TPO

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TPO (EC 1.11.1.8, Thyroid peroxidase) belongs to the family of human peroxidases. It is an important enzyme responsible for the biosynthesis of thyroid hormones and is the main autoantigen of autoimmune thyroid diseases (AITDs). The systematic name of this enzyme class is iodide:hydrogen-peroxide oxidoreductase. Other names in common use include thyroid peroxidase, iodotyrosine deiodase, iodinase, iodoperoxidase (heme type), iodide peroxidase-tyrosine iodinase, iodotyrosine deiodinase, monoiodotyrosine deiodinase, thyroperoxidase, and tyrosine iodinase. TPO is an enzyme expressed mainly in the thyroid which oxidizes iodide ions to form iodine atoms.

Structure

The full-length TPO protein consists of 933 residues and has a molecular weight of 107 kDa. Unlike other peroxidases, it is mainly an extracellular protein anchored to the apical membrane of thyrocytes via its C-terminal transmembrane domain (residues 872-933). TPO synthesizes a sequence with an N-terminal signal peptide (residues 1-108) that is cleaved upon exiting the endoplasmic reticulum to form a mature TPO. The extracellular domain of TPO consists mainly of three regions: the myeloperoxidase (MPO)-like domain (residues 142-738), complement control protein (CCP)-like domain (residues 739-795), and epidermal growth factor (EGF)-like domain (residues 796-846).

MPO occurs as a homodimer that is covalently linked by a conserved intermolecular disulfide bond (Cys319) at the dimer interface. The overall folding of the MPO is an α-helix with a small portion of the β-sheet. Each monomer has a center consisting of 5 helices and a prosthetic heme group covalently linked to Glu408 and Asp260. The CCP-like domain, also known as the Sushi domain, is based on the formation of three β-strands with hydrogen bonds on one side and two separate β-strands on the other, ultimately forming a β-sandwich arrangement. The EGF-like domain is highly conserved in evolution. There are six cysteine residues, which constitute three disulfide bonds. The overall structure is β-sheet, followed by a short loop.

Figure 1. Structure of TPO. (Williams D.E. 2018)

Catalytic Mechanism

TPO catalyzes two important reactions in the biosynthesis of thyroid hormones: iodination of tyrosine residues in thyroglobulin (Tg), and coupling of iodotyrosines to form thyroid hormones T3 and T4. This process depends on the covalent combination of the heme group and the TPO. Thionamides such as propylthiouracil (PTU) and methimazole (MMI) are the most commonly used drugs for the treatment of hyperthyroidism. These drugs serve as a priority substrate for TPO, and are iodinated by TPO. In the thyroid gland, iodide (I-) is oxidized by TPO in the presence of hydrogen peroxide and subsequently incorporated into the tyrosine residue of Tg. TPO also mediates the coupling of monomeric or diiodotyrosine residues to form T3 and T4.

Figure 2. Mechanism of action of methimasole (MMI) on TPO. (Godlewska M. 2019)

Application

TPO is the main enzyme responsible for the synthesis of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). TPO is an important autoantigen and a key antigenic target for autoantibodies (aAbs) in AITD. In AITD, many thyroid-specific autoantigen tolerances and generation of autoantibody responses are broken. Anti-TPO autoantibodies are commonly present in active form in AITD patients. Hashimoto’s thyroiditis is a typical example of AITD, whose immune response is directed against the thyroid gland, resulting in complete destruction of the thyroid gland and reduced synthesis of thyroid hormones T3 and T4. Thyroid hormones T3 and T4 affect most body systems, causing severe disease symptoms from nervous system to physical manifestations, including hypertension, seizures, muscle degeneration and diabetes. The presence of TOP's high titer autoantibodies is a hallmark of disease and is often used as a diagnostic indicator of disease.

During the maturation of TPO, its N-terminal signal peptide will be cleaved. To improve the quality of TPO preparation, TPO cDNA lacking the N-terminal signal peptide was involved and stably expressed in the Chinese hamster ovary (CHO) cell line. Studies have shown that TPO with N-terminal signal peptide deletion has the same function and immunogenicity as full-length TPO. At the same time, the researchers also designed other TPO cDNA vectors, which can obtain a large amount of soluble TPO with high biochemical and antigenic properties in mammalian cell-based protein expression system. Moreover, the authors added a leucine zipper dimerization domain at the C-terminus to stabilize the TPO dimer, and the recombinant TPO exists in both monomeric and dimeric forms, and the autoantibody Fab derived from the patient preferentially binds to the monomeric TPO.

References

1. Williams, D.E., Le, S.N., Godlewska, M., Hoke, D.E., Buckle, A.M. Thyroid peroxidase as an autoantigen in Hashimoto’s disease: structure, function, and antigenicity. Horm Metab Res, 2018, 50(12):908-921.
2. Godlewska, M., Banga, P.J. Thyroid peroxidase as a dual active site enzyme: Focus on biosynthesis, hormonogenesis and thyroid disorders of autoimmunity and cancer. Biochimie, 2019, 160:34-45.