Role of Heme in Intracellular Trafficking of Thyroperoxidase and Involvement of H2O2 Generated at the Apical Surface of Thyroid Cells in Autocatalytic Covalent Heme Binding

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 ϳ30 and ϳ80%, 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 H 2 O 2 -dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H 2 O 2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 M H 2 O 2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H 2 O 2 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) H 2 O 2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule. Thyroperoxidase (TPO)1 is a membrane-bound, glycosylated 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 (1-3 (2) showed significant differences between the pyridine hemochromogene of TPO and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. TPO, lactoperoxidase (LPO), myeloperoxidase (MPO), saliva peroxidase, eosinoperoxidase, and intestinal peroxidase belong to the mammalian peroxidase family. These proteins share related protein primary structure (10, 11) and are alike in spectral properties. Furthermore, their prosthetic heme moieties are not readily extracted by conventional approaches. Anderson et al. (12) considered that spectral similarities between LPO and other mammalian peroxidases indicated similar prosthetic heme moieties and a covalent attachment of the heme group to the protein. They suggested that the heme moiety of TPO is probably a type l heme, like LPO. This heme is covalently linked to the protein through ester bonds that link aspartate and glutamate residues (conserved in MPO, ...

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) “Peroxidase” Domains

“…This interaction, combined with an ability to bind small ligands, suggests that the CeDUOX1 1-589 heme-binding pocket has a profile similar to that of the classical mammalian peroxidases, despite the absence of some typical catalytic residues. Furthermore, the demonstration that protein covalent binding is increased upon exposure to H 2 O 2 and is thus autocatalytic, links it tightly with both LPO and TPO, both of which share this behavior (29,62). Surprisingly, identification of a dihydroxyheme derivative upon enzymatic protein digestion of CeDUOX1 suggests that the protein may interact directly with the heme through two covalent bonds.…”

Role of Peroxiredoxins in Regulating Intracellular Hydrogen Peroxide and Hydrogen Peroxide-induced Apoptosis in Thyroid Cells

“…Because H 2 O 2 can directly damage DNA (12) and other biological macromolecules (13), it has been suggested that thyrocytes should have mechanisms to control the intracellular level of H 2 O 2 . Although thyroid cells utilize several cellular defense systems against oxidative damage including antioxidant proteins, superoxide dismutase (14), catalase (15,16) and glutathione (17), the exact mechanisms involved in regulating intracellular H 2 O 2 are not known.…”

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

“…It has already been demonstrated that the attachment of heme residues to the TPO molecule is essential for delivery of TPO to the cell surface (54). The MPO molecule, as well as all other members of the mammalian peroxidase family, contains heme as a prosthetic group (43).…”