TRI-IODOTHYRONINE (T3) FORMATION WITHIN THYROGLOBULIN

CINTIA E. CITTERIO; PETER ARVAN

Lake Buena Vista, Florida

Congreso; 15th International Thyroid Congress and 85th Annual Meeting of the American Thyroid Association; 2015

American Thyroid Association

Introduction:The precursor for thyroid hormone synthesis, thyroglobulin (TG), is a large (2746 residue) homodimeric glycoprotein. Hormonogenesis involves monoiodotyrosine (MIT) and diiodotyrosine (DIT) formation by TG iodination; and DIT-DIT or MIT-DIT coupling to form thyroxine (T4) or tri-iodothyronine (T3), respectively. Evolutionary conservation at the N-terminal region of TG appears to be designed specifically for T4 formation (involving Y5), whereas the C-terminal region is enriched in T3. For reasons that remain unknown, in states in which thyroidal TSH receptors are highly activated (such as hypothyroidism caused by iodine deficiency, or hyperthyroidism of Graves' disease), T3 is formed preferentially over T4 within the thyroid gland. Methods:In mouse TG (mTG), Y2519, Y2552, and Y2744 have been suggested to engage in T3 formation (and similar residues are used in the TG of other vertebrates). Interestingly, these tyrosyl residues fall near a region known to be involved in TG homodimerization. We hypothesize that the coupling reaction(s) to form T3 within TG include both intra-monomer and inter-molecular side-chain pairing. To examine T3 formation, we have iodinated native endogenous TG, or recombinant wild-type or mutant TG, in vitro.Results/Discussion:We find that a mutant TG-Y2744C forms a novel intermolecular disulfide bond, covalently crosslinking TG homodimers providing evidence for close apposition of the 2744 side chains from two TG monomers. By contrast, neither TG-Y2552C nor TG-Y2519C forms disulfide-linked dimers, indicating that these residues do not crosslink with cognate residues in TG dimers. Upon in vitro iodination of recombinant TG, followed by Western blotting with monoclonal anti-T3, we find that single TG-Y2744C, TG-Y2552C or TG-Y2519C mutants exhibit diminished T3 formation. Remarkably, upon iodination of a double TG-Y2519C,Y2744C mutant, T3 formation is completely abolished. Conclusion:These data support the hypothesis that there are at least two primary sites of T3 formation at the C-terminus of TG. Work is now needed to understand the relative contributions of these two sites in T3 formation in pathological conditions involving hyperstimulation of thyroidal TSH receptors.