Mapping of Autoantigenic Epitopes on Recombinant Thyroid Peroxidase Fragments Using the Polymerase Chain Reaction

Abstract

Cloned cDNA templates of thyroid peroxidase (TPO) have been used in conjunction with the polymerase chain reaction (PCR) to express selected segments of the thyroid microsomal/peroxidase antigen (TMA/TPO) as recombinant protein in E. coli. Six small, different recombinant fragments averaging 120 amino acid residues and one large fragment (269 amino acids) of TPO which together encompass 80% of the extracellular region of the molecule have been produced and autoantibody (aAb) binding sites analysed by immunoblotting. A minimum of six independent, sequential antigenic determinants have been localized on the recombinant proteins and these map to the amino terminal, the central core region and the carboxyl terminal of the TPO molecule. More accurately, the six antigenic sites reside on overlapping recombinant TPO preparations termed R1a + R1b (residues 1 to 160) R1c (residues 145 to 250), R2b (residues 457 to 589), R3a (residues 577-677), R3b (residues 657-767) and R3c (residues 737-845). The large fragment of TPO termed R3 (residues 577-845) encompassing R3a, R3b and R3c also reacts with the aAbs. Different sera from patients with autoimmune thyroid disease contain antibodies to TMA/TPO which differ in their fine specificity. The use of recombinant molecular biological techniques together with PCR to prepare small segments of a large autoantigen as recombinant protein will now allow studies to progress on autoepitope mapping of the precise amino acid sequences of the TPO molecule with the use of synthetic peptides.

Search for the Autoantibody Immunodominant Region on Thyroid Peroxidase: Epitopic Footprinting with a Human Monoclonal Autoantibody Locates a Facet on the Native Antigen Containing a Highly Conformational Epitope

“…Other than the use of chimeric TPO-MPO molecules (which failed to identify the immunodominant region) (35,37), most previous attempts to identify TPO autoantibody epitopes have examined polyclonal autoantibody interactions with polypeptide fragments (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) with the inherent problem of the highly conformational nature of TPO autoantibody epitopes. However, based on the putative three-dimensional model of TPO, an r-pep fragment (TPO aa residues 742-848) (23), although not defining a precise epitope, may lie in the vicinity of K713 located in the present study.…”

“…It is, therefore, not surprising that identification of conformational, possibly discontinuous, TPO autoantibody epitopes (9 -11) on the native Ag has not been possible using synthetic peptides. Some polypeptide fragments of TPO are recognized by TPO autoantibodies in patients' sera (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). However, the wide spectrum and large size of these fragments as well as the fact that (in the native structure) polypeptides can traverse the molecule and contribute to widely separated facets on the surface of the protein have not led to deduction of the site of the immunodominant region.…”

A Conformational B-cell Epitope on the C-terminal End of the Extracellular Part of Human Thyroid Peroxidase

“…A region within amino acids 657-767 harbors a major and frequently used autoepitope (30). The last 26 amino acids of this region overlap our 20-kDa peptide.…”

Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease

“…Eight separate recombinant proteins representing the entire extracellular region of TPO were produced as glutathione-s-transferase (GST) fusion proteins in E. coli using the plasmid vector pGEX-2T [6] (Fig. 1).…”

“…Whilst considerable progress has been made in understanding and mapping the autoantibody response to TPO [1,5,6], much less is known about recognition of TPO by the pathogenic T cells in human disease. Some studies have demonstrated evidence of sensitization to thyroid microsome preparations [7].…”