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N G Morgenthaler

Publications and source records attributed to N G Morgenthaler.

40 records · Page 3Linked to original sources
PubMed1996

Binding of antithyrotropin receptor autoantibodies in Graves' disease serum to nascent, in vitro translated thyrotropin receptor: ability to map epitopes recognized by antibodies.

The binding of Graves' disease autoantibodies and xenogeneic antibodies to the human TSH receptor (TSH-R) has been studied using receptor preparations generated in an in vitro transcription and translation reaction. The complementary DNAs encoding for the full-length (764 amino acids) and the extracellular region of TSH-R (amino acids 20-414, lacking the signal sequence) were used to generate the translated receptor antigen. Stable [35S]methionine-labeled nascent protein for full-length and extracellular regions of TSH-R of approximate size 87 and 50 kDa, respectively, together with other smaller proteins were generated. The 87- and 50-kDa translated receptor proteins react by immunoprecipitation analysis with monoclonal antibodies, polyclonal antisera, and unfractionated Graves' disease serum containing autoantibody to TSH-R. The translated products of the extracellular TSH-R were examined in detail. Using three well characterized murine monoclonal antibodies whose epitopes encompass the amino-, central, and carboxyl-terminals of the extracellular region of the receptor led to immunochemical identification of the smaller translated products to derive from internal methionine start sites of TSH-R. These smaller, N-terminal-truncated translated proteins were also recognized by polyclonal antisera generated against recombinant TSH-R, thus allowing epitope mapping of some antibodies. A large proportion of Graves' disease autoantibodies (> 70%) bind to the translated extracellular region of TSH-R. This indicates that the majority of pathogenic anti-TSH-R autoantibody binds the nascent translated extracellular region of TSH-R, which is not influenced by the lack of glycosylation of the receptor.

Animals↗
PubMed1996

Human immunoglobulin G autoantibodies to the thyrotropin receptor from Epstein-Barr virus-transformed B lymphocytes: characterization by immunoprecipitation with recombinant antigen and biological activity.

The TSH receptor (TSH-R) is the target antigen for disease-related autoantibodies in Graves' disease and primary myxoedema, but the repertoire of the antibodies or the nature of the precise antigenic epitopes is not known. We have immortalized peripheral blood B cells from six different autoimmune thyroid disease patients with Epstein-Barr virus and selected IgG-producing B cells by magnetic selection on anti-IgG-coated beads. Purified recombinant insect cell-derived extracellular region of TSH-R was used to identify the positive wells for expansion in culture. Stable B cell lines (n = 11) were obtained, which after limiting dilution led to two stable B cell clones. B cell lines and clones secreted IgG antibody that were shown to react biochemically with metabolically labeled or in vitro translated, nascent extracellular region of TSH-R, giving strong, confirmatory evidence of the presence of anti-TSH-R antibody. Supernatants from lines contained thyroid-stimulating activity, thyroid-blocking activity (as assessed by inhibition of TSH-mediated cAMP stimulation), or both of these activities. Interestingly, antibodies with stimulating activity were generated from a primary myxoedema patient, and antibodies of blocking specificities were obtained from newly diagnosed thyrotoxic Graves' disease patients. Our results favor a fine balance between stimulating and blocking autoantibody activities in determining the clinical presentation observed in patients with autoimmune thyroid disease patients who have these antibodies present in their serum.

Antigens↗
PubMed1996

Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells.

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

Amino Acid Sequence↗