The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

Lee, Jaemin; Wang, Xiaofan; Jeso, Bruno Di; Arvan, Peter

doi:10.1074/jbc.m806898200

Cited by 47 publications

(43 citation statements)

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“…We previously showed that ChEL can be folded and exported as an independent secretory protein (15), yet ChEL is strongly implicated in Tg conformational stability and dimerization, allowing for Tg export from the ER (18). If we wish to understand the mechanism for Tg maturation and export, we must dissect the way in which local domain-specific folding (and folding defects) impact(s) on regional interactions within the larger Tg polypeptide.…”

Section: Discussionmentioning

confidence: 99%

“…Mouse monoclonal anti-HA epitope was from Covance (Princeton, NJ). Rabbit polyclonal anti-Tg (containing antibodies against epitopes at both N-and C-terminal regions of the protein) has been described previously (17 Mutagenesis of Mouse Tg-cDNAs encoding secretory ChEL and truncated Tg regions I-II-III (L2169X) and secretory ChEL and ChEL-myc have been described previously (14,15,18). Other constructs described in this paper were made using the QuikChange site-directed mutagenesis kit (Stratagene) using following the mutagenic primers paired with their complements: Tg-Q2139X and Q2139X in secretory II-III (5Ј-GGT-TGTGAGATGTGTGTTCTACCCTGATATATAGTAATG-CATACACAGTCTACGGAGCC-3Ј); C1973S in secretory II-III-HA (5Ј-CCAATGGGTTCTTTGAGAGTGAGCGGCT-CTGTGACAGGG-3Ј); Tg-E1192X (5Ј-GGTAGGCACCCA-GCCTGCCTGTTAAAGCCCACAGTGTCCACTGCC-3Ј); Tg-A1435X (5Ј-CCCAACCACCAGACAGGATTGACTG-GGCTGTGTGAAATGCCC-3Ј); Tg-Q1509X (5Ј-CCAGA-ATGGTCAGTACTAAGCCAGCCAGAAGAACAGGG-3Ј).…”

Section: Methodsmentioning

confidence: 99%

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Repeat Motif-containing Regions within Thyroglobulin

Lee

Arvan

2011

Journal of Biological Chemistry

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Thyroglobulin (precursor for thyroid hormone synthesis) is a large secreted glycoprotein comprising contiguous region I (multiple type-1 repeating units engaging the first ϳ1,191 residues, followed by a ϳ245-residue hinge region), regions II-III (multiple type-2 and 3 repeating units, comprising ϳ720 residues), and the C-terminal cholinesterase-like (ChEL) domain (ϳ570 residues). A signal peptide attached to ChEL makes an independent secretory protein that binds to I-II-III, stabilizing it and rescuing the secretion of I-II-III that would otherwise be trapped in the endoplasmic reticulum (ER). In this study, we found that a signal peptide attached to regions II-III also makes for an efficient secretory protein that neither demonstrably interacts nor has its secretion enhanced by the presence of secretory ChEL. By contrast, region I, either with or without the hinge region, cannot be secreted on its own and remains in the ER where it is bound to ER chaperones BiP and GRP94. Whereas ChEL can rescue secretion of I-II-III, it can rescue I-II only very weakly, and region I not at all. Yet, ChEL begins to rescue region I in cells that also co-express secretory II-III. The data suggest that conformational maturation of region I is a limiting step in the thyroglobulin maturation process, and this step is facilitated by the presence of both regions II-III and ChEL. Mutations causing hypothyroidism might induce solely local/regional misfolding or may interfere more globally by impeding interactions between regions that are required for thyroglobulin secretion.In vertebrates, thyroid hormones are first created in the apical luminal cavity of follicles of the thyroid gland, upon iodination of the thyroglobulin (Tg) 3 protein (1, 2). It is the structure of Tg (3, 4) rather than the specificity of the peroxidase (5) that is thought to catalyze the coupling of di-iodotyrosyl residues 5 and 130 at the N terminus of Tg (6, 7), forming thyroxine within the Tg molecule. Even in the endostyle of protochordates (the earliest species in which thyroid hormone synthesis is known to occur), a Tg-like protein has been reported (8), whereas no other thyroidal proteins have yet been discovered to substitute effectively for Tg in this role.The primary sequence of Tg (ϳ2,746 residues in mouse Tg after signal peptide cleavage) comprises disulfide-rich contiguous regions: region I (multiple type-1 repeating units engaging the first ϳ1,191 residues followed by a ϳ245-residue hinge region); II-III (multiple type-2 and type-3 repeating units, spanning ϳ720 residues); and the C-terminal cholinesterase-like (ChEL) domain (ϳ570 residues). The genetic origins of these units are still being discovered, but the type-1 repeat is a structural motif that is appears in evolution as a protease inhibitor (9); the type-2 and type-3 repeats have limited homology to other proteins; whereas ChEL exhibits homology to acetylcholinesterase (10 -12). Because no crystallographic data are yet available, the precise physical relationships between the Tg regions remain ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Repeat Motif-containing Regions within Thyroglobulin

Lee

Arvan

2011

Journal of Biological Chemistry

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“…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).…”

Section: Methodsmentioning

confidence: 99%

“…Concomitant with resolution of these complexes, and thereafter, there is extensive intramolecular disulfide bond maturation within internal repeat domains of the molecule (16). The oxidation of Tg monomers culminates in a transition from the "D" to oxidized "E" isoform that precedes homodimerization (17), which helps to satisfy "ER quality control" requirements that control efficiency and rate of Tg export from the ER (18).…”

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confidence: 99%

Maturation of Thyroglobulin Protein Region I

Lee

Jeso

Arvan

2011

Journal of Biological Chemistry

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Background:We have investigated which portion of thyroglobulin is engaged in its final oxidative maturation. Results: We demonstrate that terminal oxidation of thyroglobulin is linked to region I of the protein. Conclusion:Final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I. Significance: The results can explain human goitrous hypothyroidism, especially that caused by Tg-C1245R.

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“…Type 1 repetitive units present in the TG sequence have been suggested to act as binders and reversible inhibitors of the proteases implicated in TG processing (Molina, Pau, & Granier, 1996). The ACHE-homology domain is essential for protein dimerization, which plays an important role in the normal conformational maturation and intracellular transport of TG (Lee, Wang, Di Jeso, & Arvan, 2009). Cysteine residues are required in the tertiary structure of TG (Lee, Wang, Di Jeso, & Arvan, 1993).…”

Section: Discussionmentioning

confidence: 99%

Clinical and Genetic Analysis of a Compound Heterozygous Mutation in the Thyroglobulin Gene in a Chinese Twin Family With Congenital Goiter and Hypothyroidism

Liu

Zhang

et al. 2012

Twin Res Hum Genet

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Mutations in the thyroglobulin (TG) gene, which has an estimated incidence of approximately 1 in 100,000 new-borns, cause autosomal recessive congenital hypothyroidism. The mutational spectrum of the TG gene and the phenotype–genotype correlations have not yet fully been established. We report a compound heterozygous mutation in the TG gene in a Chinese twin family with congenital goiter and hypothyroidism. We also describe the gene mutation associated with the genotype–phenotype of these children with congenital goiter and hypothyroidism. The whole coding sequence of the TG gene was analyzed by direct sequence, and the identified changes in the sequence were tested for benign polymorphism by denaturing high-performance liquid chromatography screening of the mutation and sequencing 200 chromosomes from normal controls. Analysis of the TG gene of the affected twin revealed a compound heterozygous mutation, including a novel missense mutation G2687A, which is predicted to result in a glutamine to arginine substitution at codon 877, and a known nonsense mutation C7006T, predicted to result in an arginine to stop codon at codon 2317. Analysis of 200 normal chromosomes did not identify the same change in healthy subjects. This is the first report of a TG gene mutation in the Chinese Han population. Our study provides further evidence that mutations in the TG gene cause congenital goiter and hypothyroidism, demonstrates genetic heterogeneity of the mutation, and increases our understanding of phenotype–genotype correlations in congenital hypothyroidism.

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The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

Cited by 47 publications

References 43 publications

Repeat Motif-containing Regions within Thyroglobulin

Repeat Motif-containing Regions within Thyroglobulin

Maturation of Thyroglobulin Protein Region I

Clinical and Genetic Analysis of a Compound Heterozygous Mutation in the Thyroglobulin Gene in a Chinese Twin Family With Congenital Goiter and Hypothyroidism

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