Is there a double pathway for the secretion of thyroglobulin: a "short circuit" weakly glycosyl-iodinated and a "long circuit" strongly glycosyl-iodinated?

Intact rat thyroid lobes incubated in vitro release recently synthesized thyroglobulin (Tg) into the media at a faster rate than they release thyroglobulin stored in follicular structures. Differential release of this Tg fraction cannot be explained by morphological alterations in thyroid architecture during incubation. This rapidly excreted fraction exhibits a low density on rubidium chloride gradients characteristic of poorly sialylated and poorly iodinated thyroglobulin, comigrating on rubidium chloride gradients with thyroglobulin isolated from tunicamycin treated glands. This poorly sialylated and poorly iodinated thyroglobulin is itself unaffected in its density or release into the media by tunicamycin treatment. Tg isolated from the media of tunicamycin treated glands has nearly the same low iodine and low sialic acid content as rat serum thyroglobulin and does not incorporate radiolabelled glucosamine. This fraction thus appear to duplicate properties of low glycosylated-low iodinated thyroglobulin released from thyroid cells in organisms that have no follicular structures and no follicular storage process as well as from thyroid tissue in patients with thyroid disease states, particularly thyroid tumors. Thus it is proposed a "short loop" pathway of low-glycosylated low-iodinated thyroglobulin directly into circulation, that bypasses and is not stored in the follicular lumen, the "long loop".     

Hydrolysis of thyroglobulin inside isolated rat thyroid and liver lysosomes

In vitro, poorly iodinated thyroglobulin (Tg) is hydrolysed at the same rate whether it is enclosed in thyroid or in liver lysosomes, while fully iodinated Tg is degraded faster inside liver lysosomes. After in vivo TSH administration, thyroid lysosomes hydrolyse fully iodinated Tg as fast as do liver lysosomes.     

Exposure of thyroxine residues in human thyroglobulin. Two-site binding studies

Human thyroglobulin (Tg) could be adsorbed through one of its thyroxine (T4) residues by either of two T4-binding antibodies which had been covalently attached to Sepharose- CL4B . The antibodies used were (i) a purified human autoantibody specific for a T4-containing epitope in human Tg, or (ii) a rabbit antibody raised against T4 conjugated to bovine albumin side chains. Tg adsorbed by either immobilized antibody could then itself adsorb either type of antibody free in solution on to a further T4 residue. At least two T4 residues in human Tg are therefore sufficiently exposed to interact with T4-binding antibodies. Furthermore, these T4 residues are sufficiently far apart to allow the binding of two immunoglobulin molecules simultaneously. Previous observations of a marked preference by human autoantibodies for one of the T4-containing epitopes in Tg therefore reflect a higher binding energy with that epitope rather than an inability to interact with others. The T4-containing epitope which preferentially reacts with human Tg autoantibodies must therefore have a distinctive topography.     

Analysis of thyroglobulin antibody synthesis by cultured peripheral blood lymphocytes from patients with Hashimoto's thyroiditis using biotin-avidin solid phase enzyme immuno-assay

The influence of bovine thyroglobulin (Tg) and/or staphylococcus aureus cowan I (SAC) on Tg antibody synthesis has been studied using cultures of 8 Hashimoto's and 5 normal peripheral blood lymphocytes (PBL). The detection of Tg antibody in the culture supernatants was performed by sensitive biotin-avidin solid phase enzyme immunoassay. By using this technique, we were able to detect small amounts of Tg antibody synthesized by cultured Hashimoto's PBL responsive to bovine Tg and/or SAC; PBL from three out of eight patients produced increased levels of Tg antibody in the presence of 0.02 microgram/ml bovine Tg. On the other hand, PBL from two other cases among them which were unresponsive to bovine Tg alone became responsive to bovine Tg following simultaneous stimulation with SAC. PBL from the other three cases failed to respond to bovine Tg or simultaneous stimulation with bovine Tg and SAC. The former five patients had serum Tg tanned red cell hemagglutination (TGHA) titers greater than 1:409,600 except in one case and the latter had serum TGHA titers less than 1:12,800. These results indicated the presence of the different functional stages of B cells to produce Tg antibody in the circulation of Hashimoto's patients and suggested that sufficient number of lymphocytes responsive to bovine Tg are present in the circulation of Hashimoto's patients with high titers of serum TGHA.     

Iodoamino acid composition of rat thyroglobulin of varying total iodine concentration and fractions isolated by isopycnic ultracentrifugation]

Iodoaminoacid content (iodothyronines, T3 and T4, and iodotyrosines, MIT and DIT) has been determined in enzymatic hydrolysates of thyroglobulin Tg 19S of different iodine content (0.3-0.9%) isolated from equilibrium labeled rats. Preparative equilibrium centrifugation in RbCl density gradients of pure thyroglobulin was used to obtain protein fractions of largely different iodine content (0.2-1.2% I). Thin layer chromatography of total hydrolysates demonstrated that the distribution of iodoaminoacids depends on the total iodine content of each fraction. It is concluded, in agreement with previous results, that the native structure of Tg is an important factor in the regulation of hormone biosynthesis and that even at low iodination levels of Tg. T3 and T4 are synthesized.     

C-terminal extremity of ovine thyroglobulin shows strong interspecies homologies

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.     

Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure-activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix alpha16 (residues 485-503) and acidic groups at residues 505-506 in the adjacent surface loop.     

Effects of enzymatic deglycosylation of human goiter thyroglobulin on its immunochemical properties

Thyroglobulin (Tg), isolated from soluble iodoproteins by ammonium sulphate fractionation, was enzymatically deglycosylated in vitro and analyzed by polyacrylamide gel electrophoresis, double immunodiffusion and non-commercial RIA. Carbohydrate and iodine content was chemically determined. By PAAGE deglycosylated Tg (dTg) showed the appearance of a major band in the 12S region and three slower migrating bands corresponding to higher aggregates than 19S Tg. In immunodiffusion by testing native and deglycosylated Tg against anti-native Tg antiserum it was shown the appearance of a spur of native on deglycosylated Tg. By RIA of native and deglycosylated Tg against anti-deglycosylated Tg antiserum it was shown a minor binding capacity of the anti-deglycosylated antibody against native Tg at high dilutions. The results demonstrate that the enzymatic deglycosylation release almost all the carbohydrates of goiter Tg and that the removal of the carbohydrates of Tg produces a loss of antigenic determinants of the molecule.     

Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland

Summary A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.Abbreviations BSA bovine serum albumin - F BSA Fluoresceinylated BSA - GlcNAc N-Acetylglucosamine - Lac lactose - Man mannose - Man 6-P mannose-6-phosphate - MES morpholino ethanesulfonic acid - PBS phosphate buffered saline - Tg thyroglobulin     

Proteolytic processing by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin-A, the major globulin of fetal calf serum

Bovine fetuin-A is a member of a glycoprotein family with a wide spectrum of functions. Until now the bovine protein has been thought to be a single-chain protein. Recently we have shown that native bovine plasma fetuin-A partially exists as a disulfide-bridged two-chain protein with a heavy N-terminal and a lighter C-terminal chain similar to the structure of human fetuin-A homologue (alpha2HS glycoprotein), and also is partially phosphorylated at residues Ser120, Ser302, Ser305 and Ser306 (Wind et al., Anal. Biochem. 317 (2003) 26-33). Both fetuin-A modifications, the phosphorylation at the four sites as well as the proteolysis which causes longer or shorter light chains (termed lc-1 and lc-2, respectively), are probably brought about by targeted enzymatic activities which still need to be defined. In this study we show that authentic bovine fetuin-A disulfide-bridged two-chain forms, which include the original C-terminus, were liberated from the single-chain precursor by metalloproteinases MMP-3 (stromelysin-1) and MMP-7 (matrilysin), but not by elastase, cathepsin E and cathepsin G. Peptide sequencing suggested cleavage sites chiefly at the Pro277-Ser278 or Arg294-His295 peptide bonds. Fetuin-A radioactive phosphorylation in vitro by protein kinase CK2 caused (32)P incorporation into the fetuin-A light chain lc-1 but not lc-2 or the fetuin-A heavy chain, as revealed by MMP assisted proteolysis. Analysis by nanoESI-MS pinpointed phosphorylation at the native phospho-residues Ser302, Ser305 and Ser306 by increased relative abundance following in vitro phosphorylation. Moreover, CK2 phosphorylation of synthetic C-terminal fetuin-A peptides, used as effective controls to the native protein, strongly implies that CK2 is involved in the in vivo phosphorylation of fetuin-A. The phosphorylation of N-terminally truncated peptide homologs seemed highly dependent on the sequence context N-terminal of the phosphorylation sites, thus providing a likely explanation for the non-phosphorylation of the light chain lc-2 in native fetuin-A.     

Synthesis of thyroglobulin subunits in oocytes injected with thyroidal ribonucleic acid

Oocytes from Xenopus laevis were injected with RNA extracted from bovine thyroid polyribosomes. The analysis of the labelled products performed on SDS-polyacrylamide gels revealed the presence of a radioactive peak with a migration distance corresponding to a protein of about 85,000 daltons. Immunoprecipitation of the labelled products allowed us to single out this specifically induced protein. Sucrose density ultracentrifugation showed that translation of m-RNA for thyroglobulin (Tg) by oocytes did not result in the assembly of the subunits into the complete 19 S Tg molecule in the soluble phase of the homogenate.     

The acetylcholinesterase homology region is essential for normal conformational maturation and secretion of thyroglobulin

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.     

The rat asialoglycoprotein receptor binds the amino-terminal domain of thyroglobulin

We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRD(RHL-1)) and the anti-rCRD(RHL-1) antibody markedly inhibited (125)I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRD(RHL-1) was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRD(RHL-1) on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRD(RHL-1) specifically recognized a thyroglobulin fragment with an apparent M(r) of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role.     

A plasminogen-like protein, present in the apical extracellular environment of thyroid epithelial cells, degrades thyroglobulin in vitro

The prothyroid hormone, thyroglobulin (Tg), is stored at high concentrations in the thyroid follicular lumen as a soluble 19S homo-dimer and as heavier soluble (27S and 37S) and insoluble (Tgm) forms. Follicular degradation of Tg may contribute to maintaining Tg concentrations compatible with follicle integrity. Here, we report on the presence of a plasminogen-like protein in the follicular lumen of normal human thyroids and its synthesis and apical secretion by cultured epithelial thyroid cells. Since all the main luminal forms of Tg are cleaved by this plasminogen-like protein, we suggest that it contributes to Tg degradation in the follicular lumen.     

cDNA immunization of mice with human thyroglobulin generates both humoral and T cell responses: a novel model of thyroid autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.     

Identification of hormonogenic domains in the carboxyl terminal region of bovine thyroglobulin

A segment of 986 nucleotides corresponding to the 3' end of the 8.5 kb bovine thyroglobulin (Tg) mRNA has been sequenced. An open reading frame of 302 codons was found, ending with TGA and preceding an 80 nucleotide long 3' untranslated sequence. The encoded protein sequence provided the first data on the carboxyl terminal portion of Tg. Lysine was identified as the last residue. Comparison of the amino acid sequence with that of peptides known to contain thyroid hormones in the mature protein, lead to the identification of three regions involved in thyroid hormone formation. Two closely linked thyroxine- forming sites were found 182 and 196 amino acids from the carboxyl terminus respectively. The antepenultimate amino acid of the protein corresponded to the recently described triiodothyronine-forming site. Together with the previous localization of the main thyroxine-containing peptide at the amino terminus, the present results provide a map of all hormonogenic sites identified to date in Tg.     

Enhanced expression of cellular receptors for human interferon α on peripheral lymphocytes from patients with down''s syndrome

The sequence of 370 bases at the 5′-end of bovine thyroglobulin mRNA has been determine. A41 base untranslated segment was found preceeding the ATG initiator codon. It is followed by an open reading frame providing the first data on thyroglobulin primary structure. Analysis of the amino acid sequence demonstrated the presence of an 18 residue hydrophobic segment representing a putative signal peptide. Comparison of the amino terminal sequence of thyroglobulin with that of peptides known to contain thyroid hormones [7,8] demonstrated that the first tyrosine in native thyroglobulin is mainly found as thyroxine in the mature iodinated protein [8]. Our results clearly identify the amino-terminal region of thyroglobulin as an important hormonogenic domain of the protein.