Evidence for two discontinuous regions on the thyrotropin receptor involved in the binding of the monoclonal antibody 34A

The human thyrotropin receptor (hTSHR) is a major autoantigen in thyroid autoimmunity. The aim of this study was to localize the discontinuous epitope recognized by the anti-hTSHR monoclonal antibody (mAb) 34A. We used the phage-displayed peptide technology and selected thirteen 34A-specific mimotopes which could be grouped into four families according to their sequence homologies. Mimotope sequence alignments on the primary sequence of hTSHR allowed us to identify regions 88-100 (family I homologous motif TX 8 FYNL) and 276-281 (family IV homologous motif DxSYPS) as being putative parts of the discontinuous epitope recognized by the mAb 34A. Interestingly, by using the Spot method, TSH was also found to interact with peptides bearing amino acids from these two regions, suggesting their involvement in TSH/TSHR interaction. Moreover these data are in agreement with the ability of the mAb 34 to displace TSH binding to its receptor. In addition, purified IgG from nine patients with Graves' disease were able to specifically recognize family I-specific mimotopes, whereas those from healthy donors did not. Taken together, our data suggest the involvement of regions 88-100 and 276-281 in the epitope recognized by mAb 34A as well as purified IgG from patients' sera and provide a basis for rational guided mutagenesis.