Alternatively spliced form of human thyroperoxidase, TPOzanelli: Activity,intracellular trafficking, and role in hormonogenesis

Thyroperoxidase (TPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. One of the two main alternatively spliced forms of this enzyme, TPOzanelli, which is p resent in Graves's disease thyroid tissue, has a cytoplasmic domain complet ely modified. In the first stage of this study, the results of RT-PCR exper iments showed that the TPOzanelli mRNA is present in normal thyroid tissue. We then generated CHO cell lines expressing the wild-type TPO (TPO1) and t he alternatively spliced form TPOzanelli. Upon investigating a panel of 12 mAbs directed against the extracellular domain of TPO 1 and sera from patie nts with a high titer of TPO autoantibodies, we observed that (i) the three -dimensional structure of this domain is similar in both isoforms; (ii) the autoantibodies recognize TPOzanelli as well as TPO1. The results of pulse chase and cell surface biotinylation experiments showed that the TPOzanelli has a shorter half-life (7 versus 11 h) and is expressed at the cell surfa ce in lesser amounts than TPO1 (7 versus 15%). The total enzymatic activity and cell surface activity were determined in CHO cells expressing TPO1 and TPOzanelli, and TPO1 and TPOzanelli were found to have similar levels of a ctivity. It was established that approximately 20% of the TPO purified from a Graves' disease thyroid gland was precipitated by polyclonal antibodies directed against a specific part of the cytoplasmic tail of TPOzanelli. Thi s confirmed that the protein corresponding to the mRNA is present in the th yroid tissue. All in all, these results indicate that TPOzanelli can be exp ected to play a role in thyroid hormone synthesis and in thyroid autoimmuni ty.