The structure of human thyroglobulin

In the paper

“Finally, with our ELISA we could also obtain modest T4 synthesis from random tyrosine copolymers, as previously reported4 (Extended Data Figure 6e).”

What the sources say

• Supported

  The Results and Discussion report thyroxine formation from a linear random tyrosine–lysine copolymer, with coupling yields of 11% using lactoperoxidase and 15% using thyroid peroxidase. The ELISA is the citing authors’ method, not a method attributed to this source. (LLM, conf=0.92)

  cites [4] Cahnmann, H. J., Pommier, J. & Nunez, J. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl Acad. Sci. USA 74, 5333–5335 (1977). source

In the paper

“TG, a protein dimer of 600 kDa, has an unusually high number of ~ 60 disulfide bonds per monomer and 17 glycosylation sites, which confer remarkable stability and solubility to the protein1,2,9.”

What the sources say

• Partly supported

  The Introduction reports about 60 disulfide bonds per Tg monomer, and section V identifies Tg as a homodimer, but gives its mass as 660 kDa rather than 600 kDa. The glycan-dependent oxidoreductase section reports 10–15 N-linked oligosaccharides per monomer, not 17; section VI says glycans generally increase protein solubility, but does not establish that the claimed structural features confer remarkable stability and solubility on Tg. (LLM, conf=0.90)

  cites [1] Di Jeso, B. & Arvan, P. Thyroglobulin from molecular and cellular biology to clinical endocrinology. Endocr. Rev. 37, 2–36 (2016). sourceExperimental — accuracy not yet measured
• Partly supported

  A Results passage in the indexed copy describes TG as a homodimeric glycoprotein, supporting the dimer description. The passages do not give a 600 kDa mass, approximately 60 disulfide bonds per monomer, 17 glycosylation sites, or their proposed effect on stability and solubility. The indexed text also appears to contain a different article from the cited review, so those details cannot be verified against it. (LLM, conf=0.83)

  cites [2] Citterio, C. E., Targovnik, H. M. & Arvan, P. The role of thyroglobulin in thyroid hormonogenesis. Nat. Rev. Endocrinol. 15, 323–338 (2019). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [9] Heidelberger, M. The molecular weight of thyroglobulin. Science 80, 414 (1934). source

In the paper

“hormonogenic tyrosines have not been clearly identified and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4.”

What the sources say

• Partly supported

  The introductory passage (page not supplied) says thyroglobulin’s primary sequence was unknown and that its complexity prevented study of factors governing coupling. This is consistent with hormonogenic tyrosines not being clearly identified, but the paper does not specifically attribute the mechanistic limitation to a lack of three-dimensional structure. (LLM, conf=0.82)

  cites [4] Cahnmann, H. J., Pommier, J. & Nunez, J. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl Acad. Sci. USA 74, 5333–5335 (1977). sourceExperimental — accuracy not yet measured

In the paper

“Approximately 5% of the world's human population suffer from thyroid diseases, but the molecular events behind TH synthesis are yet to be completely understood6.”

What the sources say

• ?Could not verify

  The Introduction discusses thyroid dysfunction and hormone measurement, but not whether the molecular events of thyroid hormone synthesis are fully understood. A search for synthesis found a reference to a drug inhibiting synthesis, not a statement about gaps in understanding; searches for biosynthesis and molecular mechanisms likewise did not establish the claim. The indexed passages provide no page numbers. (LLM, conf=0.74)

  cites [6] Taylor, P. N. et al. Global epidemiology of hyperthyroidism and hypothyroidism. Nat. Rev. Endocrinol. 14, 301–316 (2018). sourceExperimental — accuracy not yet measured

In the paper

“As shown in Figure 2c, when we mutated all acceptors (Y24, Y2573, Y2766 and Y1310)21,13 we observed no T4 synthesis, demonstrating that there are no more than four significant hormonogenic sites in TG.”

What the sources say

• ?Not checked — no full text of the sourcecites [21] Lamas, L., Anderson, P. C., Foxy, J. W. & Dunn, J. T. Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J. Biol. Chem. 264, 13541–13545 (1989). source
• Supported

  The citation follows the list of acceptor sites; the mutation experiment and its conclusion are presented as the citing authors’ own results. The Discussion (page unavailable) identifies four corresponding hormonogenic sites in mouse Tg, while the identification section reports T4 at three of them. The source uses mouse residue numbers rather than the citing sentence’s numbering. (LLM, conf=0.84)

  cites [13] Dedieu, A., Gaillard, J.-C., Pourcher, T., Darrouzet, E. & Armengaud, J. Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J. Biol. Chem. 286, 259–269 (2011). source

In the paper

“Most of TG's disulfide bonds show clear EM density in our maps (Extended Data Figure 5a) and we found no inter-subunit disulfide bonds, in agreement with previous observations that TG is a non-covalent dimer19.”

What the sources say

• Supported

  The paper’s introductory passage states that TG self-associates noncovalently into a dimer shortly after synthesis, supporting the point attached to citation 19. The EM density and absence of inter-subunit bonds in the citing authors’ maps are their own findings, not findings attributed to this source. The same passage notes that inter-monomer disulfide bonds can form later upon oxidation. (LLM, conf=0.90)

  cites [19] Citterio, C. E., Morishita, Y., Dakka, N., Veluswamy, B. & Arvan, P. Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine. J. Biol. Chem. 293, 4860–4869 (2018). source

In the paper

“Mass spectrometry analyses of thyroid-extracted TG have identified four or more acceptor tyrosines, but the position of the donors has remained unclear13.”

What the sources say

• Partly supported

  The Identification of Iodination Sites section identifies four distinct T3 or T4 acceptor positions: Tyr 25, 993, 1310, and 2572. But the Fate of the Lost Side Chain section identifies two donor positions, Tyr 259 and Tyr 2539, so the claim that donor positions remained unclear does not describe this study's findings. (LLM, conf=0.88)

  cites [13] Dedieu, A., Gaillard, J.-C., Pourcher, T., Darrouzet, E. & Armengaud, J. Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy. J. Biol. Chem. 286, 259–269 (2011). sourceExperimental — accuracy not yet measured

In the paper

“Resolved Sites A, B and D do not show obvious structural similarity and active tyrosines are in solvent accessible and flexible regions (Figure 2a, Extended Data Figure 7a,c).”

In the paper

“Furthermore, we showed that all acceptor mutants with one hormonogenic site active at a time (Figure 3a) contribute to hormone formation and the sum of the concentrations measured individually recapitulates the total T4 amount produced by unmodified rTG.”

In the paper

“When all five proposed donors were mutated, no significant T4 formation could be detected, confirming that Site A indeed has two donor tyrosines: Y234 and Y149.”

In the paper

“In the TG monomer, the globular NTD is connected to the Core region via a linker (residues 610-620), crossing the central dimer interface and it is partially flexible (Extended Data Figure 2f).”

In the paper

“The Core contains two triplets of type-1 repeats (domain H with a very large insertion), separated by domain I, located near the C2 axis.”

In the paper

“Lysozyme (some tyrosines), SaFtsZ (no tyrosines) and T3 produce no T4 signal.”

In the paper

“Using our reaction conditions, we could only detect significant production of T4, but not of T3 (Extended Data Figure 6f-g).”

In the paper

“The heme occupancy of TPO was estimated by measuring the ratio of absorbance at 412 nm and 280 nm which was 0.2, which roughly equates to 20% occupancy30.”

What the sources say

• ?Not checked — no full text of the sourcecites [30] Guo, J., McLachlan, S. M., Hutchison, S. & Rapoport, B. 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. Endocrinology 139, 999–1005 (1998). source

In the paper

“Because in Sites A, B and D the donor backbone conformation is constrained within secondary structure elements, whereas the acceptor is in more flexible regions, the acidic Asp or Glu residues (pointing towards the solvent) could play a role in orienting the following acceptor towards the more rigid donor.”

In the paper

“The Flap extends along the minor axis of the molecule with the M domain protruding at the opposite side of the NTD and folding back onto the Arm region.”

In the paper

“The central regions of the maps, corresponding to the C-terminal ChEL domains, were well defined (~3 Å in resolution), whereas peripheral regions corresponding to the Arms were noisy and at lower resolutions (~ 6 Å).”

In the paper

“We resolved 12 previously predicted N-linked GlcNac in our maps, and four that had not been identified, at N110, N484, N1869 and N212220 (Extended Data Table 3).”

What the sources say

• ?Not checked — no full text of the sourcecites [20] Yang, S.-X., Pollock, H. G. & Rawitch, A. B. Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin. Arch. Biochem. Biophys. 327, 61–70 (1996). source

In the paper

“Within the overall TG structure, Site A is located in the NTD, Site B in the CTD, Site C is at the C-terminus across the C2 dimer interface and Site D bridges the NTD and Flap (M domain) of the other subunit in the dimer (Figure 2b).”

In the paper

“Following the domain arrangement in the context of TG's 3D structure, we defined five TG regions: NTD (N-terminal domain), Core, Flap, Arm and CTD (C-terminal domain), as indicated in Figures 1a-b.”

In the paper

“We found it contains some T4, as measured by using our T4-ELISA, but not T3 (Extended Data Figure 6).”

In the paper

“The resolution of the map is non-uniform and consequently the model has variable quality depending on the map region.”

In the paper

“In cryo-EM, TG has a bilobed shape with average dimensions of 120 x 235 Å, which correlate well with previously reported negative staining EM data15.”

What the sources say

• ?Not checked — no full text of the sourcecites [15] Berg, G., Björkman, U. & Ekholm, R. The structure of newly synthesized intracellular thyroglobulin molecules. Mol. Cell. Endocrinol. 20, 87–98 (1980). source

In the paper

“There is no preceding acidic residue in the C-terminal Site C, where both acceptor and donor (Y2766 from the two chains of the dimer) are in unstructured regions with higher intrinsic flexibility.”

In the paper

“Overall, TG's structure appears entangled and revolves around the central ChEL dimer that interacts with different regions of the Arm and the Core of the same chain and, via the E domain, with the NTD of the other chain.”

In the paper

“The Core is then connected to the Flap region, which is composed of two Ig-like domains, M and N.”

In the paper

“It is also partially degraded, as shown by SDS-PAGE (Extended Data Figure 2a).”

In the paper

“By mutagenesis of Site D, we showed that K1415 is not relevant, but D1309 is essential for hormonogenesis (Extended Data Figure 6d).”

In the paper

“LPO is as active as TPO, taking the reduced 20% heme content in our TPO into account.”

In the paper

“The best final maps we used for model building were the C2 map from the eTG dataset (overall 3.39 Å resolution) and the expanded map from the rTG dataset (overall 3.67 Å resolution).”

In the paper

“showing conformational disorder within the hormonogenic sites, making precise side chain placements difficult, but the backbone positions are resolved (rTG map).”

In the paper

“This yielded calibration curves similar to the ones provided by the manufacturer, and also showed good dynamic range (Extended Data Figure 6).”

In the paper

“Notably, the glycans linked to N2013 mediate the contact between the NTD and CTD and might contribute to the dimer stability (Extended Data Figure 5b & f, Supplementary Video 1).”

In the paper

“The C-ins1 variant, producing a putative tyrosine pair with Y341, also showed low activity, possibly because the added linker was designed to be too short for the two tyrosines to come into proximity.”

In the paper

“Therefore, TG likely contains four hormonogenic acceptor tyrosines and five donor tyrosines.”

In the paper

“We note that the involvement of glycans in TG dimer formation as revealed by the structure might explain the poor expression yields of thyroglobulin when produced in expression systems with reduced/altered glycosylation, such as insect cells, HEK293S GnT1-/- cells or HEK293T cells in the presence of kifunensine (data not shown).”

In the paper

“The solvent exposure of tyrosines is presumably compensated by the exceptional solubility and stability that allows TG to persist at high concentrations in the harsh environment of the colloid.”

In the paper

“Moreover, the complexity of the TG molecule might fulfil further important roles in endocytosis, regulation of the T3/T4 ratio, TG proteolytic processing and in the trafficking to lysosomes25,26.”

What the sources say

• ?Not checked — no full text of the sourcecites [25] Botta, R. et al. Sortilin is a putative postendocytic receptor of thyroglobulin. Endocrinology 150, 509–518 (2009). source
• ?Not checked — no full text of the sourcecites [26] Weber, J. et al. Interdependence of thyroglobulin processing and thyroid hormone export in the mouse thyroid gland. Eur. J. Cell Biol. 96, 440–456 (2017). source

In the paper

“Although the overall resolution of the expanded and re-centred map was very similar to the C2 counterpart, the local resolution and continuity of the density in the Arm region was significantly improved (Extended Data Figure 2e).”

In the paper

“We conclude that TG synthesises seven molecules of T4 per dimer, since each TG dimer contains two sites (one per monomer) of A, B and D and only one Site C.”

In the paper

“Molprobity score (percentile) | 1.85 (83rd)”

In the paper

“Nature has selected the complex and large scaffold of the TG dimer to synthesise only seven molecules of hormones, using a chemical reaction involving radicals that could be carried out by unfolded peptides or other less complex proteins.”

In the paper

“Although the reaction mechanism of T4 synthesis from polypeptide chains remains to be established in more detail, four features seem to be crucial for hormonogenesis: tyrosine pairs must be solvent exposed to be iodinated, in proximity and in roughly antiparallel orientation, and in highly mobile regions of the protein in order to allow the significant bond rearrangement resulting in T4 synthesis.”

In the paper

“We attribute this basal activity to the Y171/Y176 pair since it could be suppressed by introducing a Y171A mutation.”

In the paper

“The correct assignment of the polypeptide chain register was often helped by the presence of large aromatic side chains and disulfide bond pairs, as well as the presence of glycosylation sites at Asn residues”

In the paper

“eTG presumably contains already reacted tyrosine side chains.”

In the paper

“Residues 24 (acceptor Site A) to 29 were built tentatively into a map filtered to lower resolution using MAIN score map density modification procedure39, however their coordinates are only indicative and hence are included in the model as poly-alanine”

In the paper

“Disallowed (%) | 0”

In the paper

“Hormone synthesis from thyroglobulin (TG) occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines and is completed by TG proteolysis3.”

What the sources say

• ?Not checked — no full text of the sourcecites [3] Carvalho, D. P. & Dupuy, C. Thyroid hormone biosynthesis and release. Mol. Cell. Endocrinol. 458, 6–15 (2017). source

In the paper

“The low level of T4 production can be attributed to a lack of flexibility of the backbone near Y171 and Y176, and to their relative orientations, which is different to any of the sites in TG.”

In the paper

“Clashscore | 4.88”

In the paper

“A conserved lysine is found in proximity of the donor, and a conserved acidic residue (Glu or Asp) always precedes the acceptor 21.”

What the sources say

• ?Not checked — no full text of the sourcecites [21] Lamas, L., Anderson, P. C., Foxy, J. W. & Dunn, J. T. Consensus sequences for early iodination and hormonogenesis in human thyroglobulin. J. Biol. Chem. 264, 13541–13545 (1989). source

In the paper

“Identified crosslinked peptide candidates were filtered to an FDR of 2% on residue-pair-level using XiFDR47.”