title: Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis.
abstract: Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549-563), P14 (aa 599-617) and P18 (aa 210-225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81.5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.
authors.affiliation: Medical Centre of Postgraduate Education, Warsaw, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Gora
authors.forename: A
authors.initials: A
authors.lastname: Gardas
authors.forename: W
authors.initials: W
authors.lastname: Wiktorowicz
authors.forename: P
authors.initials: P
authors.lastname: Hobby
authors.forename: P F
authors.initials: PF
authors.lastname: Watson
authors.forename: A P
authors.initials: AP
authors.lastname: Weetman
authors.forename: B J
authors.initials: BJ
authors.lastname: Sutton
authors.forename: J P
authors.initials: JP
authors.lastname: Banga
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D001327:Autoimmune Diseases / Q000276:immunology*; D001667:Binding, Competitive; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D016056:Immunodominant Epitopes / Q000032:analysis*; D007453:Iodide Peroxidase / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000276:immunology; D040681:Structural Homology, Protein; D013959:Thyroid Diseases / Q000276:immunology*
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007106:Immune Sera; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D007453:Iodide Peroxidase