Abstract
Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.
Highlights
Hemoprotein that plays a key role in the biosynthesis of thyroid hormones
In thyroid cells, H2O2 is synthesized at the apical cell surface level and could be implicated in this covalent linking of the heme prosthetic group to TPO
To determine if the heme moiety had to be inserted into human TPO (hTPO) for its exit from the ER, we first used succinyl acetone (SA), a specific inhibitor of 5-aminolevulinic dehydratase, which catalyzes the formation of porpholibinogen from 5-aminolevulinate in the heme biosynthesis pathway [22, 23]
Summary
Hemoprotein that plays a key role in the biosynthesis of thyroid hormones. It catalyzes the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues to produce thyroxine and 3,3Ј,5-triiodothyronine [1,2,3]. To test the possibility that the heme moiety of TPO could be covalently linked to the protein through an autocatalytic process as for LPO [16], we incubated CHO-hTPO cells with or without 10 M H2O2 for 10 min or 20 M hemin for 10 min or 48 h before assaying hTPO activity (Fig. 4).
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