Cross-reactivity of a monoclonal antibody to the amino terminal region of thyrotropin receptor with the serum protein alpha(1)-antitrypsin.
In a study designed to detect the presence of soluble, secreted A subunit of thyrotropin hormone receptor (TSHR) in serum, using anti-TSHR murine antibodies (mAbs) and peptide specific antiserum for Western blotting of human serum proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) it was consistently observed that only one mAb, termed A10, reacted strongly with the 53 kd serum protein. The reaction was specific with the mAb A10 only, but not with another mAb or polyclonal antiserum. Furthermore, A10 immunoreactivity was documented in a variety of sera from healthy donors and patients, including patients whose thyroid gland was ablated during treatment for thyroid cancer. This suggests that the A10 cross-reactive protein was not derived from thyroid cells. The A10 cross-reactive protein was purified from normal serum and subjected to N-terminal sequence analysis, which identified the protein as alpha(1)-antitrypsin. Further experiments by enzyme-linked immunosorbent assay (ELISA) and the binding of antibody with deglycosylated or elastase-treated purified serum protein confirmed the cross-reactivity of mAb A10 with alpha(1)-antitrypsin. Alignment of the TSHR amino acid sequence with that of alpha(1)-antitrypsin identified five identical amino acids in a short stretch of residues 34-39 (EEDFRV) in TSHR and residues 205-210 (EEDFHV) in alpha(1)-antitrypsin. Analysis of the structural model of alpha(1)-antitrypsin revealed that these residues were exposed on the surface of alpha(1)-antitrypsin and were accessible for antibodies. Autoantibodies in patients with Graves' disease do not appear to recognize this region of the receptor and hence do not react with serum alpha(1)-antitrypsin.
Department of Biochemistry, Medical Centre of Postgraduate Education, Warsaw, Poland.
Urszula
U
Piotrowska
Grazyna
G
Adler
Andrzej
A
Gardas
Malgorzata
M
Gietka-Czernel
Marek
M
Kaniewski
J Paul
JP
Banga
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001323:Autoantibodies / Q000187:drug effects / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D008297:Male; D017434:Protein Structure, Tertiary / Q000502:physiology; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000276:immunology*; D000515:alpha 1-Antitrypsin / Q000276:immunology* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D011989:Receptors, Thyrotropin; D000515:alpha 1-Antitrypsin