Thyroid hormonogenesis. Identification of a sequence containing iodophenyl donor site(s) in calf thyroglobulin

The formation of dehydroalanine in thyroglobulin is the result of the side chain elimination of an iodophenyl group during the thyroid hormone formation from two iodotyrosyl residues. This amino acid is easily converted to labeled alanine (upon reduction with [3H] borohydride) or changed to labeled aspartic acid (upon addition of Na14CN and subsequent acid hydrolysis). The cleavage of the protein by CNBr produced many stainable electrophoretic bands, but the autoradiography indicated the presence of a much smaller number of radioactive species. Although three major species raised attention, because they could be all jointly labeled and were present in all preparations, only a species of 15,900 Da was fully studied. It was isolated and its sequence partially determined by Edman degradation. It was established that this species corresponded to the thyroglobulin fragment between methionines 2,432 and 2,578. This peptide contains two hormonogenic sites (positions 2,555 and 2,569) which are either tyrosyl residues or hormone residues arising from them, and five additional tyrosines all potentially involved as donor sites in the hormonogenesis. Upon treatment with N-chlorosuccinimide, the fragment was split into three smaller peptides of about 2,900, 8,500, and 4,600 Da containing 1, 2, and 2 tyrosyl residues, respectively. Only the 8,500-Da subfragment contained [3H]Ala. This finding strongly suggests that at least some of the tyrosines involved as donor sites in thyroid hormonogenesis are within this peptide and possibly map at positions 2,469 and/or 2,522. Moreover, at minimum levels of iodination, when thyroglobulin contains the lowest number of hormone molecules, dehydroalanine is mostly found in the 15,900-Da peptide.     

High‐resolution imaging and proteomics of peptide fragments by TOF‐SIMS

Thyroglobulin is an iodinated glycoprotein (m.w. 660 kD) required for the storage and formation of thyroid hormone. Thyroglobulin was digested by trypsin in distilled water and the resulting peptides were identified by TOF‐secondary ion mass spectrometry, using TFA as a matrix to catalyze the ionization of the peptides. Cryostate sections of pig thyroid glands were incubated with trypsin in distilled water, followed by deposition of TFA. The sections were analyzed with TOF‐secondary ion mass spectrometry, and the peptides formed were identified through comparison with the peptides of the thyroglobulin reference sample. The thyroglobulin fragments were localized in the thyroid follicle cells with a spatial resolution of 3 microns, a mass resolution m/Δm of >6000 and a mass accuracy of <60 ppm. The thyroglobulin was found localized heterogeneously in the follicle cells. The heterogeneity may be due to thyroglobulin synthesis, uptake and degradation or globules representing insoluble polymers of thyroglobulin considered to be a mechanism for storing hormone at high concentrations.     

Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.     

Cysteine proteinases mediate extracellular prohormone processing in the thyroid

Thyroglobulin, the precursor of thyroid hormones, is extracellularly stored in a highly condensed and covalently cross-linked form. Solublization of thyroglobulin is facilitated by cysteine proteinases like cathepsins B and K which are proteolytically active at the surface of thyroid epithelial cells. The cysteine proteinases mediate the processing of thyroglobulin by limited extracellular proteolysis at the apical plasma membrane, thereby rapidly liberating thyroxine. The trafficking of cysteine proteinases in thyroid epithelial cells includes their targeting to lysosomes where they become maturated before being transported to the apical plasma membrane and, thus, into the extracellular follicle lumen. We propose that thyroid stimulating hormone regulates extracellular proteolysis of thyroglobulin in that it enhances the rate of exocytosis of lysosomal proteins at the apical plasma membrane. Later, thyroid stimulating hormone upregulates thyroglobulin synthesis and its secretion into the follicle lumen for subsequent compaction by covalent cross-linking. Hence, cycles of thyroglobulin proteolysis and thyroglobulin deposition might result in the regulation of the size of the luminal content of thyroid follicles. We conclude that the biological significance of extracellularly acting cysteine proteinases of the thyroid is the rapid utilization of thyroglobulin for the maintenance of constant thyroid hormone levels in vertebrate organisms.     

Endocytosis of thyroglobulin is not mediated by mannose-6-phosphate receptors in thyrocytes. Evidence for low-affinity-binding sites operating in the uptake of thyroglobulin.

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate-(Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concentrations of thyroglobulin (20 microM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].     

Efficient thyroid hormone formation by in vitro iodination of a segment of rat thyroglobulin fused to Staphylococcal protein A.

A polypeptide of 224 amino acids from the C terminus of rat thyroglobulin fused to Staphylococcal protein A (TgC 224), containing 3 tyrosines which have been shown to be hormonogenic in vivo (Tyr-2555, -2569 and -2748), forms thyroid hormones with relatively high efficiency upon in vitro enzymatic iodination using, most likely, the hormonogenic Tyr-2555 and Tyr-2569. Acetylcholinesterase, which has sequence and structural homology with the C terminus of the thyroglobulin molecule and bovine serum albumin, used as control proteins, formed thyroid hormones with lower efficiency. These results validate our experimental approach to define the structural requirements for thyroid hormone formation using thyroglobulin fragments.     

The earliest site of iodination in thyroglobulin is residue number 5

In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The present work demonstrates that of nearly 70 tyrosyl residues present in rat thyroglobulin, there is one, residue number 5 from the NH2-terminal end, which has in vivo the highest affinity toward iodine, being the first one to be iodinated. In fact, when 6-(n-propyl)-2-thiouracil (PTU)-treated, iodine-deficient animals were injected with 125I and killed shortly after, we isolated from thyroid glands poorly iodinated thyroglobulin (about 1 iodine atom/thyroglobulin molecule), nearly 90% of the radioactivity of which was found as monoiodotyrosine. Although CNBr cleavage of this protein gave several fragments after gel electrophoresis only one of these, with apparent mass 27,000 Da, contained 125I. This fragment was isolated and fully characterized. Twelve cycles of automated Edman degradation were performed; the sequence found, i.e. N-I-F-E-X-Q-V-X-A-Q-X-L, indicated that the 27,000-Da fragment is the NH2 terminus of thyroglobulin. This portion of the polypeptide chain contains several tyrosyl residues which may well all be potentially involved in the early iodination of the protein. The observation that the removal of seven amino acids from the NH2 terminus is accompanied (at the fifth step) by the total disappearance of radioactivity in the resulting shortened peptide suggested that the fifth residue was the only one iodinated under these conditions. A second, more quantitative experiment was performed on thyroglobulin obtained from 6-(n-propyl)-2-thiouracil-treated animals whose death was postponed 24 h after the injection of 125I. In this case the radioactivity was found not only in a single CNBr fragment (27,000 Da) but also in other discrete species of lower molecular mass. The mixture of these peptides was subjected to seven steps of manual Edman degradation. Fragments before and after partial degradation were run in parallel on a polyacrylamide gel and the distribution of 125I compared. Besides some change in the background, the two profiles were identical except for the absence of the 27,000-Da species. This proves that all the 125I present in the 27,000-Da species was localized at the fifth residue, the same site at which the hormone molecule is preferentially synthesized under normal conditions. This result is not unexpected and is in accord with the known properties of thyroglobulin which has a polypeptide chain designed for efficient synthesis of the hormone even at low levels of iodination.     

H9c2 cardiomyoblasts produce thyroid hormone

Thyroid hormone acts on a wide range of tissues. In the cardiovascular system, thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Although some early reports in the literature suggested an unknown extrathyroidal source of thyroid hormone, it is currently thought to be produced exclusively in the thyroid gland, a highly specialized organ with the sole function of generating, storing, and secreting thyroid hormone. Whereas most of the proteins necessary for thyroid hormone synthesis are thought to be expressed exclusively in the thyroid gland, we now have found evidence that all of these proteins, i.e., thyroglobulin, DUOX1, DUOX2, the sodium-iodide symporter, pendrin, thyroid peroxidase, and thyroid-stimulating hormone receptor, are also expressed in cardiomyocytes. Furthermore, we found thyroglobulin to be transiently upregulated in an in vitro model of ischemia. When performing these experiments in the presence of 125 I, we found that 125 I was integrated into thyroglobulin and that under ischemia-like conditions the radioactive signal in thyroglobulin was reduced. Concomitantly we observed an increase of intracellularly produced, 125 I-labeled thyroid hormone. In conclusion, our findings demonstrate for the first time that cardiomyocytes produce thyroid hormone in a manner adapted to the cell's environment.     

Thyroid hormone synthesis and thyroglobulin iodination related to the peroxidase localization of oxidizing equivalents: studies with cytochrome c peroxidase and horseradish peroxidase

Cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP), when combined with a stoichiometric amount of H2O2, form stable compounds I which are known as FeIV Ro and FeIV o pi + structures, respectively. These compounds were assayed in the catalysis of thyroid hormone synthesis and the iodination reaction. As previously shown for the lactoperoxidase FeIV Ro compound, the CcP FeIV Ro compound was involved in the coupling and not in the iodination reactions. In contrast, the HRP FeIV o pi + compound catalyzed both iodination and hormone formation. The possible role of the different peroxidase-H2O2 compounds in the two sequential reactions, thyroglobulin iodination and thyroid hormone formation, is discussed.     

Congenital hypothyroidism, dwarfism, and hearing impairment caused by a missense mutation in the mouse dual oxidase 2 gene, Duox2

Dual oxidases generate the hydrogen peroxide needed by thyroid peroxidase for the incorporation of iodine into thyroglobulin, an essential step in thyroid hormone synthesis. Mutations in the human dual oxidase 2 gene, DUOX2, have been shown to underlie several cases of congenital hypothyroidism. We report here the first mouse Duox2 mutation, which provides a new genetic model for studying the specific function of DUOX2 in the thyroid gland and in other organ systems where it is hypothesized to play a role. We mapped the new spontaneous mouse mutation to chromosome 2 and identified it as a T>G base pair change in exon 16 of Duox2. The mutation changes a highly conserved valine to glycine at amino acid position 674 (V674G) and was named "thyroid dyshormonogenesis" (symbol thyd) to signify a defect in thyroid hormone synthesis. Thyroid glands of mutant mice are goitrous and contain few normal follicles, and anterior pituitaries are dysplastic. Serum T(4) in homozygotes is about one-tenth the level of controls and is accompanied by a more than 100-fold increase in TSH. The weight of adult mutant mice is approximately half that of littermate controls, and serum IGF-I is reduced. The cochleae of mutant mice exhibit abnormalities characteristic of hypothyroidism, including a delayed formation of the inner sulcus and tunnel of Corti and an abnormally thickened tectorial membrane. Hearing thresholds of adult mutant mice are on average 50-60 decibels (dB) above those of controls.     

False Positives in Thyroglobulin Determinations Due to the Presence of Heterophile Antibodies: An Underrecognized and Consequential Clinical Problem

ObjectiveTo report a case series of thyroid cancer patients in whom false positive results in immunometric assays for thyroglobulin (TgIMA) were caused by heterophilic antibody interference, describe the clinical scenario in which this interference should be suspected, and recommend methods to demonstrate the interference.MethodsThree patients with unexpectedly elevated thyroglobulin results (range, 1.6-75 ng/mL) were studied. In the first patient, thyroglobulin was elevated despite the presence of Tg antibody. In the second patient, suppressed thyroglobulin was higher than a recent stimulated thyroglobulin. In the third patient, thyroglobulin became detectable years after treatment and did not change after thyroid-stimulating hormone stimulation. TgIMA concentration determination was compared to determination by a mass spectrometry method (TgMS). Thyroglobulin was also remeasured after preabsorption with heterophile antibody blocking reagents and after serial dilutions.ResultsIn all cases, thyroglobulin was undetectable by TgMS. In 2 of 3 patients, dilutions provided nonlinear thyroglobulin results. After blocking agent preabsorption, thyroglobulin dropped by 35%, 45%, and 91% in the 3 samples.ConclusionFalse positive thyroglobulin concentrations from heterophilic antibody interference have significant impact on the management of thyroid cancer. Here we show that TgMS assays can be used to rule out heterophilic antibody interference. This interference should be suspected when a detectable thyroglobulin by TgIMA does not respond to thyroid-stimulating hormone or is discordant from the clinical assessment.     

Application of improved coupling assay method for peroxidase of diseased thyroids: report of three cases

Recently we have developed an assay method for peroxidase-catalyzed coupling of iodotyronine residues of thyroglobulin, which is applicable to human diseased thyroid tissues. In the present study, the assay method as well as usual peroxidase assay methods were applied to thyroids of three patients (No. 1: familial goiter with impaired thyroglobulin synthesis, No. 2: mild chronic thyroiditis, No. 3: dyshormonogenetic goiter) who showed organification of iodine with high TSH levels and low thyroid hormone levels in sera. In general, these patients showed relatively high activities measured by guaiacol oxidation assay, iodide oxidation and coupling assay compared with those of control thyroids. Iodothyronine content in thyroglobulin was very low except thyroxine in No. 2. These results indicate that factors other than peroxidase may be responsible for the cause of the hypothyroid state. The coupling assay method used here is therefore useful for the detection of the 'coupling defect' in patients in a hypothyroid state.     

Adaptive Activation of Thyroid Hormone and Energy Expenditure

The mechanisms by which thyroid hormone accelerates energy expenditure are poorly understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2 iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure during cold exposure in human newborns and other small mammals. Although BAT is not present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This article reviews current biological knowledge about D2 and adaptive T3 production and their roles in energy expenditure.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Hormone-containing peptides from normal and goiter human thyroglobulins

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.     

Thyroglobulin and the biosynthesis of thyroid hormones

Thyroglobulin (mol. wt. 660 kDa) is the specific protein of the thyroid gland in which are synthesized and stored the thyroid hormones (thyroxine and 3,5,3'-triiodothyronine). It is formed of equal-sized subunits (330 kDa) containing each identical polypeptide chains to which are associated two types of oligosaccharide units representing 8 to 10% by weight of the protein. The studies reported in this paper describe the presence in thyroglobulin of discrete hormonogenic sites. After chemical (CNBr) and enzymatic (trypsin and protease V8 of S. aureus) treatments of the protein, four different hormone-containing peptide segments have been isolated, purified and sequenced. They correspond to the hormonogenic tyrosine-containing sites of the protein. One tyrosine is located at 4 amino acid residues from the N-terminal asparagine of the chain and is a major site for thyroxine synthesis. Another one which represents the triiodothyronine site is situated 2 amino acids before the C-terminal lysine. Finally, two other sites, one of low affinity and the other of high affinity for iodine and thyroxine formation, are equally located in the C-terminal part of the chain. The hormone-forming regions localized at the opposite far ends of the thyroglobulin chain(s) likely represent zones more accessible to iodination and with a conformation suited for the coupling of iodotyrosine into iodothyronine residues and ultimately protease attack to release the free hormones into the circulation. The presence of hormonogenic sites of different affinities for iodine allows thyroglobulin to modulate adaptively its hormonogenic capacity to external iodine supply. The molecular mechanism of this process is still unknown.     

Peroxidase-catalyzed bromination of tyrosine, thyroglobulin, and bovine serum albumin: comparison of thyroid peroxidase and lactoperoxidase

A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, 10-500 microM 82Br-, tyrosine (150 microM), thyroglobulin (0.3 or 1 microM), or BSA (7.5 microM), and a source of H2O2. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 micrograms/ml), or added initially as a bolus (100 microM). With TPO, formation of organically bound 82Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of 82Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound 82Br. However, a surprisingly high percentage (20-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound 82Br as tetrabromothyronine. Peroxidase-catalyzed bromination probably depends on the oxidation of Br- to Br+ by the Compound I form of the enzyme. Since oxidation of Br- to Br+ requires a stronger oxidant than oxidation of I- to I+, our results suggest that Compound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In vivo experiments with rats on a low iodine diet injected with 82Br- showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br- in similar rats provided no evidence that Br- is a substrate for the iodide transport system of the thyroid.