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

Maturation of Thyroglobulin Protein Region I

“…Mutagenesis of Mouse Tg-cDNAs encoding secretory ChEL and truncated Tg regions I-II-III and secretory ChEL and ChEL-Myc have been described previously (17,25,26). Other constructs described in this study were made using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Inc.) using following the mutagenic primers paired with their complements: Tg-C175X (5Ј-GCCTGTCCAGGAACAGAAGC-TCATTTCCGAAGAGGACCTGTGATTTGATCTGATCC-3Ј); Tg-R277X (Myc-tagged, 5Ј-CTGGCAGATTTGAGCAGAA-GCTTATCTCTGAAGAAGACCTGTAGGCTGCCACCAGA-TTTG-3Ј); Tg-P279X (Myc-tagged, 5Ј-GGCAGATTTCGGTG-CGAGCAGAAGTTGATATCAGAAGAGGACCTTTAGACC-AGATTTGG-3Ј); Tg-G293X (Myc-tagged, 5Ј-GCCACCAGAT-TTGAGCAGAAGCTTATCTCTGAAGAAGACCTGTAGGC-TGCCACCAG-3Ј); Tg-A340X (5Ј-GGGCAGCCTCCATCTTG-CTAGTGAGATCAGTCATGTGCCTTGG-3Ј); Tg-C388S (5Ј-GGTGTCAGATCGGACACATCCTCCCCACCCAGAATCA-AGG-3Ј); Tg-C1245R (5Ј-TGCTGACTCTCCAGGCTACGAA-TCAGTGGCCC-3Ј); Tg-C1489S (5Ј-GGAGCTTTCAGCAAA-ACCCATTCTGTCACTGACTGTCAGAAGAATGAGG-3Ј); Tg-C1973S (5Ј-CCAATGGGTTCTTTGAGAGTGAGCGGCT-CTGTGACAGGG-3Ј); and Tg region I (Tg-A1435X, 5Ј-CCCAACCACCAGACAGGATTGACTGGGCTGTGTGAAA-TGCCC-3Ј).…”

“…For immunoprecipitation, anti-Tg antibody was incubated with cell or media samples overnight at 4°C, and the immunoprecipitate was recovered with protein A-agarose and washed three times in immunoprecipitation buffer. For alkylation, the immunoprecipitates were incubated with 5 mM AMS for 30 min at 30°C as described previously (26). All samples were finally boiled in SDS sample buffer with or without reducing agent (0.1 M dithiothreitol) as indicated, resolved by SDS-PAGE, and analyzed by fluorography or phosphorimaging.…”

“…After dissociation from ER oxidoreductases, Tg monomer folding proceeds during its residence in the ER, en route to final formation of roughly 60 intrachain disulfide bonds (16). This conformational maturation is easily perturbed (9) and is temperaturesensitive, tending to arrest at an immature folding intermediate known as the D-isoform (immature monomer) (26). Indeed, I-II-III contains the information needed to mature to the monomeric E-isoform (26), but features in ChEL (and not other cholinesterase superfamily members) are required to produce a stable E-isoform that is competent for export from the ER (29).…”

“…Intriguingly, ChEL domain mutants similarly impair upstream Tg folding because ChEL serves as an intramolecular chaperone for I-II-III (26). The mechanism involves stabilization of the oxidized E isoform of I-II-III, but other than requiring ChEL (26), nothing further is known about the region of Tg involved in this critical D-to-E transition.…”

Importance of molecular genetic analysis in the diagnosis and classification of congenital hypothyroidism

“…It is interesting to note that the mRNA mutants could represent adequate targets for nonsense-mediated mRNA decay (NMD) pathway, a mechanism that degrades selectively mRNAs that contain premature stop codons. It is well documented also that truncated TG devoid of the ACHE-like domain may cause TG retention in the ER and premature degradation [8]. In addition to NMD pathway and ER storage diseases as pathophysiological mechanisms in the generation of CH, these truncated mutants have impairment in thyroid hormone synthesis.…”