Amino Acid Sequence of a Tryptic Peptide of Human Thyroglobulin Reactive with Sera of Patients with Thyroid Diseases

Abstract

Autoantibodies to human thyroglobulin (hTg) are found in the sera of many patients with thyroid diseases. To localize epitopes recognized by these autoantibodies, hTg was incubated with tryspin for 4 hours at 37 degrees C under non-reducing conditions. Releasing peptides from hTg in their natural conformation. These peptides were then analyzed by western immunoblot using either autoantibodies from patients with autoimmune thyroiditis or murine monoclonal antibodies (mAb) produced against hTg. The autoantibodies reacted primarily with two low molecular weight peptides with apparent molecular weights (MWap) of 15 and 20 kDa. The pattern of tryptic peptides recognized by these autoantibodies resembled that of one of the mAbs (137C1), as shown by immunoblots in either one or two dimensional SDS-PAGE. To characterize these peptides further, they were separated by a high performance liquid chromatography (HPLC). The column separated the 4-hour tryptic digest of hTg into multiple peptide peaks. Further analysis by SDS-PAGE showed that one of these peaks contained the 15 kDa peptide. The 15 amino acid sequence at the amino-terminus of this peptide was determined. This amino acid sequence (KVPTFATPWPDFVPR) corresponds to a unique sequence near the carboxyl-terminal end of hTg, starting with amino acid 2657. The size of the peptide indicates that it extends to the carboxyl-terminal end of hTg. This fragment contains one of the antigenic sites of hTg that binds autoantibodies from patients with autoimmune thyroid disease.