Structural Studies of Thyroid Peroxidase Show the Monomer Interacting With Autoantibodies in Thyroid Autoimmune Disease.

Thyroid peroxidase (TPO) is a critical membrane-bound enzyme involved in the biosynthesis of multiple thyroid hormones, and is a major autoantigen in autoimmune thyroid diseases such as destructive (Hashimoto) thyroiditis. Here we report the biophysical and structural characterization of a novel TPO construct containing only the ectodomain of TPO and lacking the propeptide. The construct was enzymatically active and able to bind the patient-derived TR1.9 autoantibody. Analytical ultracentrifugation data suggest that TPO can exist as both a monomer and a dimer. Combined with negative stain electron microscopy and molecular dynamics simulations, these data show that the TR1.9 autoantibody preferentially binds the TPO monomer, revealing conformational changes that bring together previously disparate residues into a continuous epitope. In addition to providing plausible structural models of a TPO-autoantibody complex, this study provides validated TPO constructs that will facilitate further characterization, and advances our understanding of the structural, functional, and antigenic characteristics of TPO, an autoantigen implicated in some of the most common autoimmune diseases.

Thyroid Peroxidase Activity is Inhibited by Phenolic Compounds-Impact of Interaction.

The aim of this study was to estimate the mode of thyroid peroxidase (TPO) inhibition by polyphenols: Chlorogenic acid, rosmarinic acid, quercetin, and rutin. All the tested polyphenols inhibited TPO; the IC50 values ranged from 0.004 mM to 1.44 mM (for rosmarinic acid and rutin, respectively). All these pure phytochemical substances exhibited different modes of TPO inhibition. Rutin and rosmarinic acid showed competitive, quercetin-uncompetitive and chlorogenic acid-noncompetitive inhibition effect on TPO. Homology modeling was used to gain insight into the 3D structure of TPO and molecular docking was applied to study the interactions of the inhibitors with their target at the molecular level. Moreover, the type and strength of mutual interactions between the inhibitors (expressed as the combination index, CI) were analyzed. Slight synergism, antagonism, and moderate antagonism were found in the case of the combined addition of the pure polyphenols. Rutin and quercetin as well as rutin and rosmarinic acid acted additively (CI = 0.096 and 1.06, respectively), while rutin and chlorogenic acid demonstrated slight synergism (CI = 0.88) and rosmarinic acid with quercetin and rosmarinic acid with chlorogenic acid showed moderate antagonism (CI = 1.45 and 1.25, respectively). The mixture of chlorogenic acid and quercetin demonstrated antagonism (CI = 1.79). All the polyphenols showed in vitro antiradical ability against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS. The highest ability (expressed as IC50) was exhibited by rosmarinic acid (0.12 mM) and the lowest value was ascribed to quercetin (0.45 mM).

Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application.

Vanadium-dependent haloperoxidases in seaweeds, cyanobacteria, fungi, and possibly phytoplankton play an important role in the release of halogenated volatile compounds in the environment. These halocarbons have effects on atmospheric chemistry since they cause ozone depletion. In this chapter, a survey is given of the different sources of these enzymes, some of their properties, the various methods to isolate them, and the bottlenecks in purification. The assays to detect and quantify haloperoxidase activity are described as well as their kinetic properties. Several practical tips and pitfalls are given which have not yet been published explicitly. Recent developments in research on structure and function of these enzymes are reviewed. Finally, the application of vanadium-dependent haloperoxidases in the biosynthesis of brominated and other compounds is discussed.

Purification of recombinant human thyroid peroxidase (hTPO) from AD293 mammalian cells.

Human thyroid peroxidase (hTPO) has been secretory expressed in AD293 mammalian cells. cDNA sequence of 'Gluc' (Gaussia luciferase) protein from Gaussia princeps was incorporated at the amino terminal of hTPO gene for secretion of targeted protein outside the mammalian cells. Augmentation of TPO clone in serum free mediums was investigated and a simplified purification procedure of hTPO has been reported here. Purified hTPO was further analyzed by SDS-PAGE and immunoblotting (western blotting). The relative molecular mass of hTPO was found to be 105kDa. This is the first report with respect to cost effective and simplified purification approach to get highest yield and purity of recombinant hTPO.

Association of duoxes with thyroid peroxidase and its regulation in thyrocytes.

CONTEXT: Thyroid hormone synthesis requires H(2)O(2) produced by dual oxidases (Duoxes) and thyroperoxidase (TPO). Defects in this system lead to congenital hypothyroidism. H(2)O(2) damage to the thyrocytes may be a cause of cancer. OBJECTIVE: The objective of the study was to investigate whether Duox and TPO, the H(2)O(2) producer and consumer, might constitute a complex in the plasma membrane of human thyroid cells, thus maximizing efficiency and minimizing leakage and damage. DESIGN: The interaction between Duox and TPO was studied by coimmunoprecipitation and Western blotting of plasma membranes from incubated follicles prepared from freshly resected human thyroid tissue from patients undergoing thyroidectomy, and COS-7 cells transiently transfected with the entire Duoxes or truncated [amino (NH2) or carboxyl (COOH) terminal]. RESULTS: The following results were reached: 1) Duox and TPO from membranes are coprecipitated, 2) this association is up-regulated through the Gq-phospholipase C-Ca(2+)-protein kinase C pathway and down-regulated through the Gs-cAMP-protein kinase A pathway, 3) H(2)O(2) increases the association of Duox1 and Duox2 to TPO in cells and in membranes, and 4) truncated NH(2)- or COOH-terminal Duox1 and Duox2 proteins show different binding abilities with TPO. CONCLUSION: Coimmunoprecipitations show that Duox and TPO locate closely in the plasma membranes of human thyrocytes, and this association can be modulated by H(2)O(2), optimizing working efficiency and minimizing H(2)O(2) spillage. This association could represent one part of a postulated pluriprotein complex involved in iodination. This suggests that defects in this association could impair thyroid hormone synthesis and lead to thyroid insufficiency and cell damage.

Changes in the central component of the hypothalamus-pituitary-thyroid axis in a rabbit model of prolonged critical illness.

INTRODUCTION: Prolonged critically ill patients reveal low circulating thyroid hormone levels without a rise in thyroid stimulating hormone (TSH). This condition is labeled "low 3,5,3'-tri-iodothyronine (T3) syndrome" or "nonthyroidal illness syndrome (NTI)" or "euthyroid sick syndrome". Despite the low circulating and peripheral tissue thyroid hormone levels, thyrotropin releasing hormone (TRH) expression in the hypothalamus is reduced and it remains unclear which mechanism is responsible. We set out to study whether increased hypothalamic T3 availability could reflect local thyrotoxicosis and explain feedback inhibition-induced suppression of the TRH gene in the context of the low T3 syndrome in prolonged critical illness. METHODS: Healthy rabbits were compared with prolonged critically ill, parenterally fed animals. We visualized TRH mRNA in the hypothalamus by in situ-hybridization and measured mRNA levels for the type II iodothyronine diodinase (D2), the thyroid hormone transporters monocarboxylate transporter (MCT) 8, MCT10 and organic anion co-transporting polypeptide 1C1 (OATP1C1) and the thyroid hormone receptors alpha (TRalpha) and beta (TRbeta) in the hypothalamus. We also measured the activity of the D2 and type III iodothyronine deiodinase (D3) enzymes. RESULTS: In the hypothalamus of prolonged critically ill rabbits with low circulating T3 and TSH, we observed decreased TRH mRNA, increased D2 mRNA and increased MCT10 and OATP1C1 mRNA while MCT8 gene expression was unaltered as compared with healthy controls. This coincided with low hypothalamic thyroxine (T4) and low-normal T3 concentrations, without a change at the thyroid hormone receptor level. CONCLUSIONS: Although expression of D2 and of the thyroid hormone transporters MCT10 and OATP1C1 were increased in the hypothalamus of prolonged critical ill animals, hypothalamic T4 and T3 content or thyroid hormone receptor expression were not elevated. Hence, decreased TRH gene expression, and hereby low TSH and T3 during prolonged critical illness, is not exclusively brought about by hypothalamic thyrotoxicosis, and infer other TRH suppressing factors to play a role.

[Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland].

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.

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.

The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities.

Different haloperoxidases, one specific for the oxidation of iodide and another that can oxidize both iodide and bromide, were separated from the sporophytes of the brown alga Laminaria digitata and purified to electrophoretic homogeneity. The iodoperoxidase activity was approximately seven times more efficient than the bromoperoxidase fraction in the oxidation of iodide. The two enzymes were markedly different in their molecular masses, trypsin digestion profiles, and immunological characteristics. Also, in contrast to the iodoperoxidase, bromoperoxidases were present in the form of multimeric aggregates of near-identical proteins. Two full-length haloperoxidase cDNAs were isolated from L. digitata, using haloperoxidase partial cDNAs that had been identified previously in an Expressed Sequence Tag analysis of the life cycle of this species (1). Sequence comparisons, mass spectrometry, and immunological analyses of the purified bromoperoxidase, as well as the activity of the protein expressed in Escherichia coli, all indicate that these almost identical cDNAs encode bromoperoxidases. Haloperoxidases form a large multigenic family in L. digitata, and the potential functions of haloperoxidases in this kelp are discussed.

Vanadium haloperoxidases from brown algae of the Laminariaceae family.

Vanadium haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae--Laminaria saccharina (Linné) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40 degrees C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis-Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region.

Purification and characterisation of vanadium haloperoxidases from the brown alga Pelvetia canaliculata.

Two enzymes characterised as iodoperoxidases (PcI and PcII), with vanadium-dependent activity, have been purified from the brown alga Pelvetia canaliculata (L.) Decne et Thur. (Fucaceae, Phaeophyceae), collected in the Northern Portuguese coast, at Viana do Castelo. The relative molecular masses were 166 kDa for PcI and 416 kDa for PcII, as determined by gel filtration. SDS-PAGE shows that PcI has just one band corresponding to a subunit of 66 kDa, while PcII shows four bands (66, 72, 157 and 280 kDa). The following kinetic parameters have been determined from a steady-state analysis of the oxidation of iodide by H2O2: PcI, pHopt = 6.0, KM(I-) = 2.1 mM, KM(H2O2) = 110 microM, Ki(I-) = 127 mM; and PcII, pHopt = 6.5, KM(I-) = 2.4 mM, KM(H2O2) = 20 microM and Ki(I-) = 69 mM. These iodoperoxidases are thermostable, as also observed for vanadium bromo- and chloroperoxidases.

The greater glycan content of recombinant human thyroid peroxidase of mammalian than of insect cell origin facilitates purification to homogeneity of enzymatically protein remaining soluble at high concentration.

Structural studies on thyroid peroxidase (TPO), a major thyroid autoantigen, require milligram amounts of pure protein. We found that the human TPO ectodomain (amino acid residues 1-848) generated in insect cells did not remain in solution at high concentrations after affinity purification. In contrast, the TPO ectodomain secreted by mammalian (Chinese hamster ovary) cells, although generated to a lesser extent (1 vs. 8 mg/liter), remained in solution at high concentration (10 mg/ml) after purification to homogeneity. This purified material was well recognized by TPO autoantibodies, but lacked enzymatic activity. We attempted to restore activity by culturing the Chinese hamster ovary cells in the presence of added heme. TPO enzymatic activity was clearly detected in conditioned medium from cells cultured in hematin and hemin, but not in protoporphyrin IX (all at 1 mg/liter). Heme prosthetic group incorporation into affinity-purified TPO was highest for hematin and hemin, but unchanged for protoporphyrin IX (OD 410/280 nm ratios of 0.25, 0.23, and 0.14, respectively). Enzymatic activity was now evident with hemin (mean +/- SE, 27.2 +/- 2.6; n = 3; guaiacol units/mg protein), hematin (24.1 +/- 1.6), and, to a lesser extent, protoporphyrin IX (3.6 +/- 0.2). Culturing cells in 20 mg/liter hematin, the maximum concentration tolerated, increased enzymatic activity even further (45.6 +/- 0.6 guaiacol units/mg protein). All purified TPO preparations were homogeneous on PAGE and of similar size (105 kDa). Enzymatic deglycosylation showed a complex carbohydrate contribution of 13 kDa (unlike the 2.3 kDa in insect cell TPO). In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike TPO generated in insect cells, mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO may be useful for future structural studies.

Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves' thyroid tissue.

Milligram quantities of the human membrane autoantigen thyroid peroxidase (TPO) have been purified to a high degree of homogeneity by a combination of detergent solubilisation, monoclonal antibody affinity, and ion exchange chromatography, from pooled Graves' disease thyroid glands. The purified TPO of greater than 90% purity was enzymatically active as judged by its ability to oxidise guaiacol. Crystals of TPO have been grown from solutions of the protein solubilised in sodium deoxycholate, in the presence of ammonium sulphate. The crystals exhibited birefringence under polarised light, indicative of molecular order. Crystallisation of this large, membrane autoantigen represents the first step in delineating the complete three-dimensional structure of a human autoantigen involved in destructive thyroiditis.

Purification and characterization of a recombinant human thyroid peroxidase expressed in insect cells.

Thyroid peroxidase (TPO) is an essential enzyme for thyroid hormone biosynthesis and is an autoantigen against which antibodies are found in a number of autoimmune thyroid disorders. Large quantities of pure TPO are essential for understanding its structure and role in normal thyroid function and thyroid diseases. In this study, we describe the production of human TPO (hTPO) using a baculovirus expression vector in insect cells. TPO was sequentially extracted from insect cells using various buffers and the protein was purified to homogeneity on a C4 reversed-phase semipreparative column using high-performance liquid chromatography. The purified protein was identified as hTPO by enzyme-linked immunosorbent assay, Western blot, and amino acid sequence analyses. Carbohydrate analysis of the recombinant hTPO showed that the protein is glycosylated and mannose is the major oligosaccharide. We have extended the carbohydrate analysis by establishing the occurrence of N-acetyl galactosamine which suggested that the recombinant hTPO might contain O-glycosyl moieties. Purified hTPO reacted specifically with sera from patients with Hashimoto's thyroiditis. Crude as well as purified hTPO did not show any enzymatic activity when produced in Sf9 insect cells grown in serum free medium. In contrast, hTPO produced in the presence of 10% fetal bovine serum containing 1 microgram/ml of haematin was enzymatically active. However, the enzymatic activity of the recombinant hTPO was lower than that often found with hTPO purified from thyroid tissue. Availability of purified hTPO in relatively large quantities should allow further structural and immunological studies.

Type 3 lodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme.

Type 3 iodothyronine deiodinase (D3) catalyzes the conversion of T4 and T3 to inactive metabolites. It is highly expressed in placenta and thus can regulate circulating fetal thyroid hormone concentrations throughout gestation. We have cloned and expressed a 2.1-kb human placental D3 cDNA which encodes a 32-kD protein with a Km of 1.2 nM for 5 deiodination of T3 and 340 nM for 5' deiodination of reverse T3. The reaction requires DTT and is not inhibited by 6n-propylthiouracil. We quantitated transiently expressed D3 by specifically labeling the protein with bromoacetyl [125I]T3. The Kcat/Km ratio for 5 deiodination of T3 was over 1,000-fold that for 5' deiodination of reverse T3. Human D3 is a selenoenzyme as evidenced by (a) the presence of an in frame UGA codon at position 144, (b) the synthesis of a 32-kD 75Se-labeled protein in D3 cDNA transfected cells, and (c) the presence of a selenocysteine insertion sequence element in the 3' untranslated region of the mRNA which is required for its expression. The D3 selenocysteine insertion sequence element is more potent than that in the type 1 deiodinase or glutathione peroxidase gene, suggesting a high priority for selenocysteine incorporation into this enzyme. The conservation of this enzyme from Xenopus laevis tadpoles to humans implies an essential role for regulation of thyroid hormone inactivation during embryological development.

Topological analysis of the integral membrane protein, type 1 iodothyronine deiodinase (D1).

Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34-39. A transiently expressed D1 protein in which residues 2-25 were deleted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.

Isolation of thyroid peroxidase and lack of autoantibodies to the enzyme in dogs with autoimmune thyroid disease.

Fifty serum samples from dogs with clinical signs of hypothyroidism and autoantibodies (AA) to thyroglobulin (Tg), thyroxine, or triiodothyronine were screened for AA to thyroid peroxidase (TPO). Thyroid peroxidase is the antigen against which microsomal AA are formed in human beings with lymphocytic thyroiditis. The TPO was isolated from canine thyroid tissue, using a modification of the procedure for purifying porcine TPO. The enzyme was solubilized from the membrane, using a deoxycholate-trypsin solution, followed by ammonium sulfate precipitation and diethylaminoethyl Sephadex chromatography. Activity of TPO was determined, using an iodide oxidation assay and a guaiacol assay. A monoclonal antibody to canine Tg, coupled to an immunoaffinity column, was used to eliminate the contaminating Tg from the TPO preparation. Using the TPO preparation as an antigen, an ELISA was performed on 10 serum samples and immunoblot assays were performed on 50 canine sera. Autoantibodies to TPO were not found in any of the sera. Assays also were performed, using purified porcine and human TPO and evidence of cross-reactivity with canine TPO was not identified. The absence of AA to TPO in dogs suggests a different pathogenesis for autoimmune thyroid disease in dogs than that hypothesized for lymphocytic thyroiditis in human beings.

Isolation of thyroid peroxidase from patients with Graves' disease and comparison with animal peroxidases.

1. Human thyroid peroxidase (TPO) was isolated from 280-640 g of pooled thyroid tissue resected from patients with Graves' disease. 2. Isolation was performed by an improved and simplified method. 3. The Reinheit Zahl (A412/A280) of the final preparations was in the range of 0.16-0.32. 4. The spectroscopic and enzymatic properties of Graves' TPO were compared with those of porcine TPO and bovine LPO, revealing closer resemblance to the former. 5. Graves' TPO may provide a useful substitute for normal TPO, which is very difficult to isolate.

Different thyroid hormone-deiodinating enzymes in tilapia (Oreochromis niloticus) liver and kidney.

Enzymes catalyzing the outer ring deiodination (ORD) of iodothyronines are important for the regulation of thyroid hormone bioactivity. We have studied ORD of thyroxine (T4) and 3,3',5'-triiodothyronine (rT3) in liver and kidney microsomes of fish, i.e. tilapia (Oreochromis niloticus). Tilapia kidney contains an enzyme which resembles the mammalian selenoenzyme type I iodothyronine deiodinase (ID-I) with respect to substrate preference (rT3 > T4) and high (approximately microM) Km values, but is much less sensitive to selenocysteine (Sec)-targeted inhibitors, including 6-propyl-2-thiouracil (PTU). In contrast, tilapia liver contains an enzyme very similar to mammalian type II deiodinase (ID-II) with respect to substrate preference (T4 > rT3), low (approximately nM) Km values, and lack of sensitivity to Sec inhibitors.

Iodothyronine 5'-deiodinase is present in mouse sublingual gland.

Homogenates from male ddy mouse sublingual glands were incubated with 100 nM [125I]rT3 in the presence of 2 mM dithiothreitol (DTT). Metabolites were analyzed by HPLC or Dowex-50 minicolumn assay. 3,3'-Diiodothyronine and I- were the only appreciable products. Iodide release from rT3 was compatible with the enzymatic nature. The Km and maximum velocity from three separate determinations were 326, 356, and 629 nM and 29.8, 42.1, and 31.3 pmol I- release/mg protein.min, respectively. The deiodinase activity was DTT dependent and had higher affinity for rT3 than for T4 or T3. 6-Propyl-2-thiouracil inhibited the deiodination, which was competitively overcome by DTT. Sublingual 5'-deiodinase activity was approximately 80% of that in the liver, while the submandibular gland showed no deiodination. Our results show the presence of 5'-deiodinase (type I) in mouse sublingual gland for the first time. Selective localization and abundance of the enzyme suggest a previously unrecognized role of the sublingual gland in thyroid hormone physiology.