OR11-1 Thyroglobulin Structural Requirements in Protein Trafficking for Thyroid Hormonogenesis

In vertebrates, thyroglobulin (Tg) is secreted into the lumen of thyroid follicles to enable its iodination for the synthesis of thyroid hormone — defective Tg secretion causes hypothyroidism. A defining step in Tg secretion is export from the endoplasmic reticulum (ER), but Tg structural features needed to meet ER quality control requirements remain poorly defined. Tg primary structure is composed of Regions I, II, III, and ChEL, each engaged in intradomain disuflide bonding. Two major surprises that have recently emerged from the visualization of bovine Tg 3D-structure by cryoEM include the first identified long-range disulfide bond (C408-C608), which appears to tether a boundary separating two halves of region I, as well as the apparent absence of an evolutionarily conserved disulfide bond between C2444 and C2455 in the ChEL domain, which is known to support folding stability of region I. To understand the cell biological importance of these findings, we employed site-directed mutagenesis to determine the impact of these structural features in protein export of recombinant Tg expressed in cultured cells. To our surprise, we found that mutation of either a single Cys residue or both cysteines that contribute to the C408-C608 disulfide bridge had no discernible impact on the efficiency of Tg export. These data strongly suggest that the proximity of the two-halves of region I in the final Tg structure may be achieved by protein folding of individual repeating units of region I that must take place independently of (and thus presumably prior to) the formation of this interesting long-range disulfide bond. As ChEL supports Tg region I folding stability, we turned attention to the apparent absence of the C-2444-C2455 disulfide, which is one of the three disulfide bonds that had been thought to exist in every member of the large Cholinesterase-Like gene superfamily. Reported detection of this disulfide within human Tg, but lack of detection in bovine Tg, means either that this bond is 'structurally optional', or that this surface disulfide bond might have been broken by radiolysis under the X-ray beam used for cryoEM. Unequivocally, we found that mutagenesis of either C2444, C2455, or both, blocks Tg secretion and causes a pattern of intracellular misfolding consistent with incomplete Tg compaction indicative of lack of maturation of region I. Moreover, we found that disruption of these same residues causes misfolding and blocks secretion of an expressed isolated secretory ChEL domain, or acetylcholinesterase itself. Loss of the C2444-C2455 disulfide bridge behaves similarly to the defective folding observed in the mutant Tg encoded within cog/cog mice and rdw/rdw rats, whereas no pathogenic mutations have been reported affecting the long-range disulfide in Tg region I. Together, these results highlight the importance of ChEL domain structure in the maturation and secretion of Tg. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m.