7249 In Vivo Hormongenic Potential of the Thyroglobulin Cholinesterase-like (ChEL) Domain

Abstract Disclosure: C.E. Citterio: None. B. Morales-Rodriguez: None. P. Lee: None. R. Bhavana: None. M. Liu: None. D.P. Olson: None. X. Liao: None. S. Refetoff: None. P. Arvan: None. Vertebrate life is unsustainable without thyroid hormone. The only known precursor for thyroid hormone synthesis is thyroglobulin (Tg), composed of upstream regions I-II-III and a C-terminal cholinesterase-like (ChEL) domain. Tg protein, regulated by TSH, is secreted to the lumen of thyroid follicles, where thyroxine (T4) is formed primarily at the N-terminus, and triiodothyronine (T3) is formed primarily in the ChEL domain. Previous in vitro studies indicated that the isolated ChEL domain (following a signal peptide) is competent for dimerization, secretion, iodination, and T3 generation, but little to no T4 generation. However, the in vivo biological relevance of the C-terminal ChEL domain of Tg (in the absence of the remainder of the Tg molecule) is entirely unknown. Here, we used CRISPR/Cas9-mediated mutagenesis to create a large edition in the endogenous mouse Tg gene including all 5’ upstream regulatory elements yet encoding only the Tg-ChEL domain (Flag-tagged, immediately downstream of the endogenous signal peptide, and followed by a stop codon and strong polyadenylation signal). After confirming the mutagenesis, we bred the mice to homozygosity (and heterozygosity). We provide T4 supplementation to pregnant homozygotes and females with suckling pups but find that once the animals have weaned, the homozygous mutant mice live to adulthood without the need for thyroid hormone supplementation. We have begun initial characterization of these mice. At ∼3 months, serum T4 levels in homozygous mutant mice are below the lower limit of our assay (<1.7 µg/dL). In the setting of circulating T4 deficiency, the homozygous mice develop a goiter accompanied by high serum TSH levels (∼17,000 mU/L). Histological examination reveals significantly smaller thyroid follicles in homozygous mutant mice compared with the follicles of heterozygous controls (that exhibit a significantly lower TSH level, ∼2,000 mU/L). In homozygous mutant mice provided with T4 supplementation, immunofluorescence staining detects Flag-ChEL primarily within the thyroid follicular lumen, and biochemical analysis of thyroid lysates from these mice (by immunoblotting with anti-Flag antibodies) confirms the presence of complex (Golgi/post-Golgi modified) N-glycans, indicating that Flag-ChEL is competent for intracellular trafficking through the secretory pathway in thyrocytes in vivo. Additionally, by immunoblotting of thyroid lysates from the homozygous mice with anti-T3 mAb, we detect a T3-containing protein at the expected molecular weight of Flag-ChEL. This new mouse model establishes that in vivo the C-terminal ChEL domain of Tg is exported through the thyrocyte secretory pathway to the follicular lumen, where thyroid hormonogenesis takes place, and the mice make sufficient thyroid hormone to sustain life. Interestingly, the animals exhibit significant T4 deficiency, resulting in high TSH, and goiter. Presentation: 6/2/2024