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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.

ABSTRACT

The enzyme thyroid peroxidase (TPO) plays a central role in thyroid hormone synthesis and is the target for the autoimmune attack in lymphocytic thyroiditis. We have examined the activation of the TPO gene in cultured human thyrocytes using slotblot hybridization with a synthetic 40 mer oligonucleotide probe derived from the nucleotide sequence of the human TPO gene. The oligonucleotide probe was shown by Northern blotting to hybridize specifically to an approximately 3 kb RNA species from thyroid tissue of patients with Graves' disease, but not to RNA preparations from human or bovine retinal tissue, providing compelling evidence for the specificity of the probe for TPO mRNA. Addition of TSH (10 mU/ml) to primary thyroid cultures for 4 h led to increased TPO mRNA levels which were maximal after 48 h and significantly higher than basal even after 7 days of co-culture. Activation of TPO mRNA by TSH showed dose dependency over a wide range (0·01–100 mU/ml), with a maximal effect at 10 mU TSH/ml in cells cultured for a period of 72 h.

Comparison of TPO mRNA levels with the accumulation of thyroglobulin mRNA levels following stimulation by TSH indicated that the induction of the gene encoding thyroglobulin precedes transcription of the TPO gene. The adenylate cyclase activator forskolin (1–100 μm) mimicked TSH in increasing TPO mRNA levels whilst, in contrast, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 0·01–1 μm) led to levels of TPO mRNA that were lower than basal. Thus TSH induces a specific dose-dependent activation of TPO mRNA which is mimicked by agents which increase cyclic AMP. In contrast, TPA-induced activation of protein kinase C inhibits this response.

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.