Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate.
Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.
Rapoport
B
B
Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, California 94121, USA.
McLachlan
S M
SM
Kakinuma
A
A
Chazenbalk
G D
GD
D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D016466:CHO Cells; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A; D006224:Cricetinae; D017077:Culture Media, Conditioned; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D008358:Mannose / Q000032:analysis*; D008970:Molecular Weight; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D001323:Autoantibodies; D017077:Culture Media, Conditioned; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D003208:Concanavalin A; D008358:Mannose