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).

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.

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.

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.

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.

Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.

Obtención y caracterización de tiroperoxidasa/microsomal tiroideo / Obtention and characterization of tyroid tyroperoxidasa/microsomal

Se describe el procedimiento de aislamiento de la tiroperoxidasa humana(TPO)/ antígeno microsomal. La preparación de TPO se obtuvo de tiroides humanas de bocio tóxico-difuso (BTD), por medio de ultracentrifugación y la posterior solubilización con deoxicolato de sodio al 1 %. La concentración proteica obtenida fue de 0,1-0,3 mg/g de tejido. La obtención de TPO permitirá la validación de un inmunoensayo para la determinación de autoanticuerpos anti-TPO/antimicrosomal

Purification and partial characterization of iodothyronine 5'-deiodinase from rat liver microsomes.

We have isolated and purified iodothyronine 5'-deiodinase from rat liver microsomes to homogeneity as judged by PAGE and analytical HPLC. The enzyme progressively lost activity after solubilization, and specific activity enhancement was a modest 22-fold, but the final preparation still had substantial activity and was used for molecular characterization. The enzyme had an Mr of 56,000 with a single band in SDS-PAGE, suggesting absence of subunit structure. The high Km, and the GSH-responsive low Km, activities were co-purified, but the low Km enzyme lost GSH-responsiveness upon pretreatment with dithiothreitol (DTT) and urea. The enzyme was strongly inhibited by the iron chelator, alpha,alpha'-dipyridyl and showed a broad absorbance band at 410 nm. Spectral analysis with diethylpyrocarbonate (DEPC) revealed 5 histidine residues/mol enzyme, while enzyme activity was inhibited by DEPC in a pseudo-first order process with modification of 1 histidine residue/mol.

Studies with purified human thyroid peroxidase and thyroid microsomal autoantibodies.

We have isolated highly purified thyroid peroxidase (TPO) from human thyroid tissue to study further the relationship between TPO and the thyroid microsomal antigen that elicits the production of microsomal autoantibodies in patients with autoimmune thyroid disease. Serum samples were obtained from 24 patients with suspected autoimmune thyroid disease, and from 7 normal subjects. Microsomal autoantibodies in the patient sera, as determined by the microsomal hemagglutination assay (MCHA), varied between 1:100 and 1:102,400. Antithyroglobulin antibodies, however, were very low (less than 1:100). Binding of serum autoantibodies to purified human TPO, as determined by enzyme-linked immunosorbent assay, correlated fairly well with MCHA titers (r = 0.72; P less than 0.001). An immunoblot procedure was developed to study the binding of serum antibodies to the major active fragment of TPO (93 kDa), after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Binding under both conditions correlated very well with MCHA titers (r = 0.80-0.84; P less than 0.001). Studies were performed to determine the inhibitory effect of patient serum on the enzymatic activity of purified human TPO. A marked inhibitory effect on guaiacol activity was observed when TPO was preincubated with as little as 10 microL high titer serum. There was a significant correlation (r = 0.47; P less than 0.01) between MCHA titer and inhibitory effect. The addition of 2 micrograms purified human TPO completely or almost completely inhibited the binding of serum antibodies to thyroid microsomes (enzyme-linked immunosorbent assay) in 10 of 11 patient sera with high MCHA titers (1:25,600 or greater).

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells.

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.

Structure-activity analysis of microsomal antigen/thyroid peroxidase.

The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.

Purification of human thyroid peroxidase using ion exchange liquid chromatography.

We have utilized a one step ion-exchange (FPLC Mono Q) purification procedure for the isolation of human thyroid peroxidase (TPO). The purified TPO had the properties of a major microsomal antigen and inhibited the binding of human microsomal autoantibodies to thyroid microsomal membranes. The isolated TPO was free from thyroglobulin and showed, compared with crude microsomal proteins, a reduced background binding with control sera in enzyme-linked immunoassay (ELISA). SDS-gel electrophoresis of the isolated TPO detected one major band with an apparent molecular weight of 105 kD. The antigenicity of the protein was demonstrated by immunoblotting using sera from patients with autoimmune thyroiditis. These results demonstrate that FPLC Mono Q chromatography offers a rapid, quantitative and precise method for large scale purification of TPO with retained enzymatic and antigenic activity for use in ELISA and for further studies on the structure and function of this protein.

Solubilization of peroxidase from porcine thyroids and characterization by photoaffinity labelling.

Peroxidase was solubilized without proteolysis from porcine thyroid particulate fraction with the nonionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. The enzyme was able to catalyze the oxidation of guaiacol and the iodination of bovine serum albumin (33 atoms of iodine per molecule protein). Binding studies performed with the partially purified enzyme indicated that the substrates thyroxine (T4) and tyrosine compete for the same binding site on the enzyme. Dissociation constants of 0.9 nM and 0.5 nM were found for T4 and tyrosine, respectively. After photoaffinity labelling with underivatized 125I-labelled T4, gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 100 000 for the solubilized enzyme. The peroxidase activity and haem-absorbance peak coeluted from the Sephacryl S-1000 column. SDS-polyacrylamide gel electrophoresis under reducing conditions indicated two major radiolabelled polypeptides, Mr 83 000 and Mr 42 600, as well as a smaller peak at Mr 15 400. The 15 400 molecular weight species is probably not part of the peroxidase complex, since it could partially be removed by Sephadex G-25 prechromatography . Further analyses confirmed that the partially purified enzyme is a haemoprotein absorbing maximally at 412 nm. The Soret band is shifted to 423 nm by reducing agents and the haem-cyanide complex has a maximum absorbance at 416 nm.