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Structural Insights into Autoreactive Determinants in Thyroid Peroxidase Composed of Discontinuous and Multiple Key Contact Amino Acid Residues Contributing to Epitopes Recognized by Patients’ Autoantibodies

Department of Biochemistry and Molecular Biology (M.D., A.G., M.G.), and Department of Clinical Cytology (G.H.), Medical Centre of Postgraduate Education, 01-813 Warsaw, Poland; Division of Gene and Cell-Based Therapy (J.P.B.), King’s College London School of Medicine, London SE5 9PJ, United Kingdom; Department of Bioinformatics (D.P.), and Department of Genetics (M.G.), Institute of Biochemistry and Biophysics PAS, 02-106 Warsaw, Poland; Division of Clinical Sciences (North) (E.H.K.), University of Sheffield, Northern General Hospital, Sheffield S5 7AU, United Kingdom; and The Randall Division of Cell and Molecular Biophysics (B.J.S.), King’s College London SE1 1UL, United Kingdom

Address all correspondence and requests for reprints to: Monika Gora, Department of Biochemistry and Molecular Biology, Medical Centre of Postgraduate Education, Marymoncka 99, 01-813 Warsaw, Poland. E-mail: mgora{at}cmkp.edu.pl.

Thyroid peroxidase (TPO) is a major autoantigen of thyroid autoimmune disease, and the autoantibodies that are produced recognize two immunodominant regions (IDR) of the molecule, termed IDR-A and -B. Based upon our structural model of the TPO ectodomain, we recently identified R225 and K627 as key residues in IDR-A and -B, respectively. We report here on rational mutagenic investigations to identify additional residues surrounding R225 and K627 that affect the binding of recombinant human Fabs (rhFabs) specific for each IDR. Two residues R646 and D707 were identified from the model as promising surface-exposed amino acids adjacent to R225. Similarly, residues E604, D620, D624, and D630 were identified in the vicinity of K627. These residues were substituted in different combinations of single, double, and multiple mutations, and stably expressed in Chinese hamster ovary cells. By fluorescence-activated cell sorting and capture ELISA, we found that R225A, R646A, and D707N specifically led to the loss of binding of IDR-A rhFabs, whereas E604A, D620R, K627G, and D630N specifically abrogated the binding of IDR-B rhFabs. Further supportive evidence of the importance of these residues for the IDR epitopes was obtained with patients’ sera. We conclude that R646 and D707 together with R225 constitute a functional epitope within IDR-A, and that residues E604, D620, and D630, together with K627, constitute a functional epitope within IDR-B. This identification of key residues within the autoreactive epitopes will help in understanding the structural basis for the breakdown of immune tolerance to TPO in thyroid autoimmune disease.