Thyroid Hormone in the Making

CINTIA E. CITTERIO; PETER ARVAN

Simposio; Brehm/Kellogg Symposium 2017; 2017

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 a DIT for T3 and T4 synthesis, respectively. In mouse TG (mTG), Y2519 and Y2552 have been proposed as T3 formation sites. Based on evolutionary conservation, Y2744 is also an expected T3 hormonogenic site in mTG. Y2744 is located close to the region within the Cholinesterase-like (ChEL) domain involved in noncovalent TG homodimerization.We hypothesize that de novo T3 formation involves not only intra-monomer coupling of MIT-DIT, but also inter-monomer coupling within the TG homodimer. To confirm the sites involved in T3 formation, we bioengineered recombinant Y>F mutants, performed non-radioactive enzymatic iodinations of TG and monitored de novo T3 formation by immunoblotting with mAb anti-T3 in parallel with anti-TG Ab. To examine the potential for inter-monomer side-chain coupling, we created Y>C mutants and an N-terminally tagged version of the TG ChEL region (Flag-ChEL).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 did not alter it. Interestingly, the 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 TG-Y2552C (or even double mutants) were unable to form disulfide-linked dimers.Even as the 3D structure of TG remains unknown, these studies suggest that upstream regions of TG provide structural features that facilitate de novo T3 formation which occurs primarily within and near its ChEL domain. Whereas iodotyrosines 2519 and 2552 may indeed be involved in intramolecular coupling, our data suggests that the most evolutionarily conserved T3 formation site involves an MIT-DIT coupling of Y2744 by closely opposed TG monomers within the TG homodimer.