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
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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 11

The Tg ChEL domain functions as a molecular escort.

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The Tg ChEL domain functions as a molecular escort. (A) 293 cells were u...
(A) 293 cells were untransfected or transiently cotransfected with 0.5 μg plasmid DNA encoding I-II-III plus 2.5 μg of the constructs indicated. The cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased in complete medium for 4 hours. Cell lysates and media were immunoprecipitated with anti-Tg and analyzed by SDS-PAGE and fluorography. Note that anti-Tg recognizes both I-II-III and the ChEL protein; the ChEL-KDEL construct is selectively retained intracellularly, while secretory ChEL is released to the medium. Intracellular retention of ChEL-KDEL causes a parallel retention of I-II-III. (B) Cells cotransfected and pulse labeled as in A were chased for 5 hours before analysis as above. Note that the secretory ChEL domain bearing the cog or rdw mutation cannot support the secretion of I-II-III. The positions of molecular mass markers are shown at left.