A prion-like shift between two conformational forms of a recombinant thyrotropin receptor A-subunit module: Purification and stabilization using chemical chaperones of the form reactive with Graves' autoantibodies

A secreted recombinant TSH receptor (TSHR) ectodomain variant (TSHR-289) ne utralizes TSHR autoantibodies in Graves' disease, but is heterogeneous in c ontaining both immunologically active and inactive molecules and is also un stable. We have now purified each form of TSHR-289 using sequential affinit y chromatography with a mouse mAb (3BD10) specific for the inactive form, a nd a mAb to C-terminal His residues that recognizes both forms. The immunol ogical difference between active and inactive TSHR-289 was unrelated to pri mary amino acid sequence or carbohydrate content and was, therefore, attrib utable to its folded state. The epitopes for Graves' auto antibodies and 3B D10 overlap, and both are destroyed by denuradation. Therefore, reciprocal binding by autoantibodies and 3BD10 to conformational determinants involvin g the same TSHR segment suggests a prion-like shift between two folded stat es of the molecule. Despite purification, immunologically active TSHR-289 r emained labile, as determined by loss of autoantibody, and gain of 3BD10, r ecognition. However, using chemical chaperones we have, for the first time, been able to stabilize purified TSHR antigen in immunologically intact for m. In summary, purification of immunologically active and stable antigen in mi lligram quantities provides a powerful tool for future diagnostic and thera peutic studies in Graves' disease.