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Revisiting Iodination Sites in Thyroglobulin with an Organ-oriented Shotgun Strategy

Open AccessPublished:October 26, 2010DOI:https://doi.org/10.1074/jbc.M110.159483
      Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.

      Introduction

      Thyroglobulin (Tg)
      The abbreviations used are: Tg
      thyroglobulin
      MIT
      monoiodotyrosine
      DIT
      diiodotyrosine
      T3
      triiodothyronine
      T4
      thyroxine
      endo-GluC
      endoproteinase GluC
      HCD
      higher energy collision dissociation
      CID
      collision-induced dissociation
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      is one of the most abundant proteins produced by the thyroid gland and comprises two identical subunits of ∼330 kDa. This prohormonal glycoprotein is secreted by thyroid epithelial cells. It is stored in the lumen of thyroid follicles in a highly condensed and covalently cross-linked form with numerous disulfide bridges. There, Tg is used as a scaffold for thyroid hormonogenesis and as an iodine reservoir. Many post-translational modifications are known to occur on this protein such as glycosylation, sulfation, and iodination (
      • Deshpande V.
      • Venkatesh S.G.
      ). A signal peptide is processed during its export to the lumen. The most specific Tg post-translational modification is certainly its iodination and 3,5,3′-triiodothyronine (T3) and thyroxine (T4) thyroid hormone synthesis. Indeed, through the combined action of NADPH oxidase and thyroperoxidase on the outer surface of the thyrocyte apical membrane, iodide ions that are also transported to the follicular lumen are covalently linked to some Tg tyrosyl residues to form mono- or diiodotyrosines (MIT or DIT, respectively). Coupling of two of these DIT residues then leads to the formation of T4 at an acceptor site, whereas coupling one MIT and one DIT moiety essentially forms T3 (
      • Taurog A.
      • Dorris M.L.
      • Doerge D.R.
      ). In this reaction, the release of an iodotyrosyl moiety at the donor site leaves an “empty” side chain and the fate of this unusual residue is controversial. Some authors claim that a dehydroalanine is left instead of the tyrosine, whereas others observed pyruvic acid associated with cleavage of the polypeptide chain (
      • Dunn A.D.
      • Corsi C.M.
      • Myers H.E.
      • Dunn J.T.
      ,
      • Gavaret J.M.
      • Cahnmann H.J.
      • Nunez J.
      ,
      • Gavaret J.M.
      • Nunez J.
      • Cahnmann H.J.
      ,
      • Gentile F.
      • Ferranti P.
      • Mamone G.
      • Malorni A.
      • Salvatore G.
      ,
      • Ohmiya Y.
      • Hayashi H.
      • Kondo T.
      • Kondo Y.
      ). Finally, the protein is proteolytically processed in the lumen, followed by endocytosis in lysosomes, to release the hormones. The principal proteases involved include the cathepsins (B, D, K, L, and S, with more or less specific roles ranging from solubilizing the highly cross-linked forms of Tg to releasing the hormone moiety), aminopeptidase N and dipeptidylpeptidases (
      • Brix K.
      • Linke M.
      • Tepel C.
      • Herzog V.
      ,
      • Dunn A.D.
      • Crutchfield H.E.
      • Dunn J.T.
      ,
      • Dunn A.D.
      • Crutchfield H.E.
      • Dunn J.T.
      ,
      • Friedrichs B.
      • Tepel C.
      • Reinheckel T.
      • Deussing J.
      • von Figura K.
      • Herzog V.
      • Peters C.
      • Saftig P.
      • Brix K.
      ,
      • Jordans S.
      • Jenko-Kokalj S.
      • Kühl N.M.
      • Tedelind S.
      • Sendt W.
      • Brömme D.
      • Turk D.
      • Brix K.
      ).
      Studies of the post-translational modification of Tg began in the 1950s using radioactive iodide. Researchers attributed the radioactivity to different Tg fractions according to their sedimentation coefficient or molecular weight on SDS-PAGE gel. Later, Tg fragments obtained by proteolysis with trypsin or other proteases were separated by reversed-phase HPLC. The amount of radioactivity and the chemical form of the iodinated residues (MIT, DIT, T3, or T4) were then analyzed (
      • Gavaret J.M.
      • Dème D.
      • Nunez J.
      • Salvatore G.
      ,
      • Lejeune P.J.
      • Marriq C.
      • Rolland M.
      • Lissitzky S.
      ,
      • Turner C.D.
      • Chernoff S.B.
      • Taurog A.
      • Rawitch A.B.
      ). Most of the studies were performed on purified Tg or Tg fragments from bovine, rat, guinea pig, rabbit, or human tissues, after in vitro chemical iodination or not. Significant progress was made in the 1980s when the first polypeptide sequence was reported for Bos taurus (
      • Mercken L.
      • Simons M.J.
      • Swillens S.
      • Massaer M.
      • Vassart G.
      ). The tyrosine residues involved were then identified more accurately using Edman amino acid sequencing and, later, mass spectrometry (MS) to identify the peptides. These experiments led to the establishment of a list of a number of iodinated tyrosine residues. Nonetheless, except for peptide identification, little has been done concerning MS-based characterization of tyrosine iodination. An initial attempt was made using purified (1237–1610) bovine Tg tryptic fragment (full sequence numbering). After endoproteinase Asp-N digestion, the resulting peptides were resolved by reversed-phase liquid chromatography and analyzed by electrospray and fast atom bombardment MS (
      • Gentile F.
      • Ferranti P.
      • Mamone G.
      • Malorni A.
      • Salvatore G.
      ). The post-translational modifications of all seven tyrosine residues on this fragment were characterized at an unprecedented level of resolution, highlighting Tyr1310 as a new T3 and T4 acceptor site, and unveiling a dehydroalanine at position 1394 as a donor site residue. After proteolysis and reversed-phase separation as well, Dunn and co-workers (
      • Dunn A.D.
      • Corsi C.M.
      • Myers H.E.
      • Dunn J.T.
      ) used MS to identify another donor site (Tyr149) on bovine Tg with the presence of pyruvic acid. Finally, in a more technical study in 2005, Salek and Lehmann (
      • Salek M.
      • Lehmann W.D.
      ) tackled the problem of iodotyrosine identification. They showed that peptides containing a mono- or diiodotyrosine residue generated specific markers during collisional-induced dissociation (CID) fragmentation.
      To study post-translational modifications of Tg in the thyroid and their heterogeneity, we proposed to analyze them using the most direct strategy possible, avoiding all purification steps and their inherent bias, and without the need for radioactivity. We dissected thyroid glands from mice and directly analyzed Tg using a shotgun-based MS/MS approach. We revealed as many tyrosine positions as possible by using a combination of different proteases and different MS/MS fragmentation procedures, involving analysis with inclusion lists, and using a high-resolution LTQ-Orbitrap XL hybrid mass spectrometer. We identified 37 modified tyrosine residues in mouse tissue, whereas only 16 sites are known for human Tg. Of these, we clearly identified 4 hormonogenic sites and two donor sites by identifying a pyruvic residue instead of the initial tyrosine. The analysis of cathepsin and dipeptidyl cleavage sites provided information of what happens in the cells. Our panoramic results bring new challenging data relative to Tg post-translational modifications.

      DISCUSSION

      Tg is a polypeptide matrix necessary for the synthesis of thyroid hormones. It is a very large glycoprotein consisting of two identical chains of 330 kDa. The numerous studies carried out so far have only led to a partial and indirect survey of its degree of iodination. Table 1 shows the positions of the iodinated tyrosines in both mouse and human Tg. A more general view of the status of Tg modification has been attempted through review work and many results have been deduced similarity with other established sequences, but were not always demonstrated (
      • Gentile F.
      • Ferranti P.
      • Mamone G.
      • Malorni A.
      • Salvatore G.
      ,
      • Dunn J.T.
      • Kim P.S.
      • Dunn A.D.
      • Heppner Jr., D.G.
      • Moore R.C.
      ,
      • Kim P.S.
      • Dunn J.T.
      • Kaiser D.L.
      ,
      • Marriq C.
      • Lejeune P.J.
      • Venot N.
      • Vinet L.
      ). Palumbo et al. (
      • Palumbo G.
      • Gentile F.
      • Condorelli G.L.
      • Salvatore G.
      ) noted that of the 140 tyrosines in the Tg dimer in rats, only 25–30 are normally iodinated and a much smaller number undergo coupling to form thyroid hormones (T3 and T4). Dunn et al. (
      • Dunn J.T.
      • Anderson P.C.
      • Fox J.W.
      • Fassler C.A.
      • Dunn A.D.
      • Hite L.A.
      • Moore R.C.
      ,
      • Dunn J.T.
      • Dunn A.D.
      ) mentioned the presence of four essential hormonogenic sites in rabbit Tg. Those sites, designated A, B, C, and D, were found either experimentally or by homology when the cDNA sequences were established (
      • Mercken L.
      • Simons M.J.
      • Swillens S.
      • Massaer M.
      • Vassart G.
      ,
      • Malthiéry Y.
      • Lissitzky S.
      ,
      • Musti A.M.
      • Avvedimento E.V.
      • Polistina C.
      • Ursini V.M.
      • Obici S.
      • Nitsch L.
      • Cocozza S.
      • Di Lauro R.
      ). These sites correspond to tyrosyl numbers 5, 2553, 2746, and 1290, respectively (this numbering applies to the mature Tg rabbit molecule, omits the peptide signal, and corresponds to positions 25, 2572, 2764, and 1310 in the mouse sequence with its presequence). Sites A and B are T4 and T3 sites; D is exclusively T4, and C mainly T3. As summarized in Table 1, our proteomic approach provides the broadest panoramic survey ever achieved using native Tg. The sequence coverage for Tg is very high (89% when iodination modifications are included). In the present study, we detected mouse equivalent hormonogenic sites A, B, and D. In our case, sites B and D carry T4 and T3, whereas site A carries only T4. By analogy with other Tg mammals, site C (Tyr2764, mouse numbering) is located at the C terminus. In our proteomic strategy, the SYSK peptide containing Tyr2764 generated by trypsin was too small to be detected by the mass spectrometer unless a limited proteolysis strategy was used. As far as triiodothyronine is concerned, we demonstrated its presence at positions Tyr2572 (site B), Tyr1310 (site D), and also at Tyr993 (mouse numbering) as mentioned in Table 1. This latter position has not previously been described as a hormonogenic site. It should be noted that we did not detect triiodothyronine at position 25 (site A).
      We have already mentioned that the sequence coverage obtained for Tg was very high. We wondered why some regions were not covered and noted that most of the undetected peptides carry a potential glycosylation site as predicted by bioinformatic tools based on homology with other species. The number of tyrosyl residues detected (71 of 76), particularly the number of mono- or diiodinated tyrosines, 36 and 24, respectively, is higher than had ever been found before (
      • Palumbo G.
      • Gentile F.
      • Condorelli G.L.
      • Salvatore G.
      ). If we consider that only a few positions (4 in our study) are involved in hormonogenesis we might think that the coupling process is highly regioselective and probably driven by constraints because of the native three-dimensional structure of Tg. The abundant iodination found in mouse Tg is probably a storage process for iodine, which can be mobilized when iodine intake becomes limited. More iodination sites were found in mouse Tg compared with those previously identified in the thyroids of larger mammals. This difference may be related to the more active thyroid metabolism in smaller animals or to the greater sensitivity of our methodology.
      Dunn et al. (
      • Dunn J.T.
      • Dunn A.D.
      ) proposed a classification of iodinated tyrosyl groups into three consensus families: ((D/E)Y), ((S/T)YS)), and (EXY) motives. (i) The ((D/E)Y) di-amino acid consensus was associated with T4 at sites A, B, and D. This is also the case in mouse Tg as supported by our results. It should be noted that the novel T3 site characterized here at residue 993 belongs to this EY consensus. Moreover, this consensus sequence is found iodinated but with apparently no hormone formation at four other locations Tyr383 (DY), Tyr1115 (EY), Tyr2486 (DY), and Tyr2586 (DY). (ii) The ((S/T)YS) tri-amino acid consensus was associated with triiodothyronine synthesis at site C. In the present study, we noticed that of the 36 mono- or 24 diiodotyrosines none could be linked to this consensus group because the sequence ((S/T)YS) is only present at position 2764 (mouse numbering). (iii) The consensus sequence (EXY) appeared to favor iodination. Of 8 possibilities of this consensus in mouse Tg, 6 are iodinated sites as shown here. We can increase this figure to 9 of 12 if we include an extended ((E/D)XY) consensus sequence. This ratio is significantly higher than the mean ratio of 36 iodinated positions of 71 detectable tyrosine residues. Our results confirmed that the presence of an acidic residue located two amino acids upstream of a tyrosine promotes iodination.
      Identifying donor tyrosyl residues has been a difficult challenge for years. At donor sites, the coupling process should leave dehydroalanine, which may be converted to alanine, pyruvic acid, or acetic acid during subsequent isolation steps (
      • Gavaret J.M.
      • Nunez J.
      • Cahnmann H.J.
      ,
      • Dunn J.T.
      ). Marriq et al. (
      • Marriq C.
      • Lejeune P.J.
      • Venot N.
      • Vinet L.
      ,
      • Marriq C.
      • Lejeune P.J.
      • Venot N.
      • Vinet L.
      ) first reported a cleavage in the peptide bond with the appearance of pyruvate instead of tyrosine. In 1997, Gentile et al. (
      • Gentile F.
      • Ferranti P.
      • Mamone G.
      • Malorni A.
      • Salvatore G.
      ) noted that the identification of donor tyrosyl residues was indirect in all the cases reported so far. In their study, they claimed to provide the first direct identification of tyrosine 1375 in mature bovine Tg (not conserved in humans or mice) as a donor residue. This result was obtained by taking into account the fact that tyrosine was converted into dehydroalanine. They used electrospray MS with 250–500 ppm precision. In 1998, Dunn et al. (
      • Dunn A.D.
      • Corsi C.M.
      • Myers H.E.
      • Dunn J.T.
      ) using also electrospray MS demonstrated a cleavage in the polypeptidic chain in bovine Tg, and identified a peptide in which Tyr130 (Tyr150 in mouse numbering) was replaced by pyruvate. In our large dataset, we were unable to detect any peptide displaying a dehydroalanine modification, whereas working to 5 ppm precision, even when the parent mass list was included. We identified two donor tyrosine residues at N-terminal and C-terminal fragments of the Tg molecule with peptide bond cleavage, and demonstrated the presence of pyruvic acid replacing the tyrosine residue. Our thyroid-wide proteomic analysis suggests that the residual side chain is converted to pyruvic acid in vivo or in vitro after iodotyrosyl transfer. We do not have any evidence that this cleavage occurs in the thyroid in vivo but we wonder whether the acidic conditions used during subsequent experimental manipulations were severe enough to break the peptide bond. In any case, detection of this cleavage event provides clear evidence for donor site identification.
      Although MS is widely used in the analysis of post-translational protein modifications (
      • Jensen O.N.
      ,
      • Küster B.
      • Mann M.
      ,
      • Witze E.S.
      • Old W.M.
      • Resing K.A.
      • Ahn N.G.
      ), it has rarely been used to identify iodinated tyrosyl residues in Tg and only on fragmented Tg (
      • Dunn A.D.
      • Corsi C.M.
      • Myers H.E.
      • Dunn J.T.
      ,
      • Gentile F.
      • Ferranti P.
      • Mamone G.
      • Malorni A.
      • Salvatore G.
      ,
      • Salek M.
      • Lehmann W.D.
      ). Here, we report the first detailed analysis of the entire Tg molecule from a direct, fresh extract of the mouse thyroid gland. Using this strategy, we obtained unambiguous characterization of 71 tyrosyl residues of this huge protein, confirmed the presence of three thyroxine sites and three triiodothyronine sites, and detected one new T3 site (Tyr993). From the same dataset, we were able to characterize two hormonogenic donor tyrosines at each end of the molecule. Based on spectral count, the ratio of the number of detected spectra for modified to unmodified peptides can be calculated for each modified tyrosine site. Because each peptide has its own ionization characteristics, this ratio is not very informative per se but it could become very enlightening when comparing Tg iodination levels under different conditions such as pathophysiological states. The use of isotope synthetic peptides carrying the different iodinated forms using a stable isotope dilution strategy (
      • Keshishian H.
      • Addona T.
      • Burgess M.
      • Kuhn E.
      • Carr S.A.
      ) could also be used to record the absolute quantity of each major modification.
      The release of thyroid hormones from the Tg prohormone requires the presence of different cathepsins, B, D, and L (
      • Dunn A.D.
      • Crutchfield H.E.
      • Dunn J.T.
      ). Friedrichs et al. (
      • Friedrichs B.
      • Tepel C.
      • Reinheckel T.
      • Deussing J.
      • von Figura K.
      • Herzog V.
      • Peters C.
      • Saftig P.
      • Brix K.
      ) noted that this rather complex process involves sequential proteolytic events with some interaction with different cathepsins, B, K, and L (
      • Tepel C.
      • Brömme D.
      • Herzog V.
      • Brix K.
      ). Covalently cross-linked Tg is stored in the thyroid follicular lumen where limited proteolysis occurs. Tg processing continues under intracellular conditions after Tg fragment internalization (
      • Jordans S.
      • Jenko-Kokalj S.
      • Kühl N.M.
      • Tedelind S.
      • Sendt W.
      • Brömme D.
      • Turk D.
      • Brix K.
      ). Our organ-oriented data were obtained from a complex mixture of partially or fully degraded Tg. Novel proteomic strategies, such as those developed to label N-terminal extremities for degradosome or proteogenomic studies (
      • Baudet M.
      • Ortet P.
      • Gaillard J.C.
      • Fernandez B.
      • Guerin P.
      • Enjalbal C.
      • Subra G.
      • de Groot A.
      • Barakat M.
      • Dedieu A.
      • Armengaud J.
      ), could be developed to follow the in vivo degradation process in thyroid follicules more accurately.
      In conclusion, in this study, we established the iodination status of Tg in mice. Our direct organ to mass spectrometer shotgun approach revealed a more comprehensive set of iodination sites in Tg. Our methodology may be applied to analyze whether potential alteration in the process of Tg iodination could be detected. This study could be carried out on thyroid extracts from various mouse models, such as Tg processing or iodine uptake using genetically modified mutants. Drugs that modify the Tg iodination status could also be investigated with this type of monitoring. The approach does not require large samples. It should be straightforward in application to human thyroid punctures, which could be important for clinical studies on iodination dysfunction.

      Acknowledgments

      We thank C. Bruley (CEA-Grenoble, iRTSV) for kindly providing the IRMa 1.21.0 parser, G. Imbert and O. Pible (CEA-Marcoule, iBEB) for help with data processing, and E. Quéméneur (CEA-Marcoule, iBEB) for continuous support.

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