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

Abstract

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 amono acids ( M , = 302 773). 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.Thyroglobulin is a protein specifically synthesised by the thyroid gland, and constitutes the support for the production of the two thyroid hormones, thyroxine and triiodothyronine [l]. The existence of thyroglobulin was demonstrated a century ago [2], but its structure has been elucidated only recently. It is a dimeric glycoprotein with an M , of 660000, of two identical subunits [3, 41 encoded by a single mRNA with a sedimentation coefficient of 33 S (8500 nucleotides) [5 -71. Thyroglobulin is synthesised by the thyrocyte, then exported to the vesicular lumen where its maturation begins by the iodination of several tyrosine residues, and coupling of some of the iodotyrosine residues [8]. Then, by an endocytotic process, the molecule is absorbed into the thyrocyte where several selective cleavages occur in the lysosomes, resulting in the release of the thyroid hormones, and complete degradation of the rest of the molecule.For a thyroglobulin iodine content of 0.5% (which is rarely attained in man) a maximum of 3.5 hormonal residues per thyroglobulin molecule are formed through a reaction catalyzed by the enzyme thyroid peroxidase [9]. Four hormone-synthesis sites have been described, corresponding to four tyrosine residues in fixed positions [lo-121.The structure of human thyroglobulin seems to be responsible for the specific fixation of iodine, and the production of functional thyroid hormones. Several human pathologies are associated with an abnormal thyroid function. Since the recent demonstration of the implication of a defect in thyroglobulin gene structure in the development of congenital goitre in cattle [13], it is likely that knowledge of the structure of human thyroglobulin mRNA will help to elucidate the structural bases of human thyroid pathologies. We describe here the complete nucleotide sequence of human thyroglobulin mRNA. MATERIALS AND METHODSPreparation and sequencing of DNA cDNA fragments corresponding to human thyroglobulin mRNA were prepared from recombinant plasmids named M1-M4 and B2 -B4 (see Fig. l), as previously described [7, 141. Two additional clones, named B1 (kind gift of ...

Metastasis-association of the rat ortholog of the human epithelial glycoprotein antigen EGP314

“…Like EGP314 (Bergsagel et al, 1992;Bjork et al, 1993;Linnenbach et al, 1993;Perez and Walker, 1989;Strnad et al, 1989;Szala et al, 1990b) it is a type 1 transmembrane molecule with 12 conserved cystein residues and two N-glycosylation sites. Furthermore, an EGF-like domain and a thyroglobin-like repeat are conserved (Fornaro et al, 1995;Kiefer et al, 1991;Malthiery and Lissitzky, 1987). Also similar to EGP314 (Cooper et al, 1984;Fornaro et al, 1995), D5.7A has potential tyrosine phosphorylation sites.…”

“…Furthermore, it has been described for colon carcinoma cells (RemacleBonnet et al, 1997) that surface-bound plasmin induces selective proteolysis of IGF-binding protein-4 and thus, promotes autocrine IGF-II bio-availability. Taking into account that D5.7A/GA733 contains an IGF-binding domain homologous region (Fornaro et al, 1995;Kiefer et al, 1991;Malthiery and Lissitzky, 1987), a similar pathway of proteolytically induced accessibility is well conceivable. Thus, functional activity of D5.7A appears to depend largely on proteolytic cleavage of the molecule, which according to our ®ndings not only strengthens cell ± cell adhesion, but also provides a proliferative stimulus.…”

Delineation of Five Thyroglobulin T Cell Epitopes with Pathogenic Potential in Experimental Autoimmune Thyroiditis

“…These features, however, did not aid in the identification of pathogenic T cell epitope sites by biochemical methods, as this was hampered by the large size of the molecule (homodimeric mass ϭ 660 kDa). In addition, previous T cell epitope mapping efforts could not avail themselves of the mouse Tg (mTg) gene sequence information and used either the human (14) or the partial rat (15) Tg sequence data in EAT studies, relying on the high sequence homology among Tg from different species. Despite these difficulties, over the last decade and via a variety of methods, five immunopathogenic Tg peptides have been identified, encompassing at least six distinct T cell epitopes (16).…”

True

“…Thyroglobulin primers were custom designed according to the human thyroglobulin cDNA sequence reported by Malthiery and Lissitzky (1987) (Gene Bank accession number U93033). All primers were synthesized and purified by MWG-Biotech (Ebersberg, Germany).…”