The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone
Jaemin Lee, … , Bruno Di Jeso, Peter Arvan
Jaemin Lee, … , Bruno Di Jeso, Peter Arvan
Published July 1, 2008
Citation Information: J Clin Invest. 2008;118(8):2950-2958. https://doi.org/10.1172/JCI35164.
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Research Article Endocrinology

The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone

Abstract

Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein (“secretory ChEL”) was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism.

Authors

Jaemin Lee, Bruno Di Jeso, Peter Arvan

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Figure 1

Free Cys thiols in Tg.

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Arrested disulfide maturation of Tg bearing a mutation in the ChEL domai...
293 cells were transiently transfected with an expression vector encoding wild-type Tg, the rdw Tg mutant (G2300R), or empty vector. At 48 hours after transfection, cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased for 4 hours in the absence or presence of BFA (5 μg/ml) where indicated. At this time, chase media (lanes labeled “Tg secreted”) and cell lysates (all other lanes) were immunoprecipitated with anti-Tg. The immunoprecipitates were denatured in 2× SDS gel sample buffer lacking reducing agents and mock incubated or incubated with AMS (5 mM, 30°C for 1 hour). At the end of the incubation, samples were boiled in the presence of 20 mM DTT and analyzed by 4% SDS-PAGE and fluorography. A slowed mobility (shift up) of the Tg band after AMS is indicative of free reactive thiols in the Tg molecule that are not apparent in secreted, wild-type Tg.