Abstract
Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-beta-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannose-type structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [14C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive.
Highlights
Thyroid peroxidase (TPO)1 is the major enzyme in the biosynthesis of thyroid hormones, because it catalyzes the iodination and coupling of iodotyrosine residues on thyroglobulin to produce thyroxine (T4) and 3,3Ј,5-triiodothyronine (1–3)
TPO2 mRNA has been detected in normal and Graves thyroids (19, 20) and in a case of congenital hypothyroidism caused by a premature termination signal in the TPO gene, suggesting that TPO2 is enzymatically inactive (21)
After obtaining several clones expressing TPO2 and TPO1, we did the experiments in the presence of 10 mM sodium butyrate, which made TPO2 and TPO1 expression levels higher than those obtained without butyrate addition
Summary
Thyroid peroxidase (TPO) is the major enzyme in the biosynthesis of thyroid hormones, because it catalyzes the iodination and coupling of iodotyrosine residues on thyroglobulin to produce thyroxine (T4) and 3,3Ј,5-triiodothyronine (1–3). Distribution and delivery of TPO are apically polarized, but only ϳ30% TPO is detected at the thyrocyte surface (13) Mistargeting of another small fraction of TPO to the basolateral surface seems to occur (13, 14). This may account for the hypothetical presentation of TPO to the circulating immune system, resulting in its antigenicity and its part in autoimmune thyroid diseases. TPO2 is 57 amino acids shorter than the major protein and lacks one of the potential glycosylation sites and His-586. TPO2 mRNA has been detected in normal and Graves thyroids (19, 20) and in a case of congenital hypothyroidism caused by a premature termination signal in the TPO gene, suggesting that TPO2 is enzymatically inactive (21). We determined the effects of those structural modifications on the intracellular trafficking, the localization, and the enzymatic activity of the protein
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