title: 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 TR1.9 (Pro(715) and Asp(717)) and T13 (Lys(713), Phe(714), Pro(715), and Glu(716)). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys(713)-Asp(717) as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD.
authors.affiliation: CNRS UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Bresson
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.forename: Françoise
authors.initials: F
authors.lastname: Roquet
authors.forename: Sandra A
authors.initials: SA
authors.lastname: Rebuffat
authors.forename: Brigitte
authors.initials: B
authors.lastname: N-Guyen
authors.forename: Martine
authors.initials: M
authors.lastname: Cerutti
authors.forename: Jin
authors.initials: J
authors.lastname: Guo
authors.forename: Sandra M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: Basil
authors.initials: B
authors.lastname: Rapoport
authors.forename: Valérie
authors.initials: V
authors.lastname: Estienne
authors.forename: Jean
authors.initials: J
authors.lastname: Ruf
authors.forename: Thierry
authors.initials: T
authors.lastname: Chardès
authors.forename: Sylvie
authors.initials: S
authors.lastname: Péraldi-Roux
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001323:Autoantibodies / Q000737:chemistry*; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007118:Immunoassay; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018076:DNA, Complementary; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D007453:Iodide Peroxidase; D008239:Lysine