De Novo Tri-iodothyronine (T3) Formation

CINTIA E. CITTERIO; BALAJI VELUSWAMY; NADA DAKKA; PETER ARVAN

Victoria, B.C.

Congreso; 87th Annual Meeting of the American Thyroid Association; 2017

Introduction: De novo thyroid hormonogenesis requires iodination and coupling of two Tyr residues within Thyroglobulin (TG) - a dimeric 660kDa secretory glycoprotein. The acceptor hormonogenic site is a diiodotyrosine(DIT) and the donor is a monoiodotyrosine (MIT) or DIT for T3 and T4 synthesis, respectively. In mouse TG (mTG),Y2519 and Y2552 have been proposed as T3 formation sites, and based on evolutionary conservation, Y2744 is alsoan expected T3 hormonogenic site. Y2744 is located close to the helices within the Cholinesterase-like (ChEL)domain involved in noncovalent TG homodimerization. Methods / Case Presentation: We hypothesize that de novo T3 formation involves not only intra-monomer MIT-DIT coupling, but also inter-monomer coupling within the TG homodimer. To confirm sites involved in T3 formation, we bioengineered recombinant Y>F mutants, performed non-radioactive enzymatic iodinations of TG, and monitored denovo T3 formation by immunoblotting with mAb anti-T3 in parallel with anti-TG Ab. To examine the potential for intermonomer side-chain coupling, we created Y>C mutants and an N-terminally tagged version of the TG ChEL region(Flag-ChEL).Results / Discussion: A triple Y>F TG mutant (2519/2552/2744) and a double mutant (2552/2744) significantly decreased de novo T3 formation in TG, while a control substitution TG-Y2566F was without effect. Interestingly, Flag-ChEL by itself did not exhibit efficient T3 formation, suggesting that structural features provided by upstream regions of TG facilitate this process. TG-Y2744C formed a novel intermolecular coupling (disulfide bond), indicating intimate interaction of the 2744 side chains from opposing monomers within the TG homodimer, whereas TG-Y2519C and TGY2552C(or even double mutants) were unable to form disulfide-linked dimers. Conclusions: Even the 3D structure of TG remains unknown, these studies suggest that the most evolutionarily conserved T3 formation site involves an MIT-DIT coupling of Y2744 by closely related TG monomers within the TG homodimer. Upstream regions also stabilize the TG homodimer, and this may account for how these regions assist in the process of new T3 formation, which occurs primarily within and around the TG ChEL domain.