DE NOVO SYNTHESIS OF ACTIVE THYROID HORMONE (T3) WITHIN THYROGLOBULIN

CINTIA E. CITTERIO; PETER ARVAN

Ann Arbor, Michigan

Simposio; 2015 Spring AGEP Research Symposium; 2015

Rackham Graduate School, University of Michigan, Estados Unidos

Although thyroid hormone is the smallest chemical hormone of the endocrine system, the precursor for hormone synthesis in the thyroid gland, thyroglobulin (TG), is a huge (2746 residue) homodimeric glycoprotein. Hormonogenesis involves monoiodotyrosine (MIT) and diiodotyrosine (DIT) formation by TG iodination, followed by DIT-DIT or MIT-DIT coupling to form T4 (an inactive form of thyroid hormone) or T3 (active) respectively. In vertebrates, the amino terminal region of TG appears specialized for T4 formation, and the carboxyl terminal region specialized for T3. But TG has 76 tyrosine residues, and the selection of sites for formation of active thyroid hormone is poorly understood. Moreover, in states of iodine deficiency (affecting two billion people globally, causing mental retardation), Graves? disease (autoimmune hyperthyroidism), and other states in which thyroidal TSH receptors are highly activated, T3 is formed preferentially within the thyroid gland, by an unknown mechanism. In mouse TG (mTG), Tyrosines 2519, 2552, and 2744 are implicated in T3 formation. We hypothesize that the coupling reaction(s) engaging these residues to form T3 include one specific intramolecular (intra-monomer) and another specific intermolecular (intra-dimer) TG side-chain pairing. To test this, we have sought to develop physiologically relevant assays to examine coupling in native endogenous or recombinant TG.We find that a full-length TG bearing a Y2744C substitution forms a de novo intermolecular disulfide bond, providing strong evidence that residue 2744 side chains from opposed TG monomers can closely interact, implicating a coupling between MIT-2744 and the cognate DIT-2744 of the apposing TG monomer in the formation of T3. By contrast, neither TG-Y2552C nor TG-Y2519C formed disulfide-linked dimers, indicating that these residues do not crosslink with their cognate residues in apposing TG monomers ? thus they may be implicated in intra-monomer T3 formation. To directly evaluate T3 formation, we developed an in vitro iodination procedure for recombinant TG, and detected de novo T3 formation by Western blotting with monoclonal anti-T3 antibodies. Using this, we found that iodination of either single TG-Y2552C or TG-Y2519C or TG-Y2744C mutants partially inhibited T3 formation, whereas in a double TG-Y2519C,Y2744C mutant, T3 formation was completely abolished. These data suggest support the hypothesis that Y2744 (intermolecular), and either Y2519 or Y2552 (intramolecular) represent the two unique primary sites of T3 formation in TG. More work is needed to understand the relative contributions of these two sites to T3 formation in states of iodide deficiency, Graves´ disease, or conditions of high circulating TSH levels.