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 7

ChEL interaction improves recovery as well as secretion of Tg I-II-III.

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Oxidation state of secreted I-II-III. 293 cells were transfected either ...
(A) 293 cells were triply transfected with empty vector plus a plasmid encoding Tg region I-II-III (always 0.1 μg DNA per well) plus a plasmid encoding the secretory ChEL domain (at different DNA levels as shown). DNA in each transfection totaled 3.1 μg per well. Transfected cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased for 6 hours, at which time the cells were lysed, and both lysates and media were immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and fluorography, as shown. The position of a 76-kDa molecular mass marker is shown at left. The figure has been spliced at the position indicated by a black line (between lanes 2 and 3), but all data were derived from a single exposure of the same gel. (B) Cells were either untransfected or transfected with 0.5 μg plasmid DNA encoding I-II-III plus 2.5 μg of vector DNA or that encoding secretory ChEL. As a positive control, 2 μg of plasmid DNA encoding wild-type Tg was transfected in parallel. Cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased for either 0 or 16 hours, at which time the cells were lysed and both lysates and media immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and phosphorimaging, as shown. The intracellular band density at the 0 chase time was defined as 100%; based on this, the recovery of each band at 16 hours is shown. Total recovery of labeled I-II-III alone at 16 hours was approximately 35%, while total recovery of labeled I-II-III (cells plus media) in the presence of secretory ChEL was approximately 68%.