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Samuel Refetoff - One of the best experts on this subject based on the ideXlab platform.
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a novel mutation in the tbg gene producing partial Thyroxine Binding Globulin deficiency glencoe identified in 2 families
European thyroid journal, 2017Co-Authors: Theodora Pappa, Lars C Moeller, Deborah V Edidin, Silvana Pannain, Samuel RefetoffAbstract:Background: Thyroxine-Binding Globulin (TBG) is the major thyroid hormone transport protein in serum. Located on the long arm of the X chromosome, TBG (SERPINA7) gene mutations most commonly produce inherited partial TBG deficiency (TBG-PD). Objective: We report a novel TBG variant associated with TBG-PD identified in 2 different families of Ashkenazi origin residing in greater Chicago. Methods: Family 1: The proband was 12.6 years old when she presented for delayed puberty and was placed on L-T4. Although her serum TSH normalized, her serum T4 remained low. Affected family members had low total T4 and T3, but a normal free T4 index, even when serum TSH concentrations were normal. Family 2: A 71-year-old male presented with a history of a nonfunctioning pituitary adenoma and normal pituitary axes except for low total T4 and T3. His brother had a similar thyroid phenotype. Results: Following direct DNA sequencing, both index patients were found to carry a missense mutation in the TBG gene (c.751T>G) producing p.V215G. The proposita of family 1 was heterozygous and the proband in family 2 was hemizygous for the mutation. Isoelectric focusing showed no alteration in the TBG isoforms and in vitro expression demonstrated a TBG with reduced affinity for T4. Conclusions: We report a novel mutation in the TBG gene in 2 unrelated families that produces a molecule with reduced affinity for T4 resulting in low serum T4. However, the physical properties of the mutant molecule remained unaltered as determined by isoelectric focusing.
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complete Thyroxine Binding Globulin tbg deficiency in two families without mutations in coding or promoter regions of the tbg genes in vitro demonstration of exon skipping
The Journal of Clinical Endocrinology and Metabolism, 2002Co-Authors: Sirimon Reutrakul, Alexandra M Dumitrescu, Paolo Emidio Macchia, George Moll, H Vierhapper, Samuel RefetoffAbstract:Inherited Thyroxine-Binding Globulin (TBG) deficiency is caused by mutations in the TBG gene located on the X-chromosome. We now describe two families (K and H) with X-linked complete TBG deficiency without mutations in the coding or promoter regions of the TBG gene. The propositi of both families presented with euthyroid hypoThyroxinemia and were found to have undetectable TBG in serum. Affected females had approximately half the normal serum TBG concentration except for one woman who also had undetectable TBG (family H). All four of her children (two boys and two girls) were affected. Affected members of family K had no mutations in any of the five exons or in the minimal promoter region of the TBG gene. However, a G to A substitution, five base pairs downstream from exon 3, was associated to the phenotype of TBG deficiency (TBG-Jackson) and was not present in 100 normal alleles. In contrast to individuals without this mutation, no TBG mRNA could be detected in fibroblasts of the propositus, expressing solely TBG-Jackson. In vitro transcription of genomic DNA containing the mutant intron in an exon trapping system showed that this mutation, reducing the consensus value on the 5' donor splice site, affects the normal splicing process. The transcript of TBG-Jackson lacks exon 3 and is unstable. The deduced amino acid sequence has a frameshift and an early stop codon at position 325. Affected subject of family H had no mutations in the TBG gene including all exons, all introns, the minimal promoter, and the 3' untranslated sequence. However, an intragenic A/G polymorphism (125 bp upstream from exon 2) was identified. It allowed us to confirm a cosegregation of the phenotype to the TBG gene and to show that the single female with complete TBG deficiency was homozygous for the polymorphic TBG allele. The cause of TBG deficiency in this family remains unknown.
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characterization and primary structures of bovine and porcine Thyroxine Binding Globulin
Molecular and Cellular Endocrinology, 2002Co-Authors: O E Janssen, Samuel Refetoff, Helmut Grasberger, Harald Lahner, Sabine A Spring, B Saller, Klaus Mann, R EinspanierAbstract:Thyroxine-Binding Globulin (TBG) is the major serum transport protein for iodothyronines in most of the large, omni- or herbivorous mammals. Characterization of human TBG (hTBG), including its 20 known natural variants, allowed the identification of the ligand-Binding site and a correlation of diminished synthesis or loss of function with mutations in the TBG gene. Further refinement of the structure-function correlation, especially the high Binding affinity and heat stability, requires characterization of other mammalian TBGs, of which only rat and sheep TBG were available. We now present some of the chemical and physical properties of bovine TBG (bTBG) and porcine TBG (pTBG) and their primary structures deduced from their cDNA sequences. The serum concentrations of bTBG and pTBG estimated by Scatchard analysis of T(4)-Binding were similar to hTBG. The T(4)-Binding affinity of human, bovine and porcine TBGs were all similar, at 1.2x10(10) M(-1). However, heat stability of the animal TBGs was reduced, with a half life of denaturation of 7 min (bTBG) and 5 min (pTBG) at 55 degreeC, compared with 21 min for hTBG. Nucleotide alignment revealed identity with hTBG of 85.5% (bTBG) and 83.7% (pTBG) and amino acid identity of 82.8% (bTBG) and 82.6% (pTBG). As expected, the relevant parts of the ligand-Binding domain (amino acids 215-291, and 363-395) were highly conserved at more than 95% similarity. Comparison of the five known mammalian TBGs allows focusing of future mutagenesis experiments to further characterize the properties of the molecule.
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complete deficiency of Thyroxine Binding Globulin tbg cd buffalo caused by a new nonsense mutation in the Thyroxine Binding Globulin gene
Thyroid, 1998Co-Authors: Gisah A Carvalho, Roy E Weiss, Adrian O Vladutiu, Samuel RefetoffAbstract:Complete deficiency of Thyroxine-Binding Globulin (TBG-CD) is defined as undetectable TBG in the serum of affected hemizygous subjects. Four distinct mutations have been identified in the TBG gene that cause this phenotype: TBG-CDJ (Japan), TBG-CD6, TBG-CD5, and TBG-CD Yonago. We report a new mutation producing TBG-CD phenotype. Five family members were studied, including two affected males with undetectable TBG in serum and two obligatory heterozygote females with borderline low values. Sequencing of the exons encoding the mature protein, adjacent introns and the promoter region, revealed differences in two nucleotides compared to the common type TBG, both located in exon 3: TGG (Trp) → TAG (Stop) at codon 280 and TTG (Leu) → TTT (Phe) at codon 283. The former mutation was not previously described and the latter is a polymorphic variant. Genotyping revealed that the two affected males had the mutant and polymorphic allele and their obligatory heterozygous mothers have each a common type and a mutant alle...
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Thyroxine Binding Globulin organization of the gene and variants
Hormone Research in Paediatrics, 1996Co-Authors: Samuel Refetoff, O E Janssen, Kyoko Takeda, Yuichi Mori, Yoshiharu Murata, Yoshitaka HayashiAbstract:Thyroxine-Binding Globulin (TBG), the principal thyroid hormone transport protein in human serum, is synthesized by the liver and secreted into the bloodstream as a 54-kD acidic glycoprotein made up of a single polypeptide chain of 395 amino acids and four heterosaccharide units. The carbohydrate chains are important for the correct posttranslational folding, secretion and degradation of the molecule but are not required for hormone Binding. TBG, encoded by a single gene copy located on Xq22, consists of five exons spanning 5.5 kbp. An upstream sequence of 218 nucleotides containing a hepatocyte nuclear factor 1 Binding motif imparts to the gene a strong liver-specific transcriptional activity. Inherited TBG defects produce three phenotypes based on the level of TBG in serum of affected hemizygotes: complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). The molecular basis of the TBG defect has been identified in 12 of 16 known TBG variants. TBG-CD is caused by either premature termination of translation or an amino acid substitution resulting in failure of secretion. Point mutations resulting in single amino acid substitutions are responsible for the alteration of the properties and/or concentration of TBG-PD variants. Gene duplication and triplication has been recently identified in subjects with TBG-E.
B Kristiansson - One of the best experts on this subject based on the ideXlab platform.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:(1998). Isoforms and levels of transferrin, antithrombin, a1-antitrypsin and Thyroxine-Binding Globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 58, No. 1, pp. 55-62.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:Carbohydrate-deficient glycoprotein syndrome type I (CDGS I) is an autosomal recessive disease with multiple organ manifestations. The diagnostic biochemical marker has been typical carbohydrate-deficient isoforms of transferrin (Tf). Many other glycoproteins in blood may show similar defects, but have not been systematically studied before. Forty-eight CDGS I patients and 22 controls were examined for total concentrations and isoform distribution of Tf, antithrombin (AT), alpha(1)-antitrypsin (alpha(1)-AT) and Thyroxine-Binding Globulin (TBG), and for the level of carbohydrate-deficient transferrin (CDT). The absolute values varied with age. The most frequent persistent quantitative changes were reduced levels of AT (97%) and elevated CDT values (100%). Isoforms lacking one to eight of four to eight possible sialic acid residues were found in AT, TBG and Tf in all cases, with variable intensity and frequency, and in all except one patient in alpha(1)-AT. The isoform changes were most constant and pronounced in Tf. The other three glycoproteins showed more abnormal heterogeneity in the youngest than in the older patients. The results indicated that the biochemical defect stabilizes with age, and suggested partial hypoglycosylation rather than non-glycosylation of these glycoproteins. Analysis of Tf isoforms is still the safest diagnostic marker of CDGS I from full-term birth and over the ages.
Onno E Janssen - One of the best experts on this subject based on the ideXlab platform.
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characterization of the Thyroxine Binding site of Thyroxine Binding Globulin by site directed mutagenesis
Molecular Endocrinology, 1999Co-Authors: Christoph Buettner, Helmut Grasberger, Kristine Hermansdorfer, Bingkun Chen, Bettina Treske, Onno E JanssenAbstract:The principal transport protein for T4 in human blood, Thyroxine-Binding Globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 1010 m−1). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded β-barrel domain encompasses the ligand-Binding site. We have now characterized the T4 Binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the β-barrel motif. Mutations within this region (Val228Glu, Cys234Trp, Thr235Trp, Thr235Gln, Lys253Ala, and Lys253Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T4 Binding. However, Binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu246Thr) without compromising the proper folding of this mutan...
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modularity of serpins a bifunctional chimera possessing α1 proteinase inhibitor and Thyroxine Binding Globulin properties
Journal of Biological Chemistry, 1999Co-Authors: Helmut Grasberger, Christoph Buettner, Onno E JanssenAbstract:Abstract An exciting application of protein engineering is the creation of proteins with novel functions by the retrofitting of native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though numerous theoretical concepts deal with the delineation of structural “modules,” their potential in the design of proteins has not yet been sufficiently exploited. To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, α1-proteinase inhibitor and Thyroxine-Binding Globulin. Substitution of two of four α1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by α-backbone distance analysis, with their Thyroxine-Binding Globulin homologues resulted in a bifunctional chimera with inhibition of human leukocyte elastase and high affinity Thyroxine Binding. To our knowledge, this is the first report on a bifunctional chimera engineered from modules of homologous globular proteins. Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily.
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molecular and structural characterization of the heat resistant Thyroxine Binding Globulin chicago
Journal of Biological Chemistry, 1995Co-Authors: Onno E Janssen, Samuel Refetoff, Bingkun Chen, Christoph Buttner, Peter Christian ScribaAbstract:Abstract Thyroxine-Binding Globulin (TBG) is the main transport protein for Thyroxine (T) in blood. It shares considerable sequence homology with α-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T Binding to TBG-N and -CH revealed no significant differences in Binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T Binding capacity after incubation at 60°C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the α-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.
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in vitro expression of Thyroxine Binding Globulin tbg variants impaired secretion of tbgpro 227 but not tbgpro 113
Journal of Biological Chemistry, 1992Co-Authors: Onno E Janssen, Samuel RefetoffAbstract:Thyroxine-Binding Globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in Thyroxine (T4) Binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.
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molecular basis of inherited Thyroxine Binding Globulin defects
Trends in Endocrinology and Metabolism, 1992Co-Authors: Onno E Janssen, Richard Bertenshaw, Kyoko Takeda, Roy E Weiss, Samuel RefetoffAbstract:Thyroxine-Binding Globulin (TBG) is a liver glycoprotein that transports thyroid hormones in serum. Inherited TBG defects appear as partial or complete deficiency and TBG excess. Sequencing of the TBG gene located on the X-chromosome has revealed nucleotide substitutions in partial TBG deficiency, and substitutions or deletions in complete deficiency variants. Whereas the deduced changes of the primary structure of the protein have been sufficient to explain the observed alterations of properties in some of the TBG variants, this has not been the case in other inherited TBG defects studied at the gene level. Further analysis of these and other variants may provide helpful information on glycoprotein synthesis and processing and on protein-hormone interaction.
H Stibler - One of the best experts on this subject based on the ideXlab platform.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:(1998). Isoforms and levels of transferrin, antithrombin, a1-antitrypsin and Thyroxine-Binding Globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 58, No. 1, pp. 55-62.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:Carbohydrate-deficient glycoprotein syndrome type I (CDGS I) is an autosomal recessive disease with multiple organ manifestations. The diagnostic biochemical marker has been typical carbohydrate-deficient isoforms of transferrin (Tf). Many other glycoproteins in blood may show similar defects, but have not been systematically studied before. Forty-eight CDGS I patients and 22 controls were examined for total concentrations and isoform distribution of Tf, antithrombin (AT), alpha(1)-antitrypsin (alpha(1)-AT) and Thyroxine-Binding Globulin (TBG), and for the level of carbohydrate-deficient transferrin (CDT). The absolute values varied with age. The most frequent persistent quantitative changes were reduced levels of AT (97%) and elevated CDT values (100%). Isoforms lacking one to eight of four to eight possible sialic acid residues were found in AT, TBG and Tf in all cases, with variable intensity and frequency, and in all except one patient in alpha(1)-AT. The isoform changes were most constant and pronounced in Tf. The other three glycoproteins showed more abnormal heterogeneity in the youngest than in the older patients. The results indicated that the biochemical defect stabilizes with age, and suggested partial hypoglycosylation rather than non-glycosylation of these glycoproteins. Analysis of Tf isoforms is still the safest diagnostic marker of CDGS I from full-term birth and over the ages.
U Holzbach - One of the best experts on this subject based on the ideXlab platform.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:(1998). Isoforms and levels of transferrin, antithrombin, a1-antitrypsin and Thyroxine-Binding Globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 58, No. 1, pp. 55-62.
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isoforms and levels of transferrin antithrombin a1 antitrypsin and Thyroxine Binding Globulin in 48 patients with carbohydrate deficient glycoprotein syndrome type i
Scandinavian Journal of Clinical & Laboratory Investigation, 1998Co-Authors: H Stibler, U Holzbach, B KristianssonAbstract:Carbohydrate-deficient glycoprotein syndrome type I (CDGS I) is an autosomal recessive disease with multiple organ manifestations. The diagnostic biochemical marker has been typical carbohydrate-deficient isoforms of transferrin (Tf). Many other glycoproteins in blood may show similar defects, but have not been systematically studied before. Forty-eight CDGS I patients and 22 controls were examined for total concentrations and isoform distribution of Tf, antithrombin (AT), alpha(1)-antitrypsin (alpha(1)-AT) and Thyroxine-Binding Globulin (TBG), and for the level of carbohydrate-deficient transferrin (CDT). The absolute values varied with age. The most frequent persistent quantitative changes were reduced levels of AT (97%) and elevated CDT values (100%). Isoforms lacking one to eight of four to eight possible sialic acid residues were found in AT, TBG and Tf in all cases, with variable intensity and frequency, and in all except one patient in alpha(1)-AT. The isoform changes were most constant and pronounced in Tf. The other three glycoproteins showed more abnormal heterogeneity in the youngest than in the older patients. The results indicated that the biochemical defect stabilizes with age, and suggested partial hypoglycosylation rather than non-glycosylation of these glycoproteins. Analysis of Tf isoforms is still the safest diagnostic marker of CDGS I from full-term birth and over the ages.