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 5

ER exit of Tg region I-II-III in the absence and presence of ChEL domain.

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ER exit of Tg region I-II-III in the absence and presence of ChEL domain...
(A) 293 cells were either untransfected (control) or transiently transfected with 2 μg plasmid DNA encoding either full-length wild-type mouse Tg or Tg region I-II-III. Cells were pulse labeled and chased and samples prepared and immunoprecipitated with anti-Tg as in Figure 4, with analysis by reducing 4% SDS-PAGE and fluorography. Arrows highlight secretion (or lack thereof) from cells to media. (B) The first lane represents untransfected control cells. The remaining cells transfected to express Tg region I-II-III were pulse labeled as in A and then lysed without or with 4 hours of chasing. Tg immunoprecipitates from cell lysates and media were either mock digested or digested with endoglycosidase H for 1 hour at 37°C, before reducing 4% SDS-PAGE and fluorography. Lanes were run contiguously but have been separated for clarity. The positions of molecular mass standards are shown at left. (C) Lower panels: 293 cells were transiently transfected with 0.5 μg plasmid DNA encoding I-II-III cotransfected with 2.5 μg of plasmid DNA encoding the secretory ChEL domain or untransfected controls. Cells were pulse labeled for 30 minutes with 35S-labeled amino acids and chased for the times indicated. At each chase time, the cells were lysed and both lysates and media immunoprecipitated with polyclonal anti-Tg to recover both I-II-III and secretory ChEL proteins, as revealed by reducing 5.5% SDS-PAGE and fluorography. The upper panel represents a negative control with identical transfection of I-II-III but without cotransfection of the secretory ChEL plasmid.