Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies

Abstract

Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders.

Keywords::

• Epitope mapping
• immunodominant regions
• autoimmune thyroid diseases
• thyroid peroxidase (TPO)
• mouse monoclonal antibody

Acknowledgements

We are grateful to Dr. J. Ruf for providing the mouse anti-TPO-specific antibodies. We acknowledge Drs. A. Gardas and J. P. Banga for helpful discussions. We thank Wanda Krasuska for technical assistance.

Declaration of interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Disclosure of funding sources: This work was supported by a grant from the Medical Centre of Postgraduate Education No. 501-1-25-01-11.