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At least 19 recordsLinked to original sources
PubMed1989

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.

UNLABELLED: Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. IN CONCLUSION: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Human monoclonal thyroglobulin autoantibodies of high affinity. II. Interaction between thyroglobulin and thyroglobulin autoantibodies of different IgG subclasses.

The interaction of human thyroglobulin (Tg) autoantibodies of different IgG subclasses with Tg was investigated using four high affinity human monoclonal thyroglobulin (Tg) autoantibodies, secreted by human-mouse hybridomas, of subclasses IgG1 (kappa and lambda) and IgG2 (kappa and lambda) and an IgG4 kappa serum monoclonal Tg antibody. With exception of a low level of interference in binding between one IgG1 lambda Tg antibody and one IgG2 kappa Tg antibody (27% decrease), binding by human monoclonal Tg antibodies of one IgG subclass was unaffected by pre-incubation of 125-I Tg (or Tg on an ELISA plate) with a human monoclonal Tg antibody of a different IgG subclass. Furthermore, preincubation of Tg-coated ELISA plates with an IgG1 human monoclonal Tg antibody had little effect on binding to Tg by IgG2, IgG3 and IgG4 Tg antibodies present in the sera of 6 Hashimoto patients. Comparable observations were made using an IgG2 monoclonal Tg antibody and serum Tg antibodies of subclasses IgG1, IgG3 and IgG4. Binding of an IgG1 kappa Tg antibody was inhibited (> 80%) by pre-incubation of Tg with an IgG1 lambda Tg antibody derived by fusion of lymphocytes from the same Hashimoto patient. In contrast, pre-incubation of Tg with an IgG2 kappa Tg antibody had little effect on subsequent binding by an IgG2 lambda Tg antibody derived from lymphocytes of a different Hashimoto patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Immunization with thyroglobulin-specific cytotoxic T cell hybridoma induces anti-thyroglobulin antibodies: characteristics of monoclonal anti-thyroglobulin auto-antibody.

We have produced five monoclonal autoantibodies (mA-Abs) to thyroglobulin (Tg) and more precisely to one epitope located within the < 10-kDa pTg tryptic fragment suspension capable of inducing experimental autoimmune thyroiditis (EAT). They were selected from spleen cells from CBA/J mouse immunized with the syngeneic cytotoxic T cell hybridoma HTC2. HTC2 cells are specific for one Tg epitope located within the EAT inducer pTg tryptic fragments and are able to prevent EAT induction by pTg. The restricted specificity of the humoral response previously observed in vivo was further demonstrated and defined in vitro at the single cell level. Competitive studies for binding to pTg or to the < 10-kDa pTg tryptic fragments demonstrated that HTC2-induced anti-Tg mA-Abs recognized an epitope(s) located in the < 10-kDa pTg tryptic fragment (as did 3B8G9, one conventional anti-Tg mA-Ab we selected). We ruled out the possibility that HTC2-induced anti-Tg A-Abs belong to the group of the natural A-Abs due to the lack of recognition of actin, dsDNA, TNP-ovalbumin, tubulin, their isotypes (IgG1 or Ig2a), and their affinities (in the 10(-7) M order of magnitude). The results strengthen the hypothesis that T and B cells sharing the same specificity can express similar idiotopes on their respective receptors for antigen. They also demonstrate the existence of a regulatory idiotypic network that could explain the protection from EAT after injection of inactivated HTC2 cells or its anti-clonotypic mAb.

Animals↗
PubMed1999

[Detection of thyroglobulin autoantibodies and potential interference with serum thyroglobulin measurement].

BACKGROUND: Thyroglobulin measurement is useful for the follow up of patients subjected to total thyroidectomy for differentiated thyroid carcinoma. Thyroglobulin autoantibodies may interfere with its determination. AIM: To measure thyroglobulin autoantibodies and their interference with thyroglobulin determination. MATERIAL AND METHODS: The presence of thyroglobulin autoantibodies was investigated in 801 serum samples sent to the laboratory for measurement of thyroglobulin levels. A serum was considered positive for these autoantibodies when radioactivity corresponding to 125I-thyroglobulin bound to thyroglobulin autoantibodies, precipitated with human gamma globulin, exceeded in 1.4 times that of a negative sera pool. In positive sera, thyroglobulin autoantibody concentration was measured and its interference with thyroglobulin radioimmunoassay was assessed through a recuperation test using exogenous thyroglobulin. RESULTS: Thyroglobulin autoantibodies were detected in 149 sera (18.6%). Of these, 65 had a recuperation that fluctuated between 1 and 80%. Thyroglobulin autoantibody concentration was negatively correlated with recuperation percentages (r = -0.64; p < 0.001) but not with thyroglobulin concentrations (r = 0.08). Thyroglobulin was higher in positive sera with a recuperation over 80% than in sera with a recuperation of less than 80% (12.7 +/- 1.7 and 5.9 +/- 0.6 ng/ml, respectively; p < 0.001). CONCLUSIONS: Thyroglobulin autoantibodies interfere with thyroglobulin measurement by radioimmunoassay, sequestering variable amounts of thyroglobulin. The presence of these autoantibodies must be investigated prior to thyroglobulin determination.

Autoantibodies↗
PubMed1998

Immunoradiometric assay of thyroglobulin in patients with differentiated thyroid carcinomas: need for thyroglobulin recovery tests.

As interference from thyroglobulin autoantibodies appears to have been overcome in new commercial thyroglobulin assays by the use of monoclonal antibodies, the need for thyroglobulin recovery tests became uncertain. Sera (n=45) from patients with differentiated thyroid carcinomas were selected on the basis of a thyroglobulin recovery value below 70% in the Dynotest Tg immunoradiometric assay (Brahms) routinely used in our laboratory. Serum thyroglobulin levels were then measured using three other commercial immunoradiometric assays: thyroglobulin ERIA (Pasteur), HTGK (Sorin) and ELSA HTG (Cis Bio International). Thyroglobulin autoantibodies were measured using the Thyrak assay (Brahms). Although many patients were thyroglobulin antibodies-negative (< 200 U/ml, n=26), most immunoradiometric assays failed to detect thyroglobulin in patients with evidence of recurrence. Low thyroglobulin values associated with low thyroglobulin recovery in thyroglobulin antibody-negative patients appear to be more biologically relevant than a single low thyroglobulin value, which can lead to lack of medical intervention. We conclude that the thyroglobulin recovery test is a prerequisite for the correct interpretation of serum thyroglobulin levels determined with immunoradiometric assays in the follow-up of thyroglobulin autoantibody-negative patients treated for differentiated thyroid carcinomas.

Cell Differentiation↗
PubMed1979

[Study on enzyme immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum (author's transl)].

Enzyme-linked sandwich immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum using silicone rod and beta-D-galactosidase were studied. These methods showed excellent results in specificity, sensitivity, precision and clinical application. 1) A method using silicone rod coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin G (Fab') conjugated with beta-D-galactosidase was developed for the measurement of circulating thyroglobulin. The sensitivity of the assay with as little as 2 microliter of serum was 10.7 amoles/tube corresponding to 3.5 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The correlation coefficient between values determined by the present assay and a double-antibody radioimmunoassay was 0.99 (n = 63, p less than 0.001). Circulating thyroglobulin was detectable in 90% of 146 normal subjects, the concentration being 13.3 +/- 10.3 ng/ml (mean +/- S.D.). Interference of anti-thyroglobulin autoantibody with the assay for thyroglobulin was smaller than that in radioimmunoassay. 2) Another method using human thyroglobulin conjugated with beta-D-galactosidase and silicone rod coated with human thyroglobulin was developed for the measurement of circulating (anti-human thyroglobulin) autoantibody. The sensitivity of the assay with as little as 5 microliter of serum was 7 fmoles/tube corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The highly significant correlation was observed between the concentrations of anti-thyroglobulin autoantibody determined by the present assay and a radioimmunoassay (r = 0.80, n = 74, p less than 0.001) and also between those by the present assay and those by tanned red cell hemagglutination (r = 0.78, n = 199, p less than 0.001). No effect of thyroglobulin on the present assay was observed unless the ratio of the amount of thyroglobulin to that of (anti-human thyroglobulin) immunoglobulin G was higher than a tenth.

Autoantibodies↗
PubMed1979

Process of iodination of thyroglobulin and its maturation. II. Properties and distribution of thyroglobulin labeled in vitro or in vivo with radioiodine, 3H-tyrosine, or 3H-galactose in rat thyroid glands.

With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.

Animals↗
PubMed1979

Process of iodination of thyroglobulin and its maturation. I. Properties and distribution of thyroglobulin labeled with radioiodine in pig thyroid slices.

Pig thyroid slices were incubated with Na131I and the 17--19S 131I-labeled thyroglobulin isolated was subjected to dissociation with 0.3 mM sodium dodecyl sulphate SDS) on sucrose density gradient centrifugation and to iodoamino acid analysis. During the incubation, initially dissociable thyroglobulin was gradually altered to 0.3 mM SDS-resistant species with increasing incorporation of iodine. Microsome-bound, poorly iodinated thyroglobulin and preformed thyroglobulin were chemically iodinated and then subjected to analysis of dissociability and iodoamino acid contents with newly incorporated iodine. The results indicated that the behavior of the former thyroglobulin resembled that of 131I-thyroglobulin obtained from the slices. Then, thyroid slices were incubated for 3 min with Na131I and 3H-leucine with or without 10-min chase incubation. The sucrose density gradient centrifugation patterns of 131I and 3H-radioactivity of cytoplasmic extracts indicated that 131I-thyroglobulin is contained in particulates, especially in vesicles with low density(d=1.12) and that some of them are released into the soluble fraction within 10 min. The vesicles contained peroxidase and NADH-cytochrome c reductase, and are probably exocytotic vesicles in the apical area of cytoplasm of follicular cells. No positive evidence was obtained that plasma membranes participate in the iodination of thyroglobulin under the present experimental conditions. These results suggest that, in the incubation of thyroid slices, iodine atoms are preferentially incorporated into newly synthesized, less iodinated thyroglobulin, rather than preformed thyroglobulin, and that the iodination occurs, at least to a certain degree, in apical vesicles before the thyroglobulin is secreted into the colloid lumen.

Animals↗
PubMed1992

Endocytosis of thyroglobulin is not mediated by mannose-6-phosphate receptors in thyrocytes. Evidence for low-affinity-binding sites operating in the uptake of thyroglobulin.

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate-(Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concentrations of thyroglobulin (20 microM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].

Absorption↗
PubMed1997

Identification of the membrane receptor binding domain of thyroglobulin. Insights into quality control of thyroglobulin biosynthesis.

The last stages of thyroglobulin maturation occur in the thyroid follicular lumen and include thyroid hormone formation and glycan completion. In this compartment, newly secreted thyroglobulins interact with a thyrocyte membrane receptor that prevents their premature lysosomal transfer and degradation. Both GlcNAc moieties and thyroglobulin peptide determinants are involved in receptor interaction. Here we used monoclonal antibodies (mAbs) directed against human thyroglobulin either to inhibit (mAb78) or to enhance (mAb240) the thyroglobulin binding and to identify the region of the thyroglobulin involved in the receptor recognition. Peptides containing the mAb epitopes were obtained by immunoscreening cyanogen bromide-derived native human thyroglobulin peptides and a cDNA thyroglobulin expression library. Three peptides, localized in the thyroglobulin N-terminal domain, were obtained. Peptides N1 (Ala1148-Gln1295) and N2 (Ser789-Met1008) were recognized by mAb240 and mAb78, respectively. None of them bound the receptor. The third peptide, N3 (Ser789-Met1172), (i) overlapped all or part of the N1 and N2 peptide sequences and was recognized by both mAbs, (ii) carried two complex glycans at Asn797 and Asn928, of which a subset presented accessible GlcNAc residues, and (iii) inhibited the thyroglobulin binding to FRTL5 cell membrane preparations. The N3 peptide includes tyrosine residues that have been reported to be involved in hormone formation. These results suggest that structural modifications closely associated with hormone formation within this domain act as sensors for the receptor interaction and thus for the intrafollicular retention or lysosomal homing of the prohormone.

Amino Acid Sequence↗
PubMed1965

The production of thyroiditis and antibody following injection of unaltered thyroglobulin without adjuvant into rabbits previously stimulated with altered thyroglobulin.

Injection of rabbits with arsanil-sulfanil-thyroglobulin without adjuvant resulted in the production of hemagglutinating and precipitating antibody to native thyroglobulin and thyroiditis. Following a latent period of 1 month, these same rabbits responded to an injection of native thyroglobulin with an increase in both circulating antibody to native thyroglobulin and severity and frequency of thyroid lesions. Some rabbits initially immunized with arsanil-sulfanil-thyroglobulin also responded to a second and third monthly injection of native thyroglobulin, but the response to the third injection was usually not as good as the response to the first injection. A few of the rabbits showed a transient production of 19S antibody after the injection of native thyroglobulin was initiated. Neither circulating antibody nor thyroiditis were ever observed in rabbits injected with only native thyroglobulin without adjuvant. The correlation of the thyroiditis with the presence or absence of circulating antibody was discussed. Following a latent period after injections of aqueous arsanil-sulfanil-thyroglobulin some rabbits made a response to their own thyroglobulin released from an autotransplant of thyroid tissue.

Animals↗
PubMed1986

Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture.

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.

Animals↗
PubMedDate not supplied

An improved procedure for the preparation of the human thyroglobulin and the development of a thyroglobulin autoantibody kit.

Human thyroglobulin has various applications as a diagnosis reagent or for studying the physiopathology of the protein and hormonal biosynthesis in the thyroid gland. The previous preparation procedure for isolating the human thyroglobulin currently used in our laboratory, has some inconveniences as regards the low yield and the noxious influence of the ammonium sulfate precipitation upon its molecular integrity. Therefore, we describe an improved fractionation and purification procedure whose main steps are extraction of the thyroid tissue homogenate in 0.15 M NaCl followed by a double gel filtration on Sephadex G-200 column. In this way are obtained thyroglobulins A and B grade using the two chromatographic steps, respectively. Thyroglobulin B grade is further submitted to preparative polyacrylamide gel electrophoresis for separating some thyroglobulin isomers as recognized by other groups using the analytical ultracentrifugation procedure. The different fractionation and purification steps were checked by double diffusion in gel using rabbit anti-thyroglobulin serum and horse antihuman serum protein. The homogeneity and the molecular weight of the different fractions we evidenced were analyzed by the aid of disc and plate electrophoresis in polyacrylamide gel. The authors developed the technique for thyroglobulin autoantibody detection by the passive haemaglutination method using stabilized erythrocytes coated with thyroglobulin A-grade. Thyroglobulin B-grade used as a tracer and a reference preparation in a RIA system offered a sensitivity of 1.5 micrograms/liter for thyroglobulin detection in biological fluids.

Autoantibodies↗
PubMed1977

Properties of carbohydrate-stripped thyroglobulin. III. Solubility characteristics of thyroglobulin.

In studying the solubility characteristics of thyroglobulin, it was found that the removal of sialic acid residues from either human or hog thyroglobulin by treatment with neuraminidase markedly decreased the solubility in salt solutions. On the other hand, naturally sialic acid-rich thyroglobulin obtained from human diffuse goiter was slightly more soluble than thyroglobulin in which sialic acid content was within the normal range. However, the desialized preparations prepared from both normal and sialic acid-rich thyroglobulin showed markedly lower solubility and further, showed no significant difference in solubility between them. These results indicate that the surface charge of thyroglobulin which influenced the salting-out property depends largely on the number of sialic acid residues in a thyroglobulin molecule, not on the degree of iodination of thyroglobulin.

Amino Acids↗
PubMed1980

An approach to the structure of thyroglobulin. Hormone-forming sequences in porcine thyroglobulin.

The mixture of CNBr peptides obtained by treatment of porcine thyroglobulin with cyanogen bromide was separated into three fractions by Sephadex G-200 gel filtration in 1 M propionic acid. When one of these fractions was reduced and S-alkylated, a hormone-containing CNBr peptide was purified by filtration on Biogel A-1.5 m and ion-exchange chromatography on DEAE-Sephadex. Similarly, two other hormone-containing CNBr peptides were separated from another reduced and S-alkylated fraction by Sephacryl S-200 gel filtration and DEAE-Sephadex chromatography. The three purified CNBr peptides have the same Mr (15000), differ in amino acid composition and contain 60-70% of the hormones present in the initial thyroglobulin. Fragmentation of these peptides into smaller hormone peptides was carried out by trypsin digestion followed by gel filtration. This resulted in the purification of eight discrete hormone-containing tryptic peptides whose homogeneity was established by the study of their N-terminal and C-terminal sequences. At least four sites for the synthesis of thyroxine and two sites for the synthesis of triiodothyronine have been identified in the three CNBr peptides. A possible additional site was recovered in the form of the peptides seryl-triiodothyronine and probably seryl-thyroxine. To gain information on the number of hormone-forming sites in thyroglobulin iodinated in vivo and on the distribution of these sites in the different hormone-containing peptides isolated, estimation of thyroxine and triiodothyronine content of batches of thyroglobulin of iodine content comprised between 0.73% and 1.6% was carried out, together with the estimation of the hormone content of all the CNBr peptides separated from two preparations of thyroglobulin of different iodine contents (0.73% and 1.23%). It was shown that the number of thyroxine and triiodothyronine residues increased linearly with increasing thyroglobulin iodine content in the range 0.73-1.6% and reached six residues of thyroxine and two residues of triiodothyronine/mol protein for a thyroglobulin iodine content of 1.6% with no indication of saturation. This result is at variance with previous findings on porcine thyroglobulin iodinated in vivo which suggested that the thyroxine content reached a plateau of three to four residues/molecule for an iodine content comprised between 0.7% and 1.1%.

Amino Acid Sequence↗
PubMed1989

Towards an antigenic map of human thyroglobulin: identification of ten epitope-bearing sequences within the primary structure of thyroglobulin.

The aim of this study was to define, at the molecular level, epitopes of thyroglobulin recognized by heterologous antibodies and autoantisera. One hundred thousand clones from a lambda gt 11 human thyroid cDNA library were screened using a rabbit antibody to human thyroglobulin at a 1:2000 dilution. Twenty clones were plaque-purified to homogeneity, and characterization and sequencing of their cDNA inserts showed that they represented four distinct regions of the thyroglobulin molecule, one of them being the 22 carboxyl-terminal amino acids. Rescreening of the library with the same rabbit antibody to human thyroglobulin absorbed with peptides encoded by the carboxyl-terminal clone, led to the definition of six further epitope-bearing fragments of thyroglobulin. The ten regions that we have identified were recognized by ten further rabbit antibodies to human thyroglobulin, showing that they are representative of the repertoire of heterologous epitopes. In contrast, none of the ten heteroepitope-bearing fragments was recognized by sera from ten patients with autoimmune thyroid disease with various titres of thyroglobulin antibodies. Screening of 2 x 10(6) clones from the library using a pool of ten autoantisera (individual sera diluted to 1:1000), and of 1 x 10(6) clones using a single autoantiserum of very high antithyroglobulin titre (diluted 1:400) resulted in no thyroglobulin clones being isolated. The significance of these results to the immune process is discussed.

Amino Acid Sequence↗