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) neutralizes TSHR autoantibodies in Graves' disease, but is heterogeneous in containing both immunologically active and inactive molecules and is also unstable. We have now purified each form of TSHR-289 using sequential affinity chromatography with a mouse mAb (3BD10) specific for the inactive form, and a mAb to C-terminal His residues that recognizes both forms. The immunological difference between active and inactive TSHR-289 was unrelated to primary amino acid sequence or carbohydrate content and was, therefore, attributable to its folded state. The epitopes for Graves' autoantibodies and 3BD10 overlap, and both are destroyed by denaturation. Therefore, reciprocal binding by autoantibodies and 3BD10 to conformational determinants involving the same TSHR segment suggests a prion-like shift between two folded states of the molecule. Despite purification, immunologically active TSHR-289 remained labile, as determined by loss of autoantibody, and gain of 3BD10, recognition. However, using chemical chaperones we have, for the first time, been able to stabilize purified TSHR antigen in immunologically intact form. In summary, purification of immunologically active and stable antigen in milligram quantities provides a powerful tool for future diagnostic and therapeutic studies in Graves' disease.
Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, California 90048, USA.
G D
GD
Chazenbalk
S M
SM
McLachlan
P
P
Pichurin
X M
XM
Yan
B
B
Rapoport
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D006224:Cricetinae; D004355:Drug Stability; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D011392:Proline / Q000494:pharmacology; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000378:metabolism
D001323:Autoantibodies; D002241:Carbohydrates; D000939:Epitopes; D018828:Immunoglobulins, Thyroid-Stimulating; D008744:Methylamines; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; C094372:thyrotropin-binding inhibitory immunoglobulin; D013972:Thyrotropin; D011392:Proline; C005855:trimethyloxamine