. 1998 , Morshed et al. 2012 , Furmaniak et al. 2015 ). In particular, TSHR autoantibodies (TRAbs) bind to the receptor in such a way as to mimic TSH action and cause the hyperthyroidism of Graves’ disease ( Rees Smith et al. 1988 , Rapoport et
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Ricardo Núñez Miguel, Paul Sanders, Lloyd Allen, Michele Evans, Matthew Holly, William Johnson, Andrew Sullivan, Jane Sanders, Jadwiga Furmaniak, and Bernard Rees Smith
Stuart Baker, Ricardo Núñez Miguel, Daniel Thomas, Michael Powell, Jadwiga Furmaniak, and Bernard Rees Smith
( Tables 2 and 3 , Fig. 5 ). The 2G4 hMAB was derived from a patient with Hashimoto’s thyroiditis ( Horimoto et al. 1992 ) and is a hTPO autoantibody . In contrast, 4F5 is a mouse antibody which displays some characteristics of hTPO autoantibodies, in
Jennifer Miller-Gallacher, Paul Sanders, Stuart Young, Andrew Sullivan, Stuart Baker, Samuel C Reddington, Matthew Clue, Katarzyna Kabelis, Jill Clark, Jane Wilmot, Daniel Thomas, Monika Chlebowska, Francesca Cole, Emily Pearson, Emma Roberts, Matthew Holly, Michele Evans, Ricardo Núñez Miguel, Michael Powell, Jane Sanders, Jadwiga Furmaniak, and Bernard Rees Smith
autoantibodies (TRAb) with stimulating activity are responsible for hyperthyroidism in Graves’ disease while TRAb with blocking activity may cause hypothyroidism. Both, stimulating- and blocking-type TRAb bind to the TSHR LRD ( Rees Smith et al. 2007 , 2009
Paul Sanders, Stuart Young, Jane Sanders, Katarzyna Kabelis, Stuart Baker, Andrew Sullivan, Michele Evans, Jill Clark, Jane Wilmot, Xiaoling Hu, Emma Roberts, Michael Powell, Ricardo Núñez Miguel, Jadwiga Furmaniak, and Bernard Rees Smith
Introduction The TSH receptor (TSHR) is a major autoantigen in autoimmune thyroid disease, and TSHR autoantibodies with thyroid-stimulating (agonist) activity are responsible for the hyperthyroidism of Graves' disease ( Rees Smith et al . 1988
Ricardo Núñez Miguel, Jane Sanders, Paul Sanders, Stuart Young, Jill Clark, Katarzyna Kabelis, Jane Wilmot, Michele Evans, Emma Roberts, Xiaoling Hu, Jadwiga Furmaniak, and Bernard Rees Smith
Introduction K1–18 is a TSH receptor (TSHR) human monoclonal autoantibody (MAb; IgG1/kappa) with the ability to stimulate cAMP production in TSHR-transfected CHO cells and to inhibit TSH binding to the receptor at nanogram per millilitre
R Núñez Miguel, J Sanders, D Y Chirgadze, J Furmaniak, and B Rees Smith
of receptor mediated signal transduction. Here, we report a detailed analysis of interactions involving an autoantibody that activates the TSH receptor (TSHR) in Graves' disease ( Sanders et al . 2003 , 2007 a , Rees Smith et al . 2007 ) and a
G. C. Huang, K. S. Collison, A. M. McGregor, and J. P. Banga
ABSTRACT
Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.
G C Huang, M J Page, L B Nicholson, K S Collison, A M McGregor, and J P Banga
ABSTRACT
Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzymelinked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.
Seema Kumar, Sarah Nadeem, Marius N Stan, Michael Coenen, and Rebecca S Bahn
Graves' hyperthyroidism, a condition in which overproduction of thyroid hormones by thyroid follicular cells is stimulated by TSH receptor (TSHR) autoantibodies (TRAb). TSHR mRNA and protein are detectable in both normal and GO orbital adipose tissues
Ricardo Núñez Miguel, Jane Sanders, Jadwiga Furmaniak, and Bernard Rees Smith
) of the hTSHR in complex with the TSHR-stimulating human monoclonal autoantibody (hMAb) M22 ( Sanders et al . 2007 ) and with the TSHR-blocking hMAb K1-70 ( Sanders et al . 2011 ) are available. In addition, the crystal structure of the extracellular
