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.
Morgenthaler
N G
NG
Department of Medicine, King's College School of Medicine, London, United Kingdom.
Tremble
J
J
Huang
G
G
Scherbaum
W A
WA
McGregor
A M
AM
Banga
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D014176:Protein Biosynthesis; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018076:DNA, Complementary; D000939:Epitopes; D011989:Receptors, Thyrotropin