Evidence for the highly conformational nature of the epitope(s) on human thyroid peroxidase that are recognized by sera from patients with Hashimoto's thyroiditis.
To define the epitope(s) on human thyroid peroxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease, we constructed and screened a human TPO cDNA sublibrary containing 3.8 million random fragments of human TPO cDNA, each 200-500 basepairs in length. These fragments would code for TPO polypeptides of 66-166 amino acid residues. The validity of this approach was first tested with a murine monoclonal antibody against the denatured human thyroid microsomal antigen (TPO). Analysis of the nucleotide sequence of 14 clones selected from this library enabled molecular identification of the epitope recognized by this monoclonal antibody. In contrast to the data obtained with the monoclonal antibody, sera from patients with Hashimoto's thyroiditis containing polyclonal antimicrosomal/TPO antibodies did not recognize the TPO protein fragments generated by this library. These results differ from previous data obtained with recombinant human TPO fragments generated as bacterial fusion proteins. Our data suggest that, contrary to previous concepts, the natural B-cell epitope(s) on human TPO may be highly conformational (requiring a complex 3-dimensional structure) or may be discontinuous (formed by distant regions of the linear polypeptide chain being brought into apposition by protein folding).
Thyroid Molecular Biology Laboratory, Veterans Administration Medical Center, San Francisco, California 94121.
R
R
Finke
P
P
Seto
B
B
Rapoport
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000276:immunology; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D000939:Epitopes / Q000276:immunology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics / Q000276:immunology*; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013967:Thyroiditis, Autoimmune / Q000097:blood / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D033862:Iron-Binding Proteins; D010446:Peptide Fragments; D004247:DNA; C056658:TPO protein, human; D007453:Iodide Peroxidase