Hormone formation in the isolated fragment 1–171 of human thyroglobulin involves the couple tyrosine 5 and tyrosine 130

The thyroid couples two iodotyrosine molecules to produce thyroid hormone at the acceptor site in thyroglobulin, leaving dehydroalanine or pyruvate at the donor position. Previous work has located the acceptors but not the principal iodotyrosine donors. We incorporated [14 C]tyrosine into beef thyroid slices, isolated and iodinated the [ 14 C]thyroglobulin (Tg I), separated its Nterminal ϳ22-kDa hormone-rich peptide, and digested the latter with trypsin and endoproteinase Glu-C (EC 3.4.21.19). Nonlabeled thyroglobulin (Tg II) was isolated from the same glands and processed similarly, without iodination in vitro. Tg I was used to initially recognize pyruvate in peptide fractions, and Tg II was used to then identify its location in the thyroglobulin polypeptide chain. Sequencing of a tryptic peptide by mass spectrometry and Edman degradation showed a cleavage after Val129 . An endoproteinase Glu-C-generated peptide had the predicted molecular mass of a fragment containing residues 130 -146 with Tyr 130 replaced by pyruvate; the identification of this peptide was supported by obtaining the expected shortened fragment after tryptic digestion.14 C-labeled pyruvate was identified in the same fraction as this peptide. We conclude that Tyr 130 is an important donor of the outer iodothyronine ring. Its likely acceptor is Tyr 5 , the most important hormonogenic site of thyroglobulin, because Tyr 5 and Tyr 130 are proximate, because they are the most prominent early iodination sites in this part of thyroglobulin, and because the N-terminal region was previously found capable of forming T 4 by itself.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Tyrosine 130 Is an Important Outer Ring Donor for Thyroxine Formation in Thyroglobulin

Dunn

Corsi

Myers

et al. 1998

“…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.…”

mentioning

confidence: 99%

Repeat Motif-containing Regions within Thyroglobulin

Lee

Arvan

2011

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 ...

“…The most heavily used site of iodination and thyroxine synthesis occurs within the first 130 residues of the Tg protein (7,8). While there are other hormonogenic sites on Tg, the foregoing information leaves open the question of the biological significance of much of the remaining 2,620 residues (9, 10).…”

mentioning

confidence: 99%

The Acetylcholinesterase Homology Region Is Essential for Normal Conformational Maturation and Secretion of Thyroglobulin

Park

Arvan

2004

Secretion of thyroglobulin (Tg, a large homodimeric glycoprotein) is essential to deliver Tg to its site of iodination for thyroxine biosynthesis. An L2263P missense mutation in Tg has been proposed as the molecular defect causing congenital goitrous hypothyroidism in cog/ cog mice due to perturbed Tg homodimerization, resulting in its retention within the endoplasmic reticulum. The mutation falls within a carboxyl-terminal region of Tg with high structural similarity to the entirety of acetylcholinesterase (AChE), a secretory protein that also forms homodimers. We provide new evidence that authentic AChE and the cholinesterase-like domain of Tg share a common tertiary structure. Moreover, we find that a Tg truncation, deleted of the cholinesterase-like region (but not a comparably sized deletion of internal Tg regions), blocks Tg export. Appending to this truncation a cDNA encoding authentic AChE results in translation of a chimeric protein in which AChE is present in a native, enzymatically active (albeit latent) conformation, and this fully rescues Tg secretion. Introduction of the cog mutation inhibits AChE enzyme activity, and established denaturing mutations of AChE block secretion of the Tg. Additional studies show that the native structure of the AChE region functions as a "dimerization domain," facilitating intracellular transport of Tg to the site of thyroid hormonogenesis.