Evidence that the Complement Control Protein-Epidermal Growth Factor-Like Domain of Thyroid Peroxidase Lies on the Fringe of the Immunodominant Region Recognized by Autoantibodies

Abstract

There is no consensus regarding the location of the immunodominant region (IDR) on thyroid peroxidase (TPO) recognized by the majority of autoantibodies. Strong evidence indicates that it lies upstream of amino acid 741. However, an epitope has been localized to downstream residues 742-848 encompassing a disulfide-rich complement control protein (CCP)-like and epidermal growth factor (EGF)-like domain. To determine whether these domains comprise part of the IDR, we used a recombinant CCP/EGF-like polypeptide to screen a thyroid B-cell-derived immunoglobulin gene phage display library. Two unusual TPO autoantibodies were isolated. Neither was among the 83 clones previously obtained by panning the same library on native or denatured TPO, or TPO with the IDR masked. Fab from these clones bound native TPO, one with high affinity (Kd 6 × 10^-10 M), and both recognized TPO expressed on the surface of mammalian cells. Phage-expressing multiple copies of the antibody (multivalent), but not monovalent Fab from these clones, bound to the CCP/EGF polypeptide. Most important, inhibition of TPO binding by autoantibodies to the IDR indicated that the epitopes of the two new autoantibodies overlap with this region. The value of these two rare clones lies in the insight they provide into the location of the TPO IDR. From their binding characteristics, we deduce that the CCP/EGF-like domain lies on the fringe of the TPO immunodominant region.