Lymph node ratio independently associated with postoperative thyroglobulin levels in papillary thyroid cancer.

OBJECTIVES: To investigate the impact of the lymph node ratio (LNR) on postoperative thyroglobulin (Tg) levels in patients with papillary thyroid carcinoma (PTC). PATIENTS AND METHODS: This was a retrospective, cohort study. The association between clinicopathological variables and postoperative unstimulated Tg (uTg) levels, preablative-stimulated Tg (sTg) levels, and postablative unstimulated Tg levels was analysed. RESULTS: A total of 300 patients with PTC were identified. Multivariate logistic analysis showed that M classification (odds ratio [OR], 2.33; 95% confidence interval [CI], 1.62-3.34), and postoperative thyroid-stimulating hormone levels (OR, 1.01; 95% CI, 1.01-1.02) were independently associated with postoperative uTg levels. One hundred and sixteen patients underwent radioactive iodine (RAI) therapy. Multivariate analysis showed that LNR in the central neck (OR, 1.24; 95% CI, 1.02-1.51), LNR in the lateral neck (OR, 1.73; 95% CI, 1.09-2.77), RAI dose (OR, 1.43; 95% CI, 1.21-1.69), and M classification (OR, 1.79; 95% CI, 1.22-2.61) were independently associated with preablative sTg levels. Tumour size (OR, 1.01; 95% CI, 1.00-1.01), LNR in the central neck (OR, 1.28; 95% CI, 1.08-1.51), LNR in the lateral neck (OR, 1.66; 95% CI, 1.10-2.49), RAI dose (OR, 1.54; 95% CI, 1.34-1.79), and M classification (OR, 1.56; 95% CI, 1.12-2.19) were also independently associated with postablative uTg levels. CONCLUSION: LNR was independently associated with postoperative Tg levels in patients with PTC. Patients with high LNR were more likely to have incomplete biochemical responses after surgery.

Structural features of thyroglobulin linked to protein trafficking.

Thyroglobulin must pass endoplasmic reticulum (ER) quality control to become secreted for thyroid hormone synthesis. Defective thyroglobulin, blocked in trafficking, can cause hypothyroidism. Thyroglobulin is a large protein (~2750 residues) spanning regions I-II-III plus a C-terminal cholinesterase-like domain. The cholinesterase-like domain functions as an intramolecular chaperone for regions I-II-III, but the folding pathway leading to successful thyroglobulin trafficking remains largely unknown. Here, informed by the recent three-dimensional structure of thyroglobulin as determined by cryo-electron microscopy, we have bioengineered three novel classes of mutants yielding three entirely distinct quality control phenotypes. Specifically, upon expressing recombinant thyroglobulin, we find that first, mutations eliminating a disulfide bond enclosing a 200-amino acid loop in region I have surprisingly little impact on the ability of thyroglobulin to fold to a secretion-competent state. Next, we have identified a mutation on the surface of the cholinesterase-like domain that has no discernible effect on regional folding yet affects contact between distinct regions and thereby triggers impairment in the trafficking of full-length thyroglobulin. Finally, we have probed a conserved disulfide in the cholinesterase-like domain that interferes dramatically with local folding, and this defect then impacts on global folding, blocking the entire thyroglobulin in the ER. These data highlight variants with distinct effects on ER quality control, inhibiting domain-specific folding; folding via regional contact; neither; or both.

Serum thyroglobulin as a biomarker of iodine excess and thyroid disease occurrence in adults.

BACKGROUND: Thyroglobulin (Tg) is considered a sensitive indicator of iodine deficiency. However, the usefulness of Tg as a biomarker of excess iodine is uncertain. The present study aimed to determine the influence of different iodine intake on serum Tg levels, evaluate the influence of thyroid diseases on the distribution of Tg, and identify the factors that may affect Tg levels. METHODS: A cross-sectional survey with a total of 1208 adults was conducted in different water iodine areas in China. Urinary iodine concentration (UIC), water iodine concentration (WIC), serum Tg, thyroid-stimulating hormone (TSH), and thyroid antibodies were measured. The thyroid volumes and nodules were measured by B-scan ultrasound. RESULTS: Based on the WIC data, subjects were divided into three groups. Based on the median urinary iodine concentration (MUIC) data, the iodine levels were adequate, more than adequate, and excess for the WIC < 10 µg/L group, 10 µg/L ≤ WIC ≤ 100 µg/L g, and WIC > 100 µg/L groups, respectively. The median Tg was significantly higher in the excess iodine group than in the adequate iodine group and the more than adequate iodine group (14.6 µg/L vs.12.7 µg/L, P = 0.042; 14.6 µg/L vs.12.5 µg/L, P = 0.004). Multiple linear regression analysis showed that excess iodine intake, goitre, thyroid nodules, and hypothyroidism were significantly related to higher serum Tg levels. CONCLUSION: Serum Tg level can be a promising biomarker of excessive iodine intake, but other factors, especially the presence of thyroid disease, should be considered when using this parameter.

Site-specific N-glycosylation analysis of human thyroid thyroglobulin by mass spectrometry-based Glyco-analytical strategies.

Human thyroglobulin (Tg), which has many glycosylation sites, is an essential protein produced by the human thyroid glands. Although human Tg N-glycans play critical roles in the cellular events of the Thyroid gland, the site-specific distribution of glycan structures has not been studied in detail. This study aimed to profile human Tg N-glycosylation sites and their glycan contents by using high-throughput glyco-analytical strategies, including glycopeptide and glycan levels. The sulfated complex and hybrid type N-glycan species were determined by the analysis of the human Tg samples with HPLC-HILIC-FLD-MS/MS. It was found that all fucosylated N-glycans carried fucose residue on their N-glycan core structure. The human Tg was digested with multiple enzymes by applying both in-gel and in-solution protocols to enhance site-specific glycosylation analysis. In total, 17 out of 20 N-glycosylation sites were characterized. It was noticed that 6 N-glycosylation sites contain only high-mannose type glycans, while other regions include complex and hybrid type glycans. In addition, sulfated glycoform structures were detected at the glycopeptide level in glycosylation sites containing complex and hybrid type glycans. It is expected that the results obtained from this study will contribute to functional studies to be conducted on human Tg protein. BIOLOGICAL SIGNIFICANCE: N-glycans of human thyroglobulin modulate thyroid hormone synthesis both in vivo and in vitro. Therefore, a comprehensive analysis of the N-glycosylation sites of human thyroglobulin is essential to improve our understanding of the function of its N-glycans. The present research significantly expanded the knowledge regarding N-glycosylation profiles of human thyroid thyroglobulin protein. For instance, as highlighted here, sulfated N-glycan structures were characterized using comprehensive glyco-analytical strategies. N-glycan patterns for the sites Asn110, Asn1869, and Asn2122 were described for the first time in this current work. In addition, N-glycan structures containing core-fucosylation and bisecting types were confirmed for all determined glycosylation sites.

Cryo-EM structure of native human thyroglobulin.

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T3) and thyroxine (T4) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.

The structure of natively iodinated bovine thyroglobulin.

Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the thyroid gland, tyrosine residues within the protein become iodinated to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT). A subset of evolutionarily conserved pairs of DIT (and MIT) residues can then engage in oxidative coupling reactions that yield either thyroxine (T4; produced from coupling of a DIT `acceptor' with a DIT `donor') or triiodothyronine (T3; produced from coupling of a DIT acceptor with an MIT donor). Although multiple iodotyrosine residues have been identified as potential donors and acceptors, the specificity and structural context of the pairings (i.e. which donor is paired with which acceptor) have remained unclear. Here, single-particle cryogenic electron microscopy (cryoEM) was used to generate a high-resolution reconstruction of bovine thyroglobulin (2.3 Šresolution in the core region and 2.6 Šoverall), allowing the structural characterization of two post-reaction acceptor-donor pairs as well as tyrosine residues modified as MIT and DIT. A substantial spatial separation between donor Tyr149 and acceptor Tyr24 was observed, suggesting that for thyroxine synthesis significant peptide motion is required for coupling at the evolutionarily conserved thyroglobulin amino-terminus.

Curating the gnomAD database: Report of novel variants in the thyrogobulin gene using in silico bioinformatics algorithms.

Thyroglobulin (TG) is a large glycosylated protein of 2767 amino acids, secreted by the thyrocytes into the follicular lumen. It plays an essential role in the process of thyroid hormone synthesis. TG gene variants lead to permanent congenital hypothyroidism. In the present work, we report a detailed population and bioinformatic prediction analyses of the TG variants indexed in the Genome Aggregation Database (gnomAD). The results showed a clear predominance of nonsense variants in the European (Finnish), European (Non-Finnish) and Ashkenazi Jewish ethnic groups, whereas the splice site variants predominate in South Asian and African/African-American populations. In total, 282 novel TG variants were described (47 missense involving the wild-type cysteine residues, 177 missense located in the ChEL domain and 58 splice site variants) which were not reported in the literature and that would have deleterious effects in prediction programs. In the gnomAD population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:320. In conclusion, we provide an updated and curated reference source for the diagnosis of thyroid disease, mainly to congenital hypothyroidism due to TG deficiency. The identification and characterization of TG variants is undoubtedly a valuable approach to study the TG structure/function relations and an important tool for clinical diagnosis and genetic counseling.

Forensic Biochemical Markers to Evaluate the Agonal Period: A Literature Review.

Currently, forensic research is multidisciplinary with new methods and parameters useful to define the cause and time of death as well as survival/agony times. The identification of biochemical markers able to estimate agonal period has been studied by many forensic researchers. It is known that the estimation of agonal time in different types of death is not always easy, hence our interest in literature's data. The studies analyzed in this review confirm the important role of thanatobiochemistry for the estimation of survival times. Regardless of the death cause, the survival/agony time between the primary event and death influences markers concentrations in biological samples (e.g., blood, urine, cerebrospinal fluid). Different biomarkers can be used for qualitative evaluations in deaths with short and long agony (e.g., C-reactive protein, ferritin, GFAP, etc.). Instead, the quantitative interpretation showed limits due to the lack of reference cut-offs. Thanatobiochemistry is a useful tool to confirm what emerged from autopsies findings (macroscopic and histological analysis), but further studies are desirable to confirm the evidence emerging from our review of the literature.

Novel presentation of the c.1856A > G (p.Asp619Gly) TSHR gene-activating variant: relapsing hyperthyroidism in three subsequent generations manifesting in early childhood and an in vitro functional study.

BACKGROUND: Familial non-autoimmune hyperthyroidism is a rare disease caused by germline activating variants in the thyroid-stimulating hormone receptor (TSHR) gene. The c.1856A > G (p.Asp619Gly) pathogenic variant has been described in cases of toxic adenoma but never before, to our knowledge, in a case of familial non-autoimmune hyperthyroidism. PATIENT FINDINGS: A 3-year-old boy was admitted for acute gastroenteritis presenting with goiter and tall stature. Laboratory findings revealed peripheral hyperthyroidism and negativity for thyroid autoantibodies. Antithyroid drug treatment was effective, but relapses occurred shortly after attempts to decrease the drug dose. As the boy's father and paternal grandmother also experienced relapsing hyperthyroidism manifesting in early childhood, genetic testing of TSHR was indicated. The c.1856A > G (p.Asp619Gly) pathogenic variant was found in all three affected family members. Functional in vitro characterization of the variant verified that it enhances constitutional activation of the receptor, leading to increased production of cyclic adenosine monophosphate. Total thyroidectomy was indicated in the boy due to an unsatisfactory prognosis. Due to persistent positive thyroglobulin serum concentration, a diagnostic radioiodine scan was performed approximately 2 years later. Residual thyroid tissue was revealed; therefore, radioiodine ablative therapy was performed. Despite adequate thyroxine substitution over a long period of follow-up, TSH remained suppressed. CONCLUSIONS: Unlike Graves' disease, familial non-autoimmune hyperthyroidism cases present with antithyroid drug-dependence. Not ultrasound but positive thyroglobulin serum concentration indicated residual thyroid tissue. Early detection of residual thyroid tissue and radioiodine ablation prevented the subject from experiencing relapsing hyperthyroidism and undergoing unnecessary repeated surgery. Life-long hormone substitution should be adjusted to free thyroxine rather than TSH serum concentrations.

Cryo-EM: A new dawn in thyroid biology.

The thyroid gland accumulates the rare dietary element iodine and incorporates it into iodinated thyroid hormones, utilising several tightly regulated reactions and molecular mechanisms. Thyroid hormones are essential in vertebrates and play a central role in many biological processes, such as development, thermogenesis and growth. The control of these functions is exerted through the binding of hormones to nuclear thyroid hormone receptors that rule the transcription of numerous metabolic genes. Over the last 50 years, thyroid biology has been studied extensively at the cellular and organismal levels, revealing its multiple clinical implications, yet, a complete molecular understanding is still lacking. This includes the atomic structures of crucial pathway components that would be needed to elucidate molecular mechanisms. Here we review the currently known protein structures involved in thyroid hormone synthesis, regulation, transport, and actions. We also highlight targets for future investigations that will significantly benefit from recent advances in macromolecular structure determination by electron cryo-microscopy (cryo-EM). As an example, we demonstrate how cryo-EM was crucial to obtain the structure of the large thyroid hormone precursor protein, thyroglobulin. We discuss modern cryo-EM compared to other structure determination methods and how an integrated structural and cell biological approach will help filling the molecular knowledge gap in our understanding of thyroid hormone metabolism. Together with clinical, cellular and high-throughput 'omics' studies, atomic structures of thyroid components will provide an important framework to map disease mutations and to interpret and predict thyroid phenotypes.

Structure and genetic variants of thyroglobulin: Pathophysiological implications.

Thyroglobulin (TG) plays a main role in the biosynthesis of thyroid hormones (TH), and, thus, it is involved in a wide range of vital functions throughout the life cycle of all vertebrates. Deficiency of TH production due to TG genetic variants causes congenital hypothyroidism (CH), with devastating consequences such as intellectual disability and impaired growth if untreated. To this day, 229 variations in the human TG gene have been identified while the 3D structure of TG has recently appeared. Although TG deficiency is thought to be of autosomal recessive inheritance, the introduction of massive sequencing platforms led to the identification of a variety of monoallelic TG variants (combined with mutations in other thyroid gene products) opening new questions regarding the possibility of oligogenic inheritance of the disease. In this review we discuss remarkable advances in the understanding of the TG architecture and the pathophysiology of CH associated with TG defects, providing new insights for the management of congenital disorders as well as counseling benefits for families with a history of TG abnormalities. Moreover, we summarize relevant aspects of TH synthesis within TG and offer an updated analysis of animal and cellular models of TG deficiency for pathophysiological studies of thyroid dyshormonogenesis while highlighting perspectives for new investigations. All in all, even though there has been sustained progress in understanding the role of TG in thyroid pathophysiology during the past 50 years, functional characterization of TG variants remains an important area of study for future advancement in the field.

A novel mutation in intron 11 donor splice site, responsible of a rare genotype in thyroglobulin gene by altering the pre-mRNA splincing process. Cell expression and bioinformatic analysis.

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, two hundred twenty-seven variations of the TG gene have been identified in humans. Thyroid dyshormonogenesis due to TG gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. The purpose of the present study was to identify and characterize new variants in the TG gene. We report an Argentine patient with congenital hypothyroidism, enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, expression of chimeric minigenes as well as bioinformatics analysis were performed. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a c.3001+5G > A, whereas the paternal mutation consists of p.Arg296*. Minigen analysis of the variant c.3001+5A performed in HeLa, CV1 and Hek293T cell lines, showed a total lack of transcript expression. So, in order to validate that the loss of expression was caused by such variation, site-directed mutagenesis was performed on the mutated clone, which previously had a pSPL3 vector change, to give rise to a wild-type clone c.3001+5G, endorsing that the mutation c.3001+5G > A is the cause of the total lack of expression. In conclusion, we demonstrate that the c.3001+5G > A mutation causes a rare genotype, altering the splicing of the pre-mRNA. This work contributes to elucidating the molecular bases of TG defects associated with congenital hypothyroidism and expands our knowledge in relation to the pathologic roles of the position 5 in the donor splice site.

Structure and functional properties of Capto™ Core 700 core-shell particles.

Capto™ Core 700 is a core-shell chromatographic support with an adsorbing core contained within an inert shell layer designed to purify larger biomolecules and bioparticles in a flow-through mode. The present study aims to characterize the structure and functional properties of this resin using bovine serum albumin (BSA, Mr~65 kDa) and thyroglobulin (Tg, Mr~660 kDa) as model impurity proteins. The functionalized adsorbing core and the inert shell have the same fibrous structure typical of agarose-based beads. The resin average bead size is 90.7 µm with a range of 50-130 µm, the shell thickness is 4.18 µm with a range of 3-6 µm and a standard deviation of 0.55 µm, and the pore radius, obtained by inverse size exclusion chromatography, is 50.4 ± 1.3 nm. Both proteins present highly favorable binding isotherms with maximum binding capacities of 55 and 105 mg/mL of total bead volume for BSA and Tg, respectively. The addition of 500 mM NaCl reduces the binding capacity by less than 50%, showing the ability of the resin to operate at high salt conditions. For both proteins, the effective pore diffusivity in the core is smaller than in the shell due to additional hindrance by bound protein in the core area. Effective pore diffusivities values in the core are 1.6 × 10-7 and 0.16 × 10-7 cm2/s for BSA and Tg, respectively. The DBC10% at 2 min residence time are 24 and 2 mg/mL for BSA and Tg, respectively. This study provides qualitative and quantitative information about Capto™ Core 700 resin. This information could be used to predict and optimize the purification of large biomolecules and bioparticle in route to the establishment of more effective downstream processes.

Resuspending samples in analytical ultracentrifugation.

In analytical ultracentrifugation it is often very useful to resuspend samples in situ after sedimentation experiments for further investigation. This can be achieved by manually subjecting the entire sample cell assembly to gentle motion that causes the air bubble in the sample compartment to repeatedly move through the solution and thereby cause convection. Here we describe a cell mixing device that can accomplish the same through axial rotation and slow rocking motion. This cell mixer is low-cost, open-source, and can be easily assembled from readily available components. It can efficiently mix multiple sample cells side-by-side and may be used with various centerpiece designs.

The De Novo Detection of Anti-Thyroglobulin Antibodies and Differentiated Thyroid Cancer Recurrence.

Background: The prevalence and clinical significance of de novo detection of anti-thyroglobulin antibodies (TgAbs) during the follow-up of patients with differentiated thyroid cancer (DTC) is unknown. Methods: We utilized the National Thyroid Cancer Treatment Cooperative Study registry (1987-2012). Patients registered after 1996 (n = 3318) were analyzed. We identified 1545 subjects who had available TgAb status (TgAb cohort) between years 1996 and 2012, of whom 1325 were TgAb negative at first postoperative follow-up testing. From this initial TgAb-negative group, we excluded 513 patients: 423 patients who had less than 3 years of follow-up and/or fewer than three follow-up visits, 86 patients with persistent disease after initial treatment, and 4 patients with data entry errors. The remaining 812 patients were included for analysis, comprising the TgAb persistently negative group (defined as TgAb negative for at least 3 consecutive follow-up visits and at least 3 years of follow-up) (n = 772) and the de novo TgAb-positive group in whom TgAbs became detectable (n = 40). We then assessed whether de novo appearance of TgAb was associated with DTC structural recurrence by using the Kaplan-Meier method. Results: The de novo detection of TgAb occurred in 5% of DTC patients. Recurrence of DTC in the TgAb persistently negative group compared with the de novo TgAb-positive group did not differ significantly (9.6% vs. 15.0%, p = 0.23). Baseline characteristics, histology, history of radiation exposure, staging, and median duration of follow-up were similar between the two groups. Interestingly, in all six patients who suffered a recurrence in the de novo TgAb-positive group, the TgAbs were negative at the time of recurrence detection and became positive at a median of 2.1 (0.7-8.7) years after the structural recurrence. Conclusions: Utilizing a large North American DTC registry, we found the prevalence of de novo TgAb detection to be 5% among initially TgAb-negative patients. We did not find a statistically significant association between de novo TgAb development and DTC structural recurrence. Larger prospective studies are required to confirm these findings and further assess the significance of de novo TgAb detection in the follow-up of DTC.

Effect of Pressure Increase on Macromolecules' Adsorption in Ion Exchange Chromatography.

In this study a new method for evaluating the pressure effect on separations of oligonucleotides and proteins on an anion exchange column was developed. The pressure rise of up to 500 bar was attained by coupling restriction capillaries to the column outlet to minimize differences in pressure over the column. Using pH transient measurements it was demonstrated that no shift in ion exchange equilibria occurs due to a pressure increase. Results from isocratic and gradient separations of oligonucleotides (model compounds) were evaluated by stoichiometric displacement and linear gradient elution model, respectively. Both elution modes demonstrated that for smaller oligonucleotides the number of binding sites remained unchanged with pressure rise while an increase for large oligonucleotides was observed, indicating their alignment over the stationary phase. From the obtained model parameters and their pressure dependencies, a thermodynamic description was made and compared between the elution modes. A complementary pattern of a linear increase of partial molar volume change with a pressure rise was established. Furthermore, estimation of the pressure effect was performed for bovine serum albumin and thyroglobulin that required gradient separations. Again, a raise in binding site number was found with pressure increase. The partial molar volume changes of BSA and Tg at the maximal investigated pressure and minimal salt concentration were -31.6 and -34.4 cm3/mol, respectively, indicating a higher rigidity of Tg. The proposed approach provides an insight into the molecule deformation over a surface at high pressures under nondenaturing conditions. The information enables a more comprehensive UHPLC method development.

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.

The structure of human thyroglobulin.

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

p.L571P in the linker domain of rat thyroglobulin causes intracellular retention.

Thyroglobulin (TG), a large glycosylated protein secreted by thyrocytes into the thyroid follicular lumen, plays an essential role in thyroid hormone biosynthesis. Rattus norvegicus TG (rTG) is encoded by a large single copy gene, 186-kb long, located on chromosome 7 composed of 48 exons encoding a 8461-kb mRNA. Although the TG gene displays sequence variability, many missense mutations do not impose any adverse effect on the TG protein, whereas other nucleotide substitutions may affect its TG stability and/or TG intracellular trafficking. In order to gain a further understanding of the protein domains regulating its intracellular fate, we cloned a full-length cDNA from rTG into the pcDNA6/V5-His B expression vector. However, transient expression of the cDNA in HEK293T cells showed that the encoded protein was not a wild-type molecule, as it was unable to be secreted in the culture supernatant. Sequencing analyses revealed three random mutations, which accidentally emerged during the course of cloning: c.1712T>C [p.L571P] in the linker domain (amino acid positions 360 to 604), c.2027A>G [p.Q676R] in TG type 1-6 repeat and c.2720A>G [p.Q907R] in the TG type 1-7 repeat. Expression of cDNAs encoding a combination of two mutations [p.Q676R-p.Q907R], [p.L571P-p.Q907R] or [p.L571P-p.Q676R] indicated that any TG bearing the p.L571P substitution was trapped intracellularly. Indeed, we expressed the single point mutant p.L571P and confirmed that this point mutation was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Endo H analysis showed that the p.L571P mutant is completely sensitive to the enzyme, whereas the will-type TG acquires full N-glycan modifications in Golgi apparatus. This data suggest that the p.L571P mutant contains the mannose-type N-glycan, that was added at the first stage of glycosylation. Complex-type N-glycan formation in the Golgi apparatus does not occur, consistent with defective endoplasmic reticulum exit of the mutant TG. Moreover, predictive analysis of the 3D linker domain showed that the p.L571P mutation would result in a significant protein conformational change. In conclusion, our studies identified a novel amino acid residue within the linker domain of TG associated with its conformational maturation and intracellular trafficking.