The subunit structure of thyroglobulin

Pitt‐Rivers, Rosalind

doi:10.1042/bj1570767

Cited by 13 publications

(7 citation statements)

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“…Preparation of Tg Peptides. Peptides lacking thyroxine (T4-) were prepared as described previously (6,8,9), and the synthetic peptide Ac-STDD(T4)ASFSRAbNH2, hereafter referred to as the T4(2553) peptide, was produced on a synthesizer (9050; Milligen/Biosearch, Burlington, MA) also as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

A thyroxine-containing peptide can induce murine experimental autoimmune thyroiditis.

Hutchings

Cooke

Dawe

et al. 1992

The Journal of Experimental Medicine

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Summal"y A synthetic peptide based on a sequence containing thyroxine at position 2553 in thyroglobulin (Tg), and already shown to be recognized by two clonotypically distinct murine Tg autoreactive T cell hybridomas, can trigger primed lymph node cells to transfer thyroiditis to naive recipients. Donor lymph node cells could be prepared from mice immunized either with intact mouse Tg or with this peptide itself. After a second exposure to the priming antigen in vitro, both these populations induced 100% thyroiditis in recipient animals. The importance of the T4 residue in the development of disease was demonstrated by the failure of Tg tryptic peptides depleted of T4 to stimulate pathogenic effectors in vitro, even when the lymph node cells had been taken from mice primed with whole Tg. We conclude that this T4-containing 12mer sequence is a major thyroiditogenic epitope in CBA/J mice although we cannot exclude the possibility that there are other pathogenic epitopes present in the whole Tg molecule.

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Section: Methodsmentioning

confidence: 99%

A thyroxine-containing peptide can induce murine experimental autoimmune thyroiditis.

Hutchings

Cooke

Dawe

et al. 1992

The Journal of Experimental Medicine

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“…All steps were carried out in glass containers to avoid the irreversible losses that occur on plastic surfaces. Tg (4-20mg/ml) was reduced in 8 M-urea by using 2.43,p1 of mercaptoacetic acid/mg of Tg at pH 10.5 for 90min at room temperature, as recommended by Pitt-Rivers (1976). The pH was then lowered to 8.6 with HCI and the reduced Tg alkylated with 2.23,ug of iodoacetic acid/mg of Tg for 2h at room temperature.…”

Section: Preparation Ofhuman Thyroglobulinmentioning

confidence: 99%

Restricted antigenicity of thyroxyls in human thyroglobulin

Byfield¹,

Bond²,

Copping³

et al. 1982

Biochemical Journal

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Interactions between two human iodothyronine-binding autoantisera and three preparations of human thyroglobulin (Tg) were not proportional to the latter's thyroxyl residue content. Probably only one of the several thyroxyl-containing sites in Tg reacted with the immunoglobulins from both antisera. In the case of one of the antisera, which was thyroxine (T4)-specific, the thyroxyl residue was the immunodominant feature of the antigenic site. The other antiserum, which had a specificity for 3,5,3'-tri-iodothyronine (T3), recognized different determinants around the same thyroxyl residue, but this residue was not itself an important element of the binding site. Thus, despite the specificity for T3 free in solution, the presence of T4 in the complete antigenic site was tolerated, since other structures supplied the bulk of the binding energy. 'Specificity' of this antiserum for T3 in solution is therefore coincidental and need not be ascribed to the presence of T3 in the original immunogen. Some results obtained in these studies may be interpreted as supporting the possibility that a modified Tg was the immunogen for the generation of these naturally occurring human antisera.

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“…Preparation of Tg Peptides. HTg was reduced and alkylated with iodoacetamide in 8 M urea (25), dialyzed to remove the urea, digested with TPCK-Trypsin (Sigma Chemical Co., Poole, UK) at an enzyme :substrate ratio of 1:50 in 0.1 M ammonium bicarbonate for 4 h. The digest was lyophilized and redissolved in 20 mM sodium phosphate, pH 7.4 to 1 mg/ml and passed through an affinity matrix consisting of rabbit anti-T4 anti-bodies (Miles, Stoke Poges, UK) coupled to tosyl activated Sepharose 4B (26) . Unadsorbed peptides (T4-) were collected.…”

Section: Methodsmentioning

confidence: 99%

Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells.

Champion

Page

Parish

et al. 1991

The Journal of Experimental Medicine

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SummaryAlthough thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by .two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2 . In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule . Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-, y-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity. T he production of the thyroid hormones thyroxine (T4),t tri-iodothyronine (T3) and reverse T3 (rT3) is dependent on the organification of iodine into thyroglobulin (Tg), the major protein product of the thyroid (1, 2) . This involves thyroid peroxidase catalyzed iodination of tyrosine residues in Tg to form mono-and di-iodotyrosines and their subsequent crosslinking to form the iodothyronines T3 and T4. These mature Tg molecules are stored in a colloidal form in the lumen of thyroid follicles. Secretion of T4 and T3 involves the endocytosis and subsequent proteolysis of colloidal Tg, which releases the hormone residues for diffusion into the circulation . The recent cloning of the genes coding for Tg from several species has allowed the precise localization 1 Abbreviations used in this paper.

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The subunit structure of thyroglobulin

Abstract: Human and rat thyroglobulin were reduced and alkylated in aqueous alkaline conditions in the absence of denaturants; the product of reduction in both cases has been found to have mol.wt. about 165000, or one-quarter that of the native molecule.

Cited by 13 publications

References 15 publications

A thyroxine-containing peptide can induce murine experimental autoimmune thyroiditis.

A thyroxine-containing peptide can induce murine experimental autoimmune thyroiditis.

Restricted antigenicity of thyroxyls in human thyroglobulin

Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells.

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