HUMAN THYROID PEROXIDASE (TPO) ISOFORMS, TPO-1 AND TPO-2 - ANALYSIS OF PROTEIN EXPRESSION IN GRAVES THYROID-TISSUE

Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesi s of thyroid hormones and is an important autoantigen in autoimmune th yroid disease. Different messenger RNA species coding for TPO are pres ent in thyroid tissue, including the species coding for a 933-amino ac id protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyro id cells. In SDS-PAGE under reducing conditions, TPO appears in the th yroid microsome and purified protein preparations as a closely migrati ng double band of approximately 105 (larger form) and 100 kilodaltons (smaller form). We investigated the presence of the isoform TPO-2 poly peptide in Graves' thyroid tissue using rabbit antisera to three diffe rent synthetic peptides from exon 10 (specific for TPO-1) and a polycl onal rabbit and monoclonal anti-TPO antibody (both of which are specif ic for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purifi ed natural TPO from Graves' thyroid microsomes. Both of the purified f orms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows t hat both forms of TPO contain exon 10-encoded polypeptide of TPO-1. In terestingly, the proportion of the larger and smaller forms of TPO var ied in different Graves' thyroid microsome preparations. To investigat e the presence of the smaller TPO-2 isoform in the purified natural TP O preparation, affinity depletion of TPO-1 using the anti-exon 10 pept ide antibodies was carried out, The binding of anti-exon 10 peptide an tibodies to the immunodepleted TPO-1 fraction was considerably diminis hed in comparison to binding of polyclonal anti-TPO, suggesting the pr esence of small amounts (< 10%) of TPO-2 expressed as a protein in thy roid cells. Our results extend previous observations by showing that t he alternatively spliced form of TPO, in which exon 10 is excised, is expressed at low levels in Graves' thyroid tissue. Furthermore, we con firm that both the larger and smaller forms of TPO observed on gel ele ctrophoresis contain TPO-1, suggesting that the difference is caused b y posttranslational modifications. The presence of small amounts of TP O-2 in Graves' thyroid glands argues for its role in thyroid function, which remains to be clarified.