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Autoimmune Disease Unit, Cedars-Sinai Research Institute, and University of California School of Medicine, Los Angeles, CA 90048
Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the
humoral autoimmune response in human autoimmune thyroiditis
(Hashimoto’s thyroiditis). The majority of TPO autoantibodies in
individual patients’ sera interact 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.
Extensive biotinylation (
15 biotins/molecule protein) of lysine
residues on the surface of purified, native TPO resulted in loss of
multiple tryptic cleavage sites, as determined by analysis of tryptic
polypeptide fragments on reverse-phase HPLC. TPO was then complexed
with a monoclonal human autoantibody Fab (TR1.9) before biotinylation.
After dissociation from TR1.9, TPO was recovered by gel filtration. A
trypsin site, previously observed to be lost after TPO biotinylation,
was restored when biotinylation was performed on the TPO-TR1.9 complex.
The epitope-protected lysine (K) was present in a 30-aa TPO fragment
that, by N-terminal sequencing, was found to be K713. Altered
recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule
involving this region supported the epitope protection data. In
conclusion, we provide the first identification of an amino acid
residue (K713) comprising part of an epitope within the TPO
immunodominant region. This focal residue localizes the facet on the
large, highly complex TPO molecule that contains the immunodominant
region and provides the basis for rational guided mutagenesis studies
to more fully characterize this region.
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