Recombinant human thyroid peroxidase expressed in insect cells is soluble at high concentrations and forms diffracting crystals.
Human thyroid peroxidase (TPO), the key enzyme in thyroid hormone synthesis, can be produced in active form in the High Five insect cell line and when purified from the cell culture medium is soluble at concentrations of up to 18 mg/ml. This contrasts to a recent report in which human TPO produced in insect cells was found to be insoluble at high concentrations. Our concentrated TPO grows trigonal trapezohedral crystals of up to 0.5 mm in length in a vapour diffusion apparatus using polyethelene glycol as a precipitant. The crystals diffract X-rays to a 6 A resolution and the diffraction data from the crystals have been analysed giving unit cell dimensions. A potential molecular replacement solution has been identified using myeloperoxidase (MPO) as a phasing model. (+info
Type 1 deiodinase is stimulated by iodothyronines and involved in thyroid hormone metabolism in human somatomammotroph GX cells.
BACKGROUND: Local 5'-deiOdination of l-thyroxine (T4) to the active thyroid hormone, 3,3',5-tri-iodothyronine (T3) via two deiodinase isoenzymes (D1 and D2) has an important role for various T3-dependent functions in the anterior pituitary. However, no evidence has been presented yet for thyroid hormone inactivation via the 5-deiodinase (D3) in anterior pituitary models. METHODS: Using the human somatomammotroph cell line, GX, we analysed effects of T3 and its 5'-deiodination product, 3,5-di-iodothyronine (3,5-T2), on deiodinase activities, measuring release of iodide-125 (125I-) from phenolic-ring- or tyrosyl-ring-labelled substrates respectively. RESULTS: T3 and 3,5-T2 rapidly stimulated D1 activity in GX cells in the presence of serum in the culture medium, whereas D2 activity was not detectable under these conditions. However, when the cells were kept under serum-free conditions, specific activity of D2 reached levels similar to those of D1. With tyrosyl-ring labelled 3, 5-[125I]-,3'-T3 as substrate, a significant release of 125I- was observed in GX cell homogenates. This is comparable to the D1 activity of liver membranes, which preferentially catalyses 5'-deiodination, but to some extent also 5-deiodination, at the tyrosyl ring. CONCLUSIONS: D1 activity of human GX cells is increased by T3 and 3,5-T2. Inactivation of T3 in the anterior pituitary might occur by deiodination at the tyrosyl ring via D1, thus terminating the stimulatory thyroid hormone signal in human somatomammotroph cells. (+info
Maximal number of hormonogenic iodotyrosine residues in thyroglobulin iodinated by thyroid peroxidase.
Almost non-iodinated human goiter thyroglobulin has been iodinated in vitro by thyroid peroxidase to levels as high as 75 iodine atoms per mol of protein. The following results were obtained. 1. The iodine distribution obtained in vitro with human thyroglobulin strongly ressembles that obtained in vivo for rat thyroglobulin. Thus the distribution of iodine seems to depend essentially on the structure of thyroglobulin and on the reactivity of the different tyrosine residues. 2. Although the number of hormone residues increased with iodination the highest efficiency of hormone synthesis was obtained in a very narrow range of iodination: in vitro (40%) between 25 and 30 iodine atoms, and in vivo (48%) between 10 and 20 atoms. This result suggests that the tyrosines which are coupled with a high efficiency are iodinated sequentially. 3. Maximal thyroxine content was found to be lower than approximately 3 mol/mol of thyroglobulin. This result might mean that the two 12-S subunits of thyroglobulin are not identical and that one of them is able to produce 2 mol of hormone while the second only 1 mol. (+info
Kinetics of thyroglobulin iodination and of hormone synthesis catalysed by thyroid peroxidase. Role of iodide in the coupling reaction.
The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the same enzyme (thyroid peroxidase), have been compared. Thyroxine synthesis always began after a lag period of 3-5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non-iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catlyzes thyroid hormone synthesis at a slower rate than iodination. The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained. 1. Iodotyrosine residues which can couple to form thytoxine are made considerably before coupling occurs. 2. H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues. 3. In addition a strict requirement for iodide was needed for coupling; the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase. (+info
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.
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. (+info
Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase.
Although thyroid hormones are critically important for the coordination of morphogenic processes in the fetus and neonate, premature exposure of the embryo to levels of the hormones present in the adult is detrimental and can result in growth retardation, malformations, and even death. We report here that the pregnant rat uterus expresses extremely high levels of the type 3 iodothyronine deiodinase (D3), which inactivates thyroxine and 3,3', 5-triiodothyronine by 5-deiodination. Both D3 mRNA and activity were present at the implantation site as early as gestational day 9 (E9), when expression was localized using in situ hybridization to uterine mesometrial and antimesometrial decidual tissue. At later stages of gestation, uterine D3 activity remained very high, and the levels exceeded those observed in the placenta and in fetal tissues. After days E12 and E13, as decidual tissues regressed, D3 expression became localized to the epithelial cells lining the recanalized uterine lumen that surrounds the fetal cavity. These findings strongly suggest that the pregnant uterus, in addition to the placenta, plays a critical role in determining the level of exposure of the fetus to maternal thyroid hormones. (+info
Expression of type 2 iodothyronine deiodinase in hypothyroid rat brain indicates an important role of thyroid hormone in the development of specific primary sensory systems.
Thyroid hormone is an important epigenetic factor in brain development, acting by modulating rates of gene expression. The active form of thyroid hormone, 3,5,3'-triiodothyronine (T3) is produced in part by the thyroid gland but also after 5'-deiodination of thyroxine (T4) in target tissues. In brain, approximately 80% of T3 is formed locally from T4 through the activity of the 5'-deiodinase type 2 (D2), an enzyme that is expressed mostly by glial cells, tanycytes in the third ventricle, and astrocytes throughout the brain. D2 activity is an important point of control of thyroid hormone action because it increases in situations of low T4, thus preserving brain T3 concentrations. In this work, we have studied the expression of D2 by quantitative in situ hybridization in hypothyroid animals during postnatal development. Our hypothesis was that those regions that are most dependent on thyroid hormone should present selective increases of D2 as a protection against hypothyroidism. D2 mRNA concentration was increased severalfold over normal levels in relay nuclei and cortical targets of the primary somatosensory and auditory pathways. The results suggest that these pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some interneurons of the cerebral cortex were also labeled. Therefore, the T3 target cells, mostly neurons, are dependent on local astrocytes for T3 supply. (+info
Risk of iodine-induced thyrotoxicosis after coronary angiography: an investigation in 788 unselected subjects.
In this study, the risk of iodine-induced thyrotoxicosis in unselected patients from an iodine-deficient area was investigated. The patients were consecutively enrolled. Thyroid hormone values and urinary iodine excretion were determined before, as well as 1, 4 and 12 weeks after iodine contamination by coronary angiography. Two of 788 unselected patients developed hyperthyroidism within 12 weeks. The two patients did not belong to a risk group for iodine-induced thyrotoxicosis (i.e. old people, patients with goiter or possible thyroid autonomy, low TSH). Both patients had normal TSH levels at baseline and ultrasound of the thyroid was without evidence of nodules. The study shows that in euthyroid unselected patients from an iodine-deficient area short-term iodine contamination by contrast media rarely leads to hyperthyroidism. On account of these facts, prophylactic therapy, e.g. by perchlorate or thiamazole, is not generally recommended, because the risk of side-effects is perhaps even greater than the risk of iodine-induced thyrotoxicosis. (+info