Thyroglobulin structure and function: recent advances.

Thyroglobulin is a large-size iodoglycoprotein specific to thyroid tissue and is the substrate for the synthesis of thyroid hormones, thyroxine and 3,5,3'-triiodothyronine. Recent studies, which greatly benefited from recombinant DNA methodologies, improved the knowledge of several structural features of this dimeric protein and permitted insights into some structure-function relationships. Analysis-function of the primary structure of the human thyroglobulin monomer revealed several main characteristics: 1) 3 types of internal homologies; 2) extensive homology with the bovine thyroglobulin monomer and known partial sequences in the thyroglobulins of other mammalian species; 3) significant homologies with 2 other non-thyroid proteins (acetylcholinesterase and the invariant chain of the Ia class II histocompatibility antigen); 4) a terminal localization of the hormonogenic sites at both ends of the monomer. Current studies aim at determining conformational characteristics, understanding the molecular mechanisms of thyroid hormone formation and unraveling those interactions which in the thyroid cell and the thyroid follicle will permit this large pro-hormone to synthesize and release a few small thyroid hormone molecules. A more precise knowledge of this molecule in higher vertebrates and during evolution would impart valuable information concerning thyroid pathology, since thyroglobulin has been implicated in some genetic and in autoimmune thyroid diseases.

Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA.

The mRNA encoding human thyroglobulin has been cloned and sequenced. It is made up of a 8301-nucleotide segment encoding a preprotein monomer of 2767 amino acids, flanked by non-coding 5' and 3' regions of 41 and 106 nucleotides, respectively. This preprotein consists of a leader sequence of 19 amino acids, followed by the sequence of the mature monomer, corresponding to a polypeptide of 2748 amino acids (Mr = 302773). On its amino-terminal side, 70% of the monomer is characterized by the presence of three types of repetitive units. In contrast, the remaining 30% of the protein is devoid of repetitive units. This last region however shows an interesting homology (up to 64%) with the acetylcholinesterase of Torpedo californica. The sites of thyroid hormones synthesis are clustered at both ends of the thyroglobulin monomer. By contrast, the potential glycosylation sites are scattered along the polypeptide chain.

[C-terminal extremity of ovine thyroglobulin shows strong interspecies homologies]. / L'extrémité C-terminale de la thyroglobuline ovine présente de fortes homologies interspécifiques.

We report on the 466 nucleotides long, extreme 3' end of the ovine thyroglobulin (Tg) mRNA. The nucleotide sequence and the deduced carboxyl terminal region of the protein have been compared with those reported in other species. Comparison with hog peptides known to contain all the triiodothyronine (T3) of the mature Tg suggests that the antepenultimate amino acid, a tyrosine residue could be involved in hormone formation. This also agree with the data reported for bovine and murine Tg.

Characterization of a hormonogenic domain from human thyroglobulin.

A polypeptide domain of molecular mass near 22 kDa was purified from CNBr-digest of iodine poor human thyroglobulin (hTgb). This fragment represents the N-terminal part of the hTgb molecule and consequently contains the preferential hormonogenic tyrosine 'acceptor' of the protein. This fragment could correspond to the non-iodinated and unreduced form of the thyroxinyl-containing 26 kDa peptide previously purified from reduced and iodinated hTgb. This 22 kDa fragment is capable by itself, i.e. independently of the remaining hTgb molecule, of synthesizing thyroxine with a high efficiency after in vitro iodination. Its study should constitute a valuable way to identify at least one of the hormonogenic tyrosine 'donor' residues of hTgb.

Idiotypic analysis of five xenogeneic antisera to anti-human thyroglobulin monoclonal antibodies.

Five xenogeneic anti-idiotypic antisera (anti-id) were produced against individual BALB/c-derived monoclonal antibodies (mAb) which bound to different peptidic determinants on the human thyroglobulin molecule (Tg). Idiotypic analysis performed using sensitive radioimmunoassays revealed that: (1) the anti-id highly precipitated their homologous ligands; (2) two anti-id displayed minor cross-reactivities with one or two heterologous mAb; (3) each unlabelled homologous mAb was able to inhibit the idiotype binding of the corresponding anti-id; (4) no significant inhibition of homologous idiotype binding was observed with large excess of heterologous mAb; (5) efficient inhibition of mAb binding to Tg was observed only when homologous anti-id served as inhibitor. The data support the conclusion that xenogeneic anti-id may detect on their corresponding ligands individual idiotypic specificities that can be located at the mAb-combining site. Such reagents may constitute appropriate probes for further studies on anti-Tg autoimmunity.

Interaction of highly purified thyroid peroxidase with anti-microsomal antibodies in autoimmune thyroid diseases.

Among the several antigens involved in autoimmune thyroid diseases, the microsomal antigen has been recently identified as the human thyroid peroxidase (TPO). The availability of highly purified TPO and anti-TPO monoclonal antibody (mAb) allowed us to study in more details anti-TPO autoimmune antibodies (aAb) and their relationship with anti-microsomal aAb. Only sera with anti-microsomal aAb, as assayed by passive hemagglutination, highly immunoprecipitated purified TPO; anti-thyroglobulin aAb did not contribute to this effect. IgG binding to TPO and inhibition of mAb binding to TPO were also observed using anti-microsomal positive and anti-thyroglobulin negative sera. The correlation between anti-TPO and anti-microsomal aAb titers was found significant (p less than 0.02). These data suggest that anti-microsomal and anti-TPO immunoreactivity are presented by the same aAb. It could not be ruled out, however, that some of the anti-microsomal aAb detected by passive hemagglutination could be directed against antigens different from TPO. The use of highly purified TPO and anti-TPO mAb would allow large scale studies of anti-TPO aAb, necessary for investigating their pathological significance in thyroid disorders.

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases.

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.

Quantification of thyroglobulin messenger RNA by in situ hybridization in differentiated thyroid cancers. Difference between well-differentiated and moderately differentiated histologic types.

Thyroglobulin messenger RNA (mRNA) was located and quantified in tissue sections of differentiated human thyroid cancers by in situ hybridization using cloned complementary DNA probes. The cells of the well-differentiated follicular and papillary forms contained similar levels of thyroglobulin mRNA, corresponding to about 2000 copies per cell. In contrast, cells of moderately differentiated thyroid cancers contained about two to three times less thyroglobulin mRNA. It was also found that thyroglobulin mRNA was present almost exclusively in polyribosomes under the form of heavy polyribosomes actively synthesizing thyroglobulin. It is suggested that in situ hybridization method allows localization of specific mRNA in differentiated thyroid cancers and correlation with the level of differentiation of the cells.

Use of guanidine hydrochloride in the purification by reversed-phase high-performance liquid chromatography of thyroxinyl- and triiodothyronylpeptides derived from thyroglobulin.

When peptides containing thyroid hormones are first solubilized in 6 M guanidine hydrochloride they can be perfectly separated by reversed-phase high-performance liquid chromatography on an octadecylsilica column using conventional elution conditions (trifluoroacetic acid-acetonitrile).

Various expressions of a unique anti-human thyroglobulin antibody repertoire in normal state and autoimmune disease.

Polyclonal anti-human thyroglobulin (hTgb) antibodies (Ab) were purified from sera of rabbits immunized with human thyroglobulin, normal humans and patients suffering from Graves' disease, Hashimoto's thyroiditis and thyroid carcinoma. The avidity of the various Ab preparations for hTgb ranged from 0.3 X 10(10) -2.2 X 10(10) M-1. By using well characterized mouse monoclonal antibodies (mAb) directed against hTgb, it was shown that the fine specificities of induced anti-hTgb Ab in rabbits, natural Ab in normal subjects and autoantibodies in diseased patients were similar; however, they differed from that of rabbit anti-bovine and anti-porcine thyroglobulin Ab which were able to inhibit the hTgb binding of only a few of the mAb. Anti-hTgb in rabbits and in patients with thyroid carcinoma varied from those in normal subjects only by uniformly elevated serum titers. In contrast, patients with Graves' disease and Hashimoto's thyroiditis showed an increased concentration essentially restricted to Ab reacting with few of the antigenic determinants recognized by the mAb. Our data suggest that the repertoire of anti-hTgb Ab is similar in mouse, rabbit and human. Furthermore, the finding of identical fine specificities for anti-hTgb Ab in normal and pathological conditions implies that autoantibodies are produced in normal subjects and held to a low level by regulatory processes which fail with respect to selected epitopes in autoimmune diseases.

Sequence of the 5'-end quarter of the human-thyroglobulin messenger ribonucleic acid and of its deduced amino-acid sequence.

Thyroglobulin, the dimeric glycoprotein (19 S, 2 X 330 kDa), specific to the thyroid gland, is the support for thyroid hormone synthesis. Elucidation of the mechanism for thyroid hormone synthesis requires the knowledge of the primary sequence of the protein. In this paper the sequence of the first coding 2190 nucleotides from the 5' end of the human mRNA is presented. This was obtained by sequencing two previously described overlapping clones and by construction and sequencing of a single-stranded cDNA corresponding to the 5' end of the mRNA. The nucleotide sequence represents a quarter of the human thyroglobulin mRNA, from which a polypeptide sequence of 730 amino acids at the NH2-terminal end of the monomer has been deduced. This sequence shows a repetition of five highly conserved motifs each of approximately 50 amino acids, the analysis of which allowed us to establish a consensus sequence. We have also demonstrated (a) the hormonogenic tyrosine residue recently described in the mature protein, which is located four amino acids after the NH2-terminal Asn; (b) a prepeptide signal of thyroglobulin secretion comprising 19 amino acids preceding the Asn residue, the NH2-terminal residue of the mature protein and (c) a six-signal tripeptide (Asn-Xaa-Thr or Ser) of N-glycosylation of the chain.

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin.

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.

Localization of the thyroglobulin gene by in situ hybridization to human chromosomes.

The human thyroglobulin gene was mapped by in situ hybridization whereby a 3H-labeled recombinant plasmid DNA containing a fragment of 2.3 kilobases of human thyroglobulin gene was hybridized to human chromosome preparations. A high proportion (25%) of hybridized metaphases exhibited silver grains at the distal portion of the long arm of chromosome 8. Analysis of the grain position at this site indicated that the chromosomal localization of the human thyroglobulin gene was 8q242-8q243.

[Thyroglobulin and the biosynthesis of thyroid hormones]. / La thyroglobuline et la biosynthèse des hormones thyroídiennes.

Thyroglobulin (mol. wt. 660 kDa) is the specific protein of the thyroid gland in which are synthesized and stored the thyroid hormones (thyroxine and 3,5,3'-triiodothyronine). It is formed of equal-sized subunits (330 kDa) containing each identical polypeptide chains to which are associated two types of oligosaccharide units representing 8 to 10% by weight of the protein. The studies reported in this paper describe the presence in thyroglobulin of discrete hormonogenic sites. After chemical (CNBr) and enzymatic (trypsin and protease V8 of S. aureus) treatments of the protein, four different hormone-containing peptide segments have been isolated, purified and sequenced. They correspond to the hormonogenic tyrosine-containing sites of the protein. One tyrosine is located at 4 amino acid residues from the N-terminal asparagine of the chain and is a major site for thyroxine synthesis. Another one which represents the triiodothyronine site is situated 2 amino acids before the C-terminal lysine. Finally, two other sites, one of low affinity and the other of high affinity for iodine and thyroxine formation, are equally located in the C-terminal part of the chain. The hormone-forming regions localized at the opposite far ends of the thyroglobulin chain(s) likely represent zones more accessible to iodination and with a conformation suited for the coupling of iodotyrosine into iodothyronine residues and ultimately protease attack to release the free hormones into the circulation. The presence of hormonogenic sites of different affinities for iodine allows thyroglobulin to modulate adaptively its hormonogenic capacity to external iodine supply. The molecular mechanism of this process is still unknown.

Precursor-product relationship between the 26-kDa and 18-kDa fragments formed by iodination of human thyroglobulin.

At moderate iodination levels (20 iodine at atoms/molecule), human thyroglobulin (hTgb) produces after reduction a thyroxinyl-peptide of 26 kDa which represents the N-terminal part of the protein. At higher iodination levels, the 26-kDa peptide is accompanied by another T4-containing peptide of 18 kDa. A precursor-product relationship between the 26- and 18-kDa fragments was demonstrated by the study of the tryptic fragments of both hormonopeptides. In addition, comparison with the protein sequence deduced from the nucleotide sequence of the 5'-end of hTgb mRNA demonstrated that the N-terminal region of Htgb from which are issued the 26-kDa peptide and its 18-kDa derivative is especially sensitive to proteolysis. This character is possibly related with a facilitated release of thyroid hormones in vivo.

The relationships between ribosomal genes and fibrillar centers in thyroid cells cultivated in vitro.

Despite the fact that the fibrillar centers of the nucleolus and the chromosomal nucleolar organisers (NORs) are similarly stained with the NOR-silver technique, there remain some questions about the identification of fibrillar centers as NORs. The distinct delineation of the fibrillar centers in porcine thyroid cells allowed us to determine whether there was a numerical equivalence or correlation between fibrillar centers and NORs. Hybridization in situ and silver staining performed on pig chromosomes showed that pairs 8 and 10 contained rDNA sites. Silver staining of thyroid cells in electron microscopy showed that the fibrillar centers and their surrounding layer of dense fibrils were the sites of silver deposit. Chromatin fibers were demonstrated within the fibrillar centers through the aid of the osmiumammine reaction and with the oxidized diaminobenzidine technique. It was observed that in cultured thyroid cells the fibrillar centers could be identified in the light microscope as argyrophilic spherules, and easily counted. The number of fibrillar centers was variable according to culture conditions. In cells cultured for 5 hr, the mean number of fibrillar centers was 1.7. After 5 days of culture, the number of fibrillar centers increased, reaching a mean value of 5.93. When thyroid cells were stimulated with thyrotropin, the number of fibrillar centers again increased to a mean value of 7.54. These results demonstrate that the relationship between fibrillar centers and NORs is not a simple proportionality: the number of fibrillar centers increases with increased cellular activity. These data imply that in active cells each NOR may pass through several fibrillar centers.

[Relation between the iodination of human thyroglobulin and the cleavage of the hormone peptide 26K N-terminal]. / Relation entre l'iodation de la thyroglobuline humaine et le clivage du peptide hormonogénique 26K N-terminal.

At moderate iodination levels (about 20 iodine atoms/mol) human thyroglobulin yields after reduction and alkylation a hormone (T4)-containing N-terminal peptide of 26K. Further iodination of the thyroglobulin in vitro results in the cleavage of this part of the molecule into smaller peptides of 22K and 18K. A precursor-product relationship between the 26K peptide segment and the latter was established by showing an identical N-terminal T4-containing sequence in the 3 peptides. Cleavage of peptide bonds in the 26K segment to give the smaller fragments could possibly be related to the formation of another hormone residue.