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 2

Arrested disulfide maturation of Tg bearing a mutation in the ChEL domain.

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Oxidation state of secreted Tg. At each chase time, cells were lysed in ...
(A) 293 cells were transiently transfected with vector DNA encoding wild-type Tg (bearing a C-terminal myc-6xHis epitope tag that does not block Tg secretion; ref. 37) or empty vector (control [Con]). Transfected cells were then pulse labeled for 30 minutes with 35S-labeled amino acids and chased at 37°C for up to 4 hours in the absence or presence of BFA (5 μg/ml) where indicated. At each chase time, cells were lysed, immunoprecipitated with anti-Tg, and analyzed by nonreducing 4% SDS-PAGE and fluorography. A region of the gel enriched in disulfide-linked Tg adducts A, B, and C (28) is indicated. Two additional forms of Tg monomer — folding intermediate D and the E band representing fully oxidized mature Tg — are shown. (B) Results of an experiment identical to the one represented in A, except that the cells were chased at either 25°C (left) or 20°C (right). At each time, chase medium was also collected (as indicated), but at these temperatures the media contained no labeled Tg. (C) Results of an experiment identical to the one represented in A, except that cells were transfected with plasmid DNA encoding cog (L2263P) or rdw (G2300R) mutations within the Tg ChEL domain, and the cell lysis buffer included 20 mM N-ethylmaleimide. No detectable Tg was secreted, so analysis of the supernatant is not shown.