Sequencing of the variant thyroxine-binding globulin (TBG)-Quebec reveals two nucleotide substitutions.

Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1987 a variant TBG was discovered in an infant born in Quebec, following an investigation prompted by the finding of low blood thyroxine (T4) level on screening for neonatal hypothyroidism. This variant, TBG-Quebec, has cathodal shift on isoelectric focusing, reduced affinity for thyroxine, and markedly reduced stability. The latter property of the variant molecule is probably responsible for the partial TBG deficiency. We now report the results of sequencing of the entire coding region and exon-intron junctions of TBG-Quebec, which revealed two nucleotide substitutions; one, located in exon 3, changes the normal codon 283 of TTG (leucine) to that of TTT (phenylalanine), and the other, in exon 4, results in the replacement of the normal histidine-331 (CAT) by tyrosine (TAT). Allele-specific amplification (ASA) confirmed the cosegregation of the two nucleotide substitutions with the TBG-Quebec phenotype in individual members of this family. The substitution in codon 283, but not that in codon 331, has been previously described and, when occurring alone, does not alter the properties of the gene product. Thus, it appears that the replacement of histidine-331 by tyrosine is responsible for the observed altered properties of TBG-Quebec. However, the question of whether substitution of both amino acids is necessary for expression of the variant phenotype has yet to be answered.     

Sequence of the variant thyroxine-binding globulin (TBG) in a Montreal family with partial TBG deficiency

Summary The variant thyroxine-binding globulin in a family from Montreal (TBG-M) has a reduced affinity for thyroxine, shows a slight cathodal shift on isoelectric focusing, and has an increased susceptibility to inactivation by heat and acid. We present the molecular basis for TBG-M, deduced by sequencing the entire 1245-bp coding regions and intron/exon junctions of the TBG gene of an affected hemizygous male. A single nucleotide substitution in the codon for amino acid 113 of the mature protein (GCC to CCC) was found, resulting in the replacement of alanine by proline. The mutation was confirmed by allele-specific amplification of genomic DNA from the propositus and three other affected family members. Since point mutations throughout the molecule have been shown to alter the properties of variant TBG_S, and because amino acid substitutions with proline are known to impair stability and function of proteins, the replacement of alanine 113 by proline provides a logical explanation for the observed properties of TBG-M.Presented in part at the 72nd Annual Meeting of the Endocrine Society in Atlanta, Georgia, 1990     

The genetic polymorphism of the thyroxine-binding globulin (TBG)

Summary Hereditary deficiency of the thyroxine-binding globulin (TBG) has been described in 17 families, in 12 of which it is X-chromosomal inherited. Different modes of transmission can be assumed for some of the other families but are not yet warranted. Genetic elevation of TBG has been described in 7 families. This trait is presumably uniformly X-chromosomal inherited.

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Zusammenfassung Ein erblicher Mangel von thyroxinbindendem Globulin (TBG) ist bisher bei 17 Familien beschrieben worden, von denen bei 12 Familien eine X-chromosomale Vererbung angenommen werden kann. Bei einigen der übrigen Familien muß eine andersartige Vererbung angenommen werden, doch konnte kein anderer Erbgang bisher gesichert werden. Eine genetische Vermehrung des thyroxinbindenden Globulin wurde bei 7 Familien beschrieben. Dieses Merkmal wird vermutlich in allen Fällen X-chromosomal vererbt.

     

Genetic studies on human thyroxine-binding globulin (TBG)

Summary An enzyme immunoassay technique combined with Western blotting is described to demonstrate thyroxine-binding globulin (TBG) by isoelectric focusing in thin-layer polyacrylamide gels with 8mol/l urea. Quantitative evaluation was by laser densitometry. No genetic charge variants of TBG were encountered in a sample of 840 unrelated individuals from southwestern Germany. There was no correlation between structural and quantitative variations in the TBG protein. Results from a family with quantitative TBG deficiency strongly support the postulated X-linked mode of inheritance. The method described can be considered as an additional diagnostic tool in thyroid evaluation.     

A senescence up-regulated protein: the rat thyroxine-binding globulin (TBG)

Thyroxine-binding globulin (TBG), the major carrier of thyroid hormones in human serum, was thought to be absent in most species, including rodents. We demonstrated recently that in fact the rat possesses a TBG gene, virtually non-expressed in young adults, bu actively transcribed during post-natal development. We now find that the TBG gene is als increasingly re-expressed during senescence. Evidence is presented suggesting that physiologically decreased thyroid hormone levels, characteristic of neonates and of ageing rats, might consitute a common factor inducing up-regulation of TBG in both developmental and ageing processes. Rat TBG is to our knowledge the first biochemical ‘positive’ (i.e increasing) marker of non-pathological senescence, expressed at both biosynthetic and bloodstream levels.     

Cloning of a complementary deoxyribonucleic acid coding for human thyroxine-binding globulin (TBG): existence of two TBG messenger ribonucleic acid species possessing different 3'-untranslated regions

An adult human liver cDNA library constructed in expression vector, bacteriophage lambda gt11, was screened with polyclonal antibody directed against human T4-binding globulin (TBG). TBG cDNA cloned in the present study was 944 nucleotides in length. It contained approximately 70% of the coding region and complete 3'-untranslated region. When the sequence was compared with that of TBG cDNA recently cloned by I. L. Flint, T. J. Bailey, T. A., Gustafson, B. E. Markham, and E. Morkin, the 3'-untranslated region of our cDNA was 231 nucleotides shorter than their cDNA. These results indicated that two TBG mRNAs with different length of 3'-untranslated regions may exist in human liver. Indeed, Northern blot analysis revealed that two TBG mRNAs differing in the length approximately 200 base pairs were present in normal human liver as well as in human hepatoma cell line (HepG2). It was demonstrated that this size difference was due to the length of 3'-untranslated region by hybridization with a probe specific to the longer 3'-end. Together with the sequence data, it was suggested that these two TBG mRNA species may be produced by alternative processing and polyadenylation at two different sites.     

In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113.

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.     

Human thyroxine-binding globulin (TBG): Heterogeneity within individuals and among individuals demonstrated by isoelectric focusing

Isoelectric focusing of human plasma samples labeled in vitro with [¹²⁵I]-thyroxine reveals considerable biochemical and genetic variation in thyroxine-binding globulin. (1) In all individuals tested, at least three primary isoelectric bands are seen in the pH range of 4.2 to 4.5, with additional bands at lower pH ranges. Similar patterns are produced by plasma from nonhuman primates. These band differences appear to be the result of differences in sialic acid content. TBG produces a single electrophoretic band on standard polyacrylamide gel electrophoresis. (2) Genetically determined, X-linked differences in electrophoretic mobility of TBG are observed in several human populations. Female homozygotes or male hemizygotes for the TBG slow variant (TBG-S) produce band patterns shifted by 0.5 pH unit cathodal to the common pattern (TBG-C). Female heterozygotes produce patterns with six-plus bands, representing the simple sum of the common and slow types. This difference is not the result of differences in sialic acid content. The gene frequency of this variant is 10% in American Blacks. (3) In pregnant women additional anodal bands are observed, giving the impression of a shift, by integral steps, in the pattern relative to the nonpregnant type. This shift is abolished by mild neuraminidase treatment.This work was supported by a grant from the O'Brien family of Houston, Texas.     

Genetic polymorphism of thyroxin-binding globulin (TBG) in the Pacific area.

Human plasma samples, radiolabeled with [125I]thyroxin, from the Asian, Pacific, and Australian area have been subjected to isoelectric focusing to reveal genetic variation in thyroxin-binding globulin (TBG). A genetically determined electrophoretic slow variant, TBG S, indistinguishable from the variant found in black Africans, has been observed with a frequency of 1%-10% in all Melanesian and Polynesian populations studied. The TBG S variant is present also with low frequency in Micronesians and in some Indonesian populations. However, East Asians, Indians, and Australian Aborigines were found to be monomorphic.     

Thyroxine binding globulin (TBG) assay by radial immunodiffusion.

Thyroxine-binding globulin was measure by radial immuno-diffusion using ten microliters of serum. The mean value in a normal group was 1.95 +/- 0.38 mg/dl. The values obtained by this method were in good agreement with the thyroxine-binding capacity determined by paper electrophoresis (r = 0.956).     

Studies on human thyroxine-binding globulin. 8. Isoelectric focusing evidence for microheterogeneity of thyroxine-binding globulin

Highly purified thyroxine-binding globulin from pooled human serum homogeneous by conventional criteria, subjected to isoelectric focusing in polyacrylamide gels in a pH gradient from 3–6, produced a pattern of at least nine stainable protein bands. All of these bands appeared to bind thyroxine. Completely desialylated thyroxine-binding globulin subjected to isoelectric focusing produced the same number and pattern of bands located at a different area in the pH gradient. Thyroxine-binding globulin purified from the serum of a single donor was subjected to isoelectric focusing. This thyroxine-binding globulin had the same pattern of protein bands with the exception that one of the major bands seen in the thyroxine-binding globulin from pooled serum was absent. Several possible explanations for these phenomena are discussed.     

Characterization of the thyroxine-binding site of thyroxine-binding globulin by site-directed mutagenesis.

The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 10(10) 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 beta-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 beta-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 mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within the beta-barrel. The importance of the binding site's aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe249 in the middle of strand 3B completely abolished T4 binding, while the substitution of several other phenylalanines had no effect.     

Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.     

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.