This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Prentice, L. Articles by Smith, B. R. Search for Related Content PubMed PubMed Citation Articles by Prentice, L. Articles by Smith, B. R.

Thyrotropin (TSH) Receptor Autoantibodies Do Not Appear to Bind to the TSH Receptor Produced in an in Vitro Transcription/Translation System

FIRS Laboratories, RSR Ltd. (L.P., J.F.S., M.P., R.K., J.S., Y.O., J.F., B.R.S.), Llanishen, Cardiff, United Kingdom CF4 5DU; the Department of Surgery, Railway District Hospital, (D.J.), Poznan, Poland; and the Department of Medicine, University of Wales College of Medicine (M.P., R.K., J.S., Y.O., J.F., B.R.S.), Cardiff, United Kingdom

Address all correspondence and requests for reprints to: Dr. Bernard Rees Smith, FIRS Laboratories, Parc Ty Glas, Llanishen, Cardiff, United Kingdom CF4 5DU.

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.

This article has been cited by other articles:

				N. G. Gavalas, E. H. Kemp, K. J. E. Krohn, E. M. Brown, P. F. Watson, and A. P. Weetman The Calcium-Sensing Receptor Is a Target of Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2107 - 2114. [Abstract] [Full Text] [PDF]

				R. Metcalfe, N. Jordan, P. Watson, S. Gullu, M. Wiltshire, M. Crisp, C. Evans, A. Weetman, and M. Ludgate Demonstration of Immunoglobulin G, A, and E Autoantibodies to the Human Thyrotropin Receptor Using Flow Cytometry J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1754 - 1761. [Abstract] [Full Text] [PDF]

				R. A. Ajjan, E. H. Kemp, E. A. Waterman, P. F. Watson, T. Endo, T. Onaya, and A. P. Weetman Detection of Binding and Blocking Autoantibodies to the Human Sodium-Iodide Symporter in Patients with Autoimmune Thyroid Disease J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 2020 - 2027. [Abstract] [Full Text]

				J. Sanders, Y. Oda, S. Roberts, A. Kiddie, T. Richards, J. Bolton, V. McGrath, S. Walters, D. Jaskolski, J. Furmaniak, et al. The Interaction of TSH Receptor Autoantibodies with 125I-Labelled TSH Receptor J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3797 - 3802. [Abstract] [Full Text] [PDF]

				Y. Oda, J. Sanders, S. Roberts, M. Maruyama, A. Kiddie, J. Furmaniak, and B. Rees Smith Analysis of Carbohydrate Residues on Recombinant Human Thyrotropin Receptor J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2119 - 2125. [Abstract] [Full Text]

				B. Rapoport, G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies Endocr. Rev., December 1, 1998; 19(6): 673 - 716. [Abstract] [Full Text]

				Y. Osuga, S.-G. Liang, J. S. Dallas, C. Wang, and A. J. W. Hsueh Soluble Ecto-Domain Mutant of Thyrotropin (TSH) Receptor Incapable of Binding TSH Neutralizes the Action of Thyroid-Stimulating Antibodies from Graves' Patients Endocrinology, February 1, 1998; 139(2): 671 - 676. [Abstract] [Full Text] [PDF]