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J. Biol. Chem., Vol. 279, Issue 37, 39058-39067, September 10, 2004

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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^*

Damien Bresson¶, Martine Pugnière¶, Françoise Roquet, Sandra A. Rebuffat, Brigitte N-Guyen, Martine Cerutti||, Jin Guo**, Sandra M. McLachlan**, Basil Rapoport**, Valérie Estienne, Jean Ruf, Thierry Chardès||, and Sylvie Péraldi-Roux

From the 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, ^||INRA-CNRS FRE 2689, Station de Recherche de Pathologie Comparée, 30380 St. Christol-lez-Alès, France, the ^**Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California School of Medicine, Los Angeles, California 90048, and U555 INSERM, Faculté de Médecine, Laboratoire de Biochimie Endocrinienne et Métabolique, 13385 Marseille Cedex 5, France

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, Lys⁷¹³ and Tyr⁷⁷², 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 (Pro⁷¹⁵ and Asp⁷¹⁷) and T13 (Lys⁷¹³, Phe⁷¹⁴, Pro⁷¹⁵, and Glu⁷¹⁶). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys⁷¹³-Asp⁷¹⁷ as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD.

Received for publication, April 7, 2004 , and in revised form, May 17, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a fellowship from the CNRS and the Région Languedoc-Roussillon. Current address: La Jolla Institute for Allergy and Immunology, Dept. of Developmental Immunology-3, 10355 Science Center Drive, San Diego, CA 92121.

^¶ Both authors contributed equally to this work.

To whom correspondence should be addressed: CNRS UMR 5160, Centre de Pharmacologie et Biotechnologie pour la Santé, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier, Cedex 5, France. Tel.: 33-467-548-604; Fax: 33-467-548-610; E-mail: sylvie.roux{at}ibph.pharma.univ-montp1.fr.

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