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
J. Guo et al., 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, J IMMUNOL, 166(2), 2001, pp. 1327-1333
Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral
autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditi
s). The majority of TPO autoantibodies in individual patients' sera interac
t with a restricted immunodominant region on TPO. Although this region can
be mapped, previous studies have failed to localize its position on the TPO
molecule. We, therefore, used a footprinting approach that can localize a
highly conformational, discontinuous epitope on a very large molecule. Exte
nsive biotinylation (similar to 15 biotins/molecule protein:) of lysine res
idues on the surface of purified, native TPO resulted in loss of multiple t
ryptic cleavage sites, as determined by analysis of tryptic polypeptide fra
gments on reverse-phase HPLC. TPO was then complexed with a monoclonal huma
n autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR
1.9, TPO was recovered by gel filtration. A trypsin site, previously observ
ed to be lost after TPO biotinylation, was restored when biotinylation was
performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was pr
esent in a 30-aa TPO fragment that, by N-terminal sequencing, was found to
be K713. Altered recognition by TR1.9 of a TBO-myeloperoxidase chimeric mol
ecule involving this region supported the epitope protection data. In concl
usion, we provide the first identification of an amino acid residue (K713)
comprising part of an epitope within the TPO Immunodominant region. This fo
cal residue localizes the facet on the large, highly complex TPO molecule t
hat contains the immunodominant region and provides the basis for rational
guided mutagenesis studies to more fully characterize this region.