SYNTHESIS OF TRI-IODOTHYRONINE (T3) WITHIN THYROGLOBULIN

CINTIA E. CITTERIO; PETER ARVAN

Ann Arbor, Michigan

Simposio; Brehm/Kellogg Trainee Symposium 2015; 2015

Kellogg Eye Center, University of Michigan.

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 in vertebrates involves monoiodotyrosine (MIT) and diiodotyrosine (DIT) formation by TG iodination, followed by DIT-DIT coupling to form T4 (an inactive form of thyroid hormone) or MIT-DIT coupling to form T3 (which, upon liberation to the bloodstream, is bioactive). TG has 76 tyrosine residues, and molecular basis for site selection for active thyroid hormone formation remains poorly understood ? nevertheless, the amino terminal region of TG appears evolutionarily designed for T4 formation, whereas the carboxyl terminal region of TG is enriched in T3. Importantly, for unknown reasons, in states in which thyroidal TSH receptors are highly activated, such as hypothyroidism caused by iodine deficiency (affecting two billion people globally) or hyperthyroidism caused by autoimmune (Graves?) disease, T3 is formed preferentially over T4 within the thyroid gland. In mouse TG (mTG), Y2519, Y2552, and Y2744 have been reported to engage in T3 formation; these residues fall within a region of TG known to be involved in homodimerization. We hypothesize that 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.We find that mutant TG-Y2744C forms a novel intermolecular disulfide bond, covalently crosslinking TG homodimers ? providing evidence that the side chains of residue 2744 from apposed TG monomers may intimately interact ? strongly implicating coupling between MIT-2744 and DIT-2744 in the formation of T3 within the TG homodimer. (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.) Upon in vitro iodination of recombinant TG, followed by SDS-PAGE and Western blotting with monoclonal anti-T3, we found that single TG-Y2744C and TG-Y2552C or TG-Y2519C mutants each exhibited diminished T3 formation. Remarkably, upon iodination of a double TG-Y2519C,Y2744C mutant, T3 formation was completely abolished. These data support the hypothesis that there are at least two primary sites of T3 formation at the carboxy-terminus of TG: an intermolecular coupling at Y2744 and an intramolecular coupling that appears to involve Y2519 and Y2552. Work is now needed to understand the relative contributions of these two sites in T3 formation during iodide deficiency, Graves´ disease, or in other conditions involving hyperstimulation of thyroidal TSH receptors.