 -775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.Human thyroid peroxidase (TPO), 1 described previously as the "thyroid microsomal antigen" (1), is a membrane-bound enzyme expressed at the apical pole of thyrocytes (2). TPO generates the functional form of thyroglobulin by iodination and coupling of tyrosine residues (3). During autoimmune thyroid diseases (AITD), TPO represents a major target for the immune system (4, 5), leading to high titer TPO-specific autoantibodies (aAbs) in the sera of patients suffering from Hashimoto's thyroiditis and Graves' disease. Besides their role as efficient and early diagnostic markers of AITD, TPO-specific aAbs also act as effector molecules either through modulating antigen presentation to T cells or by mediating thyroid destruction after complement activation or antibody-dependent cell cytotoxicity (6 -12). Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an EGF-like domain, from the N-to the Cterminal extremities (13). Although the structure of each domain has been elucidated in part by three-dimensional modeling (13-16), the full three-dimensional structure of TPO remains unknown, even though low resolution crystals have been obtained (17,18). The flexibility observed for the hinge regions probably make difficult the exact positioning of each domain in relation to the others (15).These observations denote a highly complex structure of TPO, thus explaining the reason why TPO aAbs from patients' sera preferentially recognize discontinuous epitopes on . Different approaches have been used to determine the epitopic regions recognized by anti-TPO aAbs from ...

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.…”

“…Human recombinant anti-TPO scFv antibody B 4 was selected in our laboratory using a phage-display library and expressed as IgG 1 in baculovirus/insect cells system by Dr M Cerutti as previously described (Bresson et al, , 2003. Sera from patients suffering from Graves' disease were obtained from Dr B Guerrier (Guy de Chauliac Hospital, Montpellier, France) and Dr L Baldet (Lapeyronie University Hospital, Montpellier, France).…”

“…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 ).…”

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

“…Total cell proteins were extracted from CHO cells as previously described (12). Cell surface protein was isolated using the Cell Surface Protein Isolation Kit (Pierce) according to the protocol provided by the manufacturer.…”

“…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.…”