sodium-dodecyl-sulfate and Graves-Disease

A thyrotropin (TSH) binding inhibiting protein (TBIP) that inhibits TSH binding to the TSH receptor, as determined by the TSH receptor assay, was purified from human and porcine thyroid. The soluble fraction (100,000 x g supernatant of Graves' thyroid homogenate) was precipitated with ammonium sulfate between 1.75 to 2.5 mol/L. TBIP was eluted by 0.5 mol/L sodium chloride (NaCl) containing 20 mmol/L Tris buffer, pH 7.5 from a Q-sepharose column. The unbound fraction from concanavalin A (Con A) and blue-sepharose was gel-filtered using sephadex G-100, and finally purified by Resource Q column chromatography. Purified TBIP was confirmed as a single protein band of 17 kDa. The TBI activity in the purified TBIP was significantly decreased by either etnylene glycol tetraacetate (EGTA) (1 mmol/L) or antibody to calmodulin (CaM) in the TSH receptor assay. The TBIP was confirmed immunologically as CaM by the Ouchterlony method using antibody for CaM. These findings demonstrated that the TBIP purified from human and porcine thyroids was, in fact, CaM. We examined the effects of TBIP purified from human thyroid on bovine TSH (bTSH) or thyroid stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cells (PTC). TBIP itself did not increase basal levels of cAMP production, but inhibited bTSH (100 mU/L)-stimulated cAMP production. However, TBIP did not inhibit cAMP production stimulated by TSAb-IgG and various thyroid stimulators (GTPgammaS, forskolin and pituitary adenylate cyclase-activating polypeptide [PACAP, 27 and 38 amino acids]). Authentic CaM purified from bovine brain behaved in a manner similar to that of TBIP. These data showed that CaM differentially affects thyroid stimulation by TSH and TSAb in intact thyroid cell experiments.

Topics: Animals; Antibodies; Calmodulin; Cattle; Chromatography; Chromatography, Gel; Concanavalin A; Cyclic AMP; Dextrans; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Graves Disease; Humans; Immunodiffusion; Iodine Radioisotopes; Protein Binding; Receptors, Thyrotropin; Sepharose; Sodium Dodecyl Sulfate; Swine; Thyroid Gland; Thyrotropin

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves'

Topics: Alternative Splicing; Animals; Antibodies, Monoclonal; Base Sequence; Blotting, Western; DNA Primers; Electrophoresis, Polyacrylamide Gel; Graves Disease; Humans; Immune Sera; Kidney; Laryngeal Neoplasms; Molecular Sequence Data; Peroxidase; Rabbits; Recombinant Fusion Proteins; Sodium Dodecyl Sulfate; Thyroid Gland

We have tested for serum antibodies reactive with 1D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to 1D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-1D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with 1D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against 1D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both 1D and the 64-kDa eye muscle protein were incubated with CHO (1D) cell membrane, reactivity against 1D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of 1D.

Topics: Adolescent; Adult; Aged; Animals; Antibodies; Blotting, Western; CHO Cells; Cricetinae; Electrophoresis, Polyacrylamide Gel; Eye Diseases; Female; Fluorescent Antibody Technique; Graves Disease; Humans; Male; Membrane Proteins; Middle Aged; Recombinant Proteins; Sodium Dodecyl Sulfate; Thyroiditis, Autoimmune; Transfection