Identification of localized autoantibody epitopes in thyroid peroxidase.
Recent reports have disagreed on the nature of the autoantibody epitopes in thyroid peroxidase (TPO). We used immunoprecipitation of recombinant human TPO constructs to determine if localized autoantibody binding sites exist in this autoantigen. In vitro transcription and translation of TPO cDNA fragments yielded 35S-labeled products consisting of either full-length protein (933 amino acids) or N-terminal peptides of 631, 455, and 120 amino acids. Immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the Hashimoto's sera consistently precipitated the full-length and the 631 amino acid products, but not the shorter N-terminal peptides. An additional construct resulting in a full-length TPO peptide with an internal deletion of amino acids 4-455 was also made, and this product was also precipitated by the Hashimoto's sera. A fusion protein consisting of maltose binding protein followed by amino acids 456-933 of human TPO was produced in Escherichia coli and subjected to Western blot analysis using the Hashimoto's sera. The Hashimoto's sera reacted with the MalTose binding protein TPO (MBP/TPO) fusion protein, but not a control fusion protein (MBP/LacZ alpha). Together, these results indicate the presence of localized autoantibody epitopes in the portion of the human TPO molecule from amino acids 456 to 933, with at least one binding site located between amino acids 456 and 631.
Maastricht
J
J
Department of Medicine, University of Michigan Medical School, Ann Arbor 48109-0666.
Koenig
R J
RJ
Kaplan
M M
MM
Arscott
P
P
Thompson
N
N
Baker
J R
JR
D001323:Autoantibodies / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology*; D011233:Precipitin Tests; D013967:Thyroiditis, Autoimmune / Q000201:enzymology; D014162:Transfection
D001323:Autoantibodies; D000939:Epitopes; D007453:Iodide Peroxidase