title: Thyrotropin (TSH) receptor autoantibodies do not appear to bind to the TSH receptor produced in an in vitro transcription/translation system.
abstract: An in vitro transcription/translation (TnT) system was used to produce 35S-labeled full-length TSH receptor (TSHR) and TSHR extracellular domain (TSHRex). The interaction of the labeled proteins with TSHR autoantibodies in Graves' sera was then studied using an immunoprecipitation assay. In the assay, 35S-labeled TSHR or TSHRex were incubated with test sera, and any immune complexes formed were precipitated with protein A-Sepharose (in the case of mouse monoclonal antibodies, antimouse IgG-agarose was used). Rabbit antibodies to the TSHR and a mouse monoclonal antibody precipitated as much as 50% of the 35S-labeled TSHR preparations compared with about 2% for normal rabbit serum and 4% for a control monoclonal antibody. However, none of 34 Graves' sera (TSHR autoantibody levels ranging from 14-95% inhibition of [125I]TSH binding) were able specifically to immunoprecipitate 35S-labeled TSHR or TSHRex. These negative findings were confirmed by analysis of the immunoprecipitates on SDS-PAGE followed by autoradiography. Our results indicate that the TnT system is not useful for producing labeled TSHR preparations that can bind TSHR autoantibodies well. This is in contrast to TnT produced 35S-labeled glutamic acid decarboxylase, thyroid peroxidase, and 21-hydroxylase, which react well with their respective autoantibodies. One main difference between these 3 autoantigens and the TSHR is that the receptor is highly glycosylated, and this extensive glycosylation may be of critical importance for correct folding of the receptor. Consequently, the inability of the TnT system to glycosylate proteins could explain in part why TnT-produced 35S-labeled TSHR and TSHRex do not bind TSHR autoantibodies.
authors.affiliation: FIRS Laboratories, RSR Ltd, Llanishen, Cardiff, United Kingdom.
authors.forename: L
authors.initials: L
authors.lastname: Prentice
authors.forename: J F
authors.initials: JF
authors.lastname: Sanders
authors.forename: M
authors.initials: M
authors.lastname: Perez
authors.forename: R
authors.initials: R
authors.lastname: Kato
authors.forename: J
authors.initials: J
authors.lastname: Sawicka
authors.forename: Y
authors.initials: Y
authors.lastname: Oda
authors.forename: D
authors.initials: D
authors.lastname: Jaskolski
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
authors.forename: B R
authors.initials: BR
authors.lastname: Smith
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D004704:Endocrinology / Q000379:methods; D006111:Graves Disease / Q000097:blood / Q000276:immunology; D006801:Humans; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins; D013972:Thyrotropin / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin