Identification of antigenic epitopes of thyroperoxidase, thyroglobulin and interleukin-24. Exploration of cross-reactivity with environmental allergens and possible role in urticaria and hypothyroidism.

BACKGROUND: Human proteins such as interleukin-24 (IL24), thyroperoxidase (TPO) and thyroglobulin (Tg) are targets of IgE or IgG autoantibodies. Why these proteins are recognized by autoantibodies in some patients with chronic spontaneous urticaria (CSU) or hypothyroidism is unknown. OBJECTIVE: Through in silico analysis, identify antigen patches of TPO, Tg and IL24 and compare the sequences of these human proteins with some prevalent allergens. METHODS: The amino acids sequences of IL24, thyroperoxidase and thyroglobulin were compared between them and with 22 environmental allergens. Phylogenetic studies and multiple pairing were carried out to explore the degree of protein identity and cover. The proteins without 3D structure reported in the database, were modeled by homology with "Swiss Modeller" and compared through PYMOL. Residues conserved and accessible to the solvent (rASA> 0.25) were located in the 3D model to identify possible areas of cross-reactivity and antigen binding. RESULTS: We build a 3D model of the TPO and thyroglobulin protein base on proteins closely related. Five epitopes for TPO, six for IL24 and six for thyroglobulin were predicted. The amino acid sequences of allergens from different sources (Dermatophagoides pteronyssinus, Blomia tropicalis, Betula verrucosa, Cynodon dactylon, Aspergillus fumigatus, Canis domesticus, Felis domesticus) were compared with human TPO, Tg and IL24. The cover and alignments between allergens and human proteins were low. CONCLUSION: We identify possible linear and conformational epitopes of TPO, Tg and IL24 that could be the target of IgE or IgG binding in patients with urticaria or hypothyroidism; These epitopes do not appear to be present among common environmental allergens, suggesting that autoreactivity to these human proteins are not by cross-reactivity.

Dynamic mRNA distribution pattern of thyroid hormone transporters and deiodinases during early embryonic chicken brain development.

Maternal thyroid hormones (THs) are important in early brain development long before the onset of embryonic TH secretion, but information about the regulation of TH availability in the brain at these early stages is still limited. We therefore investigated in detail the mRNA distribution pattern of the TH activating type 2 and inactivating type 3 deiodinases (D2 and D3) and the TH transporters, organic anion transporting polypeptide 1c1 (Oatp1c1) and monocarboxylate transporter 8 (Mct8), in chicken embryonic brain as well as in retina and inner ear from day 3 to day 10 of development. Oatp1c1, Mct8 and D3 are expressed in the choroid plexus and its precursors allowing selective uptake of THs at the blood-cerebrospinal fluid-barrier with subsequent inactivation of excess hormone. In contrast, the developing blood-brain-barrier does not express Oatp1c1 or Mct8 but appears to be a site for TH activation by D2. Expression of D3 in several sensory brain centers may serve as protection against premature TH action. Expression of D2 and Mct8 but not D3 in the developing pituitary gland allows accumulation of active THs even at early stages. Mct8 is widely expressed in gray matter throughout the brain. This is the first comprehensive study on the dynamic distribution pattern of TH-transporters and deiodinases at stages of embryonic brain development when only maternal THs are available. It provides the essential background for further research aimed at understanding early developmental processes depending on maternal THs.

Iodotyrosine deiodinase is the first mammalian member of the NADH oxidase/flavin reductase superfamily.

The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deiodinase, was solubilized from porcine thyroid microsomes by limited proteolysis with trypsin. The resulting protein retained deiodinase activity and was purified using anion exchange, dye, and hydrophobic chromatography successively. Peptide sequencing of the final isolate identified the gene responsible for the deiodinase. The amino acid sequence of the porcine enzyme is highly homologous to corresponding genes in a variety of mammals including humans, and the mouse gene was expressed in human embryonic kidney 293 cells to confirm its identity. The amino acid sequence of the deiodinase suggests the presence of three domains. The N-terminal domain provides a membrane anchor. The intermediate domain contains the highest sequence variability and lacks homology to structural motifs available in the common databases. The C-terminal domain is highly conserved and resembles bacterial enzymes of the NADH oxidase/flavin reductase superfamily. A three-dimensional model of the deiodinase based on the coordinates of the minor nitroreductase of Escherichia coli indicates that a Cys common to all of the mammal sequences is located adjacent to bound FMN. However, the deiodinase is not structurally related to other known flavoproteins containing redox-active cysteines or the iodothyronine deiodinases containing an active site selenocysteine.

Role of spatiotemporal expression of iodothyronine deiodinase proteins in cerebellar cell organization.

Thyroid hormones (TH) play a crucial role in various developmental processes in all vertebrates. The expression of a number of thyroid hormone responsive genes is of critical importance in processes like cell maturation and migration. Since these genes are mostly regulated by binding of the receptor-active TH (T(3)) to the thyroid hormone receptor, the availability of this T(3) is indispensable for correct brain lamination. One important way to regulate local TH availability is via the ontogenetic changes in activating and inactivating iodothyronine deiodinases. The current study was set up to investigate the distribution of type I, type II and type III (D1, D2 and D3) iodothyronine deiodinase protein in the chicken cerebellum at two important developmental ages, namely embryonic day 18 when cerebellar cell migration is fully in progress, and 1 day posthatch, when cerebellar maturation is mostly finished. The results show that the deiodinase proteins are divergently expressed in the cerebellar cell population. D1 and D3 are expressed in the granule cells at E18, whereas D2 is found mostly in the molecular layer and the Purkinje cells at that time. One day posthatch, the expression of D1 is limited to the mature granule cells and that of D3 to the Purkinje cells exclusively, whereas D2 remains clearly present in the molecular layer. Comparison of the deiodinase protein distribution with the expression of TH-responsive proteins involved in cell migration (reelin, disabled protein 1 and tenascin-C) allows speculating about the effect of this spatiotemporal distribution pattern on cerebellar cell communicative pathways.

Deiodinase type II and tissue specific mRNA alternative splicing in the Australian lungfish, Neoceratodus forsteri.

Deiodinase type II metabolises the prohormone T4 (thyroxine) into the biologically active hormone T3 (3,5,3'-triiodothyronine), at the cellular level in extrathyroidal target tissues. In juvenile lungfish, Neoceratodus forsteri, a typical deiodinase type II is present in most tissues. We have identified the full length of a 1.8 kb deiodinase type II mRNA in liver, and a truncated (1.3 kb) version in brain. Both mRNAs have two in frame UGA codons, but only the liver form has a predicted SECIS structure (form 1) in its 3'-UTR. We also report the presence of additional different length transcripts of deiodinase II mRNA, i.e., 3, 4, and 8 kb, in liver, and 8 kb in kidney, heart, and gill tissues. Expression of the longer (approximately 8 kb) transcript is very low. Real-time PCR confirmed the low expression of transcripts in all tissues, suggested by the Northern blot analysis.

Expression of type II iodothyronine deiodinase in brain tumors.

Type II iodothyronine deiodinase (DII) messenger ribonucleic acid (mRNA) and its activity have been demonstrated in human normal brain. Although DII activity has been demonstrated in brain tumors, expression of DII mRNA has not been studied in these tumors. To investigate the mechanisms involved in the expression of DII activity in brain tumors, we studied DII mRNA and DII activity in astrocytoma (two cases), glioblastoma (three cases), and oligodendroglioma (one case). DII mRNA, the size of which was indistinguishable from that in control cerebral cortical tissue, was demonstrated in all of the brain tumors tested, although the intensity of the hybridization signal showed wide variation among the tumors. DII activity was also detected in all tumors. DII mRNA and DII activity were highest in the tissue from oligodendroglioma. A significantly positive correlation was observed between DII mRNA and DII activity in these tumors (r = 0.94; P < 0.01), suggesting that DII expression in brain tumors is regulated at the pretranslational level. The present results demonstrate, for the first time, that DII mRNA as well as DII activity are expressed in brain tumors, and that DII mRNA is significantly correlated with DII activity in those tissues.

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase.

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Structure of the human type I iodothyronine 5'-deiodinase gene and localization to chromosome 1p32-p33.

The human type I iodothyronine 5'-deiodinase gene encodes a member of the family of selenocysteine-containing deiodinases. These enzymes catalyze the activation of the prohormone thyroxine to 3,3',5-triiodothyronine or the degradation of thyroxine and triiodothyronine to inactive metabolites. Here we report the isolation of two genomic type I 5'-deiodinase clones from a chromosome 1-specific gridded cosmid library, the localization of the gene to chromosome 1p32-p33 by fluorescence in situ hybridization, and the determination of the complete structure of the 17.5-kb gene.

Cloning of a cDNA for the type II iodothyronine deiodinase.

Three types of iodothyronine deiodinase have been identified in vertebrate tissues. cDNAs for the types I and III have been cloned and shown to contain an inframe TGA that codes for selenocysteine at the active site of the enzyme. We now report the cloning of a cDNA for a type II deiodinase using a reverse transcription/polymerase chain reaction strategy and RNA obtained from Rana catesbeiana tissues. This cDNA (RC5'DII) manifests limited but significant homology with other deiodinase cDNAs and contains a conserved in-frame TGA codon. Injection of capped in vitro synthesized transcripts of the cDNA into Xenopus laevis oocytes results in the induction of deiodinase activity with characteristics typical of a type II deiodinase. The levels of RC5'DII transcripts in R. catesbeiana tadpole tail and liver mRNA at stages XII and XXIII correspond well with that of type II deiodinase activity but not that of the type III activity in these tissues. These findings indicate that the amphibian type II 5'-deiodinase is a structurally unique member of the family of selenocysteine-containing deiodinases.

Tumor necrosis factor-alpha and interferon-gamma modulate gene expression of type I 5'-deiodinase, thyroid peroxidase, and thyroglobulin in FRTL-5 rat thyroid cells.

Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) have many effects on a number of cell types, including thyrotrophs. In the present study, we used FRTL5 cells, a cultured rat thyroid follicular cell line, to examine the effects of IFN-gamma and TNF-alpha on type I 5'-deiodinase (5'D-I) activity and 5'D-I, thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression. Incubation of FRTL5 cells with the highest concentrations of TNF-alpha and IFN-gamma tested (1000 ng/ml or 1000 U/ml, respectively) for 72 h in the presence and absence of TSH had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived FRTL-5 cells, TNF-alpha and IFN-gamma resulted in a small but dose-dependent decrease in 5'D-I activity. TNF-alpha or IFN-gamma blocked the TSH- or cAMP-induced rise in 5'D-I activity. 100 ng/ml TNF-alpha and 100 U/ml IFN-gamma completely blocked the TSH- or cAMP-induced rise in 5' D-I activity. However, when cells were incubated with TNF-alpha and IFN-gamma, in combination, there was a marked decrease in 5'D-I activity, with TNF-alpha (25 ng/ml) plus IFN-gamma (25 U/ml) completely blocking the TSH-induced rise in 5'D-I activity. Northern blot analyses were performed to examine the effect of TNF-alpha and IFN-gamma on 5'D-I gene expression. TNF-alpha had little effect on 5'D-I messenger RNA (mRNA) levels, while IFN-gamma resulted in a modest decrease in 5'D-I mRNA levels in TSH-deprived cells, and in TSH-stimulated FRTL-5 cells. However, when TNF-alpha and IFN-gamma were added in combination there was a marked decrease in 5'D-I gene expression with TNF-alpha (50 ng/ml) plus IFN-gamma (50 U/ml) decreasing 5'D-I mRNA levels by 89 percent in TSH-deprived cells. In TSH-stimulated cells incubated with 500 ng/ml TNF-alpha plus 500 U/ml IFN-gamma, 5'D-I mRNA levels were almost undetectable. We also examined the effect of IFN-gamma and TNF-alpha on TPO and Tg gene expression. As observed with 5'D-I mRNA levels, there was a synergistic effect of IFN-gamma and TNF-alpha on the inhibition of basal and TSH-stimulated TPO and Tg gene expression. These findings indicate that TNF-alpha and IFN-gamma in combination have a marked inhibitory effect on thyroid function, which is consistent with a decrease in thyroid hormone synthesis and metabolism.

Investigation of type I and type III iodothyronine deiodinases in rat tissues using N-bromoacetyl-iodothyronine affinity labels.

In the present study the hypothesis was tested that N-bromoacetyl-3,3',5-[125I]triiodothyronine (BrAc[125I]T3) is a useful affinity label for both type I and type III iodothyronine deiodinases (ID-I and ID-III). Therefore, the microsomal fractions of various rat tissues were tested for ID-I and ID-III activities, and microsomal proteins were labeled with BrAc[125I]T3 and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I activities were found in liver, kidney and thyroid, and high ID-III activities in brain, in particular fetal brain, and placenta. SDS-PAGE of BrAc[125I]T3-labeled microsomes showed a prominent radioactive approximately 27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a approximately 32 kDa protein (p32) in brain, in particular fetal brain, and placenta. A good correlation was found between the affinity labeling of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents ID-III or a subunit thereof. After treatment of microsomes with 0.05% deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by BrAc[125I]T3, indicating that p32 is a transmembrane protein. Although 3,3',5'-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily labeled with BrAc[125I]rT3. Labeling of p32 in rat brain microsomes by BrAc[125I]rT3 was not affected by addition of 100 microM unlabeled thyroxine (T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Selenium deficiency and thyroid hormone metabolism and function].

Type I 5'-deiodinase is a Se-containing enzyme. If Se is deficient, the deiodinase activity would be inhibited, the level of circulation T4 will be elevated, and the concentration T3 in peripheral tissues will be decreased. Se deficiency will also accelerate the iodine depletion of thyroid and may even exacerbate some detrimental effects of iodine deficiency. Possibly Se deficiency is involved in the occurrence and development of iodine deficient disorders. Keshan disease, with Se deficiency as the major cause, was also observed a change of thyroid hormone metabolism. The change of respiratory enzyme activities in myocardium of Keshan disease is in the way somewhat like that of hypothyroidism caused by iodine deficiency. The metabolic change of thyroid hormone after Se deficiency or iodine deficiency may be related to the occurrence of Keshan disease.

Hormone-nuclear receptor interactions in health and disease. The iodothyronine deiodinases and 5'-deiodination.

Two types of iodothyronine deiodinase (ID-I and ID-II) catalyse the 5'-deiodination of thyroxine (T4) to produce the biologically active triiodothyronine (T3). Under normal circumstances ID-I in liver and kidney provides the main source of T3 to the circulation, whilst ID-II is largely responsible for local T3 production in the CNS, brown adipose tissue and pituitary. In some circumstances ID-II in brown adipose tissue and ID-I in the thyroid may provide a significant source of plasma T3, and ID-I in the pituitary may be important for local T3 production in this gland. The IDs thus play a pivotal role in controlling the supply of T3 to the nuclear receptors. ID-I is a selenoenzyme and, although ID-II activity is reduced in selenium deficiency, this is a consequence of increased plasma T4 concentration, rather than ID-II activity being directly dependent on selenium. Changes in 5'-deiodination occur in a number of situations such as poor nutrition, illness, iodine and selenium deficiency, and drug therapy. In iodine deficiency these changes appear to have evolved to ensure that the plasma T3 level is maintained and also to provide the brain with a degree of protection from hypothyroxinaemia. Relatively little is known about the importance of selenium deficiency on thyroid function in humans but, in combination with iodine deficiency, selenium deficiency may prove to be a contributing factor in the pathogenesis of myxodematous cretinism. The changes that occur in ID-I and ID-II in illness produce abnormalities in thyroid function tests which, although of no direct clinical significance, may lead to interpretative problems.

Postnatal changes in lambs of two pathways for thyroxine 5'-monodeiodination in brown adipose tissue.

We have recently shown that ovine fetal brown adipose tissue (BAT) contains two distinct iodothyronine 5'-monodeiodinase (5'MDI) activities, one with a high Km (type I) and another with a low Km (type II). Both activities increased to maximum levels near term (150 days gestation). BAT plays a major role in neonatal temperature regulation in lambs, and available evidence suggests that BAT 5'MDI activity is closely linked to thermogenic capacity. To better characterize the changes in 5'MDI after birth, we studied both type I and type II 5'MDI in lamb BAT from the time of birth to 30 days of postnatal age. Type I 5'MDI activity [pmol 3,5,3'-triiodothyronine (T3).mg protein-1.h-1] showed no significant changes during the first 11 days after birth [newborn (NB), 95 +/- 16; 1 day, 83 +/- 20; 3-4 days, 80 +/- 11; 10-11 days, 92 +/- 28]. Activity decreased significantly at 30 days (24 +/- 8.9, P less than 0.05). On the other hand, the type II 5'MDI activity (fmol I- released.mg protein-1.h-1) increased significantly (P less than 0.01) during the first 4 days, (NB, 348 +/- 23; 1 day, 679 +/- 37; 3-4 days, 785 +/- 199), decreased toward NB values (401 +/- 87) at 10-11 days of age, and fell to 66 +/- 31 at 30 days (P less than 0.05 vs. NB). Kinetic analysis of BAT type II thyroxine 5'MDI revealed a rise in maximum velocity from NB to 1 and 3-4 days of age without a change in the enzymatic activity Km.(ABSTRACT TRUNCATED AT 250 WORDS)

The actin cytoskeleton mediates the hormonally regulated translocation of type II iodothyronine 5'-deiodinase in astrocytes.

Thyroid hormone, specifically thyroxine, alters cytoskeletal organization in astrocytes by modulating actin polymerization and, in turn, regulates the turnover of the short-lived membrane protein, type II iodothyronine 5'-deiodinase. In the absence of thyroxine, approximately 35% of the total cellular actin is depolymerized, and greater than 90% of the deiodinase is found in the plasma membrane and not associated with the cytoskeleton. Addition of thyroxine promotes actin polymerization and decreases the depolymerized actin to approximately 10% of the total actin pool, induces binding of the deiodinase to F-actin, and promotes rapid internalization of the enzyme. These data provide direct evidence that the actin cytoskeleton participates in the inactivation pathway of the deiodinase by translocating this short-lived plasma membrane protein to an internal membrane pool.

A study of hepatic low Km iodothyronine 5'-monodeiodinase.

To document the presence of a low Km rT3 and T4-5'-monodeiodinase (5'MDL; Km in nanomolar concentrations) in the liver and to study its characteristics in comparison with the high Km 5'MD (5'MDH; Km in micromolar concentrations), we incubated rat liver microsomal protein (20 micrograms for rT3 substrate and 200 micrograms for T4 substrate) with 125I-labeled rT3 or T4 and dithiothreitol (DTT; up to 20 mM) for 5 min (for rT3) or 30-120 min (for T4) and determined the amount of 125I liberated during incubation. Pilot studies had shown that the activity of rT3 5'MDH is markedly (greater than or equal to 85%) inhibited in the presence of 2 M NaCl, while the rT3 5'MDL is essentially unaffected, and both low and high Km T4 5'MD are minimally (approximately 20%) inhibited. The representative kinetics of various substrates studied were: Km, 13 nM for rT3 5'MDL, 640 for rT3 5'MDH, 26 for T4 5'MDL, and 3620 for T4 5'MDH; maximum velocity, 0.28 nmol/h.mg protein for rT3 5'MDL, 46 for rT3 5'MDH, 0.002 for T4 5'MDL, and 0.46 for T4 5'MDH. Propylthiouracil and iopanoate inhibited all enzymic activities studied. The relative Ki values (micromolar concentrations) for propylthiouracil were: 7.1 for rT3 5'MDL, 1.5 for rT3 5'MDH, 24 for T4 5'MDL, and 40 for T4 5'MDH; those for iopanoate were 0.4 for rT3 5'MDL, 18 for rT3 5'MDH, 7.0 for T4 5'MDL, and 4.0 for T4 5'MDH. DTT was a potent stimulator of enzyme activities studied; its dose (millimolar concentrations) that caused a 50% maximal stimulation was 0.04 for rT3 5'MDL, 1.0 for rT3 5'MDH, 0.025 for T4 5'MDL, and 0.035 for T4 5'MDH. T4 inhibited rT3 5'-monodeiodination and vice versa. The Ki of T4 was 1.3 microM for rT3 5'MDL and 2.0 for rT3 5'MDH, while that of rT3 was 0.4 for T4 5'MDL and 0.6 for T4 5'MDH. We examined the activity of the hepatic 5'MDL (rT3, 0.5 nM; DTT, 0.06 nM; 2 M NaCl) and 5'MDH (rT3, 0.5 microM; DTT, 20 mM; no NaCl) in groups (six animals per group) of rats that were saline treated (control), thyroidectomized, or hyperthyroid (given T3, 20 micrograms/day for 5 days). The relative values for 5'MDL were (mean +/- SD) 17 +/- 3.0, 4.0 +/- 2.0 (P less than 0.01), and 24 +/- 2.0 (P less than 0.01) pmol/h.mg protein, respectively, whereas those for 5'MDH were 13 +/- 4.0, 3.0 +/- 1.6 (P less than 0.01), and 25 +/- 1.0 (P less than 0.01) nmol/h.mg protein, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

A circulating thyroid peroxidase-like substance in healthy human peripheral blood.

Circulating thyroid peroxidase (TPO)-like substance in healthy human peripheral blood was studied to clarify the immunological role of TPO. By using a highly sensitive radioimmunoassay system combined with murine monoclonal antibodies, TPO-like substance was measurable in 6 out of 84 sera. Characterization of this circulating substance by gel filtration revealed that the molecular weight of a major peak corresponded to that of trypsinized TPO. From these findings it is possible that peripheral blood lymphocytes are exposed to a low level of TPO as well as to thyroglobulin.