Comparison of the action of the sera of pregnant women and of trophoblastic beta 1-glycoprotein in a number of immunological tests

The effects of blood sera of pregnant women in the I, II and III trimesters (SPWI, SPWII, SPWIII) were compared with those of purified preparations of trophoblastic beta 1-glycoproteid (TBG) on phytohemagglutinin (PHA) induced proliferative response of lymphocytes of the intact donors and on the production of a factor inhibiting the migration of leucocytes (FIM-L). SPWI and purified TBG preparations in doses corresponding to its level in SPWI were shown to produce similar effects. With the increase of TBG concentration, however, the inhibiting effect of its preparations on the proliferation of lymphocytes increased much more intensively than that of SPWII and SPWIII containing the same dose of this protein. The stimulating effect of TBG on the production of FIM-L increased with the dose as well while SPWII and SPWIII did not exert any effect on the migration of leucocytes. A suggestion is put forward that SPWII and SPWIII contain factors modifying the effect of TBG.     

Molecular cloning and primary structure of rat thyroxine-binding globulin

Rat thyroxine-binding globulin (TBG) cDNAs were isolated from a rat liver cDNA library by using a human TBG cDNA as a probe. From two overlapping cDNA inserts, an aligned cDNA sequence of 1714 nucleotides was obtained. There was 70% homology with human TBG cDNA over the span of 1526 nucleotides. In order to confirm that the cloned cDNA encodes rat TBG and to localize the NH2-terminal amino acid of the mature molecule, the protein was purified by affinity chromatography and subjected to direct protein microsequencing. The NH2-terminal amino acid sequence was identical with that deduced from the nucleotide sequence. The rat TBG cDNA sequenced consisted of a truncated leader sequence (35 nucleotides), the complete sequence encoding the mature protein (1194 nucleotides) and the 3'-untranslated region (485 nucleotides), containing two polyadenylation signals. It was deduced that rat TBG consists of 398 amino acids (Mr = 44,607), three NH2-terminal residues more than human TBG, with which it shares 76% homology in primary structure. Of the six potential N-glycosylation sites, four are located in conserved positions compared to human TBG. Northern blot analysis of rat liver revealed an approximately 1.8-kilobase TBG mRNA. Its amount increased markedly following thyroidectomy and decreased with thyroxine treatment in a dose-dependent manner.     

Effect of estrogen on the synthesis and secretion of thyroxine-binding globulin by a human hepatoma cell line, Hep G2

Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.     

Effect of tunicamycin and monensin on secretion of thyroxine-binding globulin by cultured human hepatoma (Hep G2) cells

We have reported in the preceding paper that human hepatoma (Hep G2) cells synthesize thyroxine-binding globulin (TBG). In this paper, we evaluated the kinetics of secretion of the protein and the effects produced by the ionophore monensin and the glycosylation inhibitor tunicamycin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. TBG appeared in the medium after 10 min, and 50% of the protein was secreted after 45 min. After 2 h, more than 85% of TBG had been released. The rate of secretion of TBG was much slower than that of albumin, 50% of which was secreted after 20 min. Monensin, 1 microM, caused a marked delay in TBG secretion, with 50% released after 80 min. After 2 h, less than 60% had been released and a plateau was approached. Endoglycosidase H (endo H) treatment of intracellular and secreted TBG showed no alteration in the rate of conversion of TBG oligosaccharide units from high-mannose type (endo H-sensitive) to complex type (endo H-resistant), thus suggesting that monensin impeded the exit of TBG from the Golgi apparatus without affecting the terminal glycosylation of the protein. Tunicamycin, 5 micrograms/ml, completely blocked glycosylation and markedly affected TBG secretion, almost doubling the time required for the secretion of 50% of the protein. The effect was specific for TBG, since it was not observed in the case of albumin. After 2 h, only 56% of the protein had been released. Analysis of intracellular and extracellular immunoprecipitated products revealed the presence of aggregates (Mr greater than 100,000). The lack of carbohydrates, although not preventing TBG secretion, had marked quantitative effects, and increased the susceptibility to aggregation.     

TBG-dependency of age related variations of thyroxine and triiodothyronine.

Thyroxine (T4), triiodothyronine (T3) and thyroxine-binding globulin (TBG) were determined in healthy individuals ranging in age from newborn to 95 years. T4: 10.25 +/- 1.62 microng/100 ml, T3: 1.62 +/- 0.35 ng/ml and TBG: 1.34 +/- 0.15 mg/100 ml, were found elevated until puberty compared to a middle age group with T4: 7.27 +/- 2.26 microng/100 ml, T3: 1.15 +/- 0.24 ng/ml and TBG: 0.98 +/- 14 mg/100 ml. T4 and T3 followed almost TBG concentration. In old age is dissociation between T4: 5.79 +/- 1.56 microng/100 ml, T3: 0.79 +/- 0.21 ng/ml and TBG: 1.28 +/- 0.15 mg/100 ml was found. Except for old age the ratio T4/TBG and T3/TBG minimized the age dependent variation of T4 and T3 and reduced the coefficient of variance from 26% to 17.7% for T4 and from 26.5 to 25% for T3. Age reduction of T4/TBG is 15% and of T3/TBG 13% respectively more pronounced than for T4 and T3 alone. These data indicate: 1) age related variations of T4 and T3 due to age dependency of TBG, 2) deviation of T4 and T3 values in old age from that expected by their TBG levels and 3) the importance of the routine use of hormone/TBG ratio.     

Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.     

Immunochemical analysis of lectin receptors in the structure of trophoblastic beta 1-glycoprotein and pregnancy-associated alpha 2-glycoprotein

Trophoblastic beta-glycoprotein (TBG), pregnancy-associated alpha 2-glycoprotein (AGP) and alpha-fetoprotein (AFP) bind to concanavalin A, phytohemagglutinin P and pea lectin. Lentil lectin interacts with TBG only, whereas peanut lectin and castor bean lectin are characterized by affinity to AGP. Hence, TBG, AGP and AFP contain the following carbohydrate components: alpha-D-mannose, alpha-D-glucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. In addition, AGP contains beta-D-galactose.     

Characterization of a major development-regulated serum thyroxine-binding globulin in the euthyroid mouse.

We confirm our finding of a major development-regulated thyroxine-binding globulin (TBG) in the serum of the euthyroid mouse and investigate a number of its binding, structural and regulatory properties. Between 16 days foetal and 60 days postnatal life, the thyroxine (T4)- and tri-iodothyronine (T3)-binding activities of the sera show a striking ontogenic pattern: the binding is 2-3 times higher in foetuses than in mothers, then further increases after birth, reaching between 3 and 5 days maximum values which are 7-8 times higher than the adult ones. This pattern is not correlated with the ontogenesis of the acknowledged specific (transthyretin, TTR) and non-specific (albumin, alpha 1-foetoprotein) thyroid-hormone carriers of the mouse sera. PAGE studies demonstrate that the protein responsible for the elevated binding of the perinatal period is an alpha 1-globulin, with a migration similar to that of human and rat TBGs. Scatchard analysis is consistent with the notions that the T4-binding sites of TBG have high association constants, about two orders of magnitude above the T4 sites of TTR (10(9) M-1 as against 10(7) M-1) and low capacities (37 and 4 nmol/g of serum proteins in pups and adults respectively). Isoelectric focusing (i.e.f.) demonstrates that mouse TBG is a microheterogeneous protein separable, as a function of the pH gradient, in up to 10-12 isoforms, Marked shifts of the relative abundance of isoforms in the course of development are evidenced. The modulation of the TBG binding activity by non-esterified fatty acids (NEFA) and the control of its synthesis by the thyroid status are also reported. Mono- and poly-unsaturated NEFAs are strong inhibitors of the TBG, although they affect TTR less readily. On the other hand, the biosynthesis and/or secretion of TBG, but not of TTR, is under thyroid-hormone control, experimental hypothyroidism inducing a marked increase of the serum TBG. The TBG of mouse behaves as a highly significant parameter of development, pointing to a likely important function of the protein in the process of maturation. Our finding of major TBGs in both euthyroid rats and mice suggests that TBG is more widely spread than was thought until now, but difficult to detect in certain species outside definite maturation stages.     

A thyroxine-binding globulin of major biological significance in the serum of mice: demonstration and ontogenesis

We demonstrate in the mouse serum a hitherto unrecognized major thyroxine binding globulin (TBG), analogous to human TBG or to the recently discovered rat TBG. Our demonstration is based on equilibrium dialysis, electrophoresis, immunoelectrodiffusion and autoradiography techniques. Mouse TBG displays a remarkable ontogenic pattern, with 2-3 times higher activity in foetal than in maternal serum, and a further dramatic increase after birth. Between 1 and 5 days, the T4 binding to serum reaches peak levels 7-10 times more elevated than those measured in normal or pregnant adults. We also present for the first time the ontogenesis of the thyroxine binding prealbumin (TBPA), considered until now as the only specific T4 carrier of the murine species. We show that throughout development it is the TBG, not the TBPA, which crucially governs the level of the T4-serum interactions.     

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.     

Clinical study on increased serum thyroxine-binding globulin in cancerous state

Serum thyroxine-binding globulin (TBG) in 169 patients with various cancers was determined by radioimmunoassay (RIA). Eleven patients showed a high serum TBG level (greater than 35 micrograms/ml). Two of them had been treated with estrogen for prostate cancer. One patient had high serum TBG with serum hepatitis. Another 8 cases had normal liver function and also normal levels serum estrogen. Thus, about 4.7% (8/169) of the cancer patients had high serum TBG and mild hyperthyroxinemia caused by unknown mechanisms. The high TBG level in these patients continued until just before death, or in some cases decreased to normal after removal of cancer tumors by operation. Cancer is occasionally associated with an increase in serum TBG. Although the mechanism is not clear, the increased TBG in the cancerous state in interesting and has significance as a tumor marker.     

Concentration of thyroxine-binding globulin: value of direct assay.

The concentration of thyroxine-binding globulin (TBG) in the serum can now be measured by direct assays that are simple and inexpensive. Comparison of a direct measurement of TBG concentration with a widely used indirect method (Thyopac-3) showed that the indirect method was inaccurate when TBG concentrations were high. This will result in an increase in the derived free thyroxine index (FTI), so that euthyroid patients with a raised TBG concentration may be at risk of being labelled thyrotoxic. Correction of serum total thyroxine (T4) concentration according to the actual TBG concentration (T4:TBG ratio) provided a better correlation with thyroid state than FTI.     

A variant of porcine thyroxine-binding globulin has reduced affinity for thyroxine and is associated with testis size

The field of genomics applies the dissection of genetic differences toward an understanding of the biology of complex traits. Quantitative trait loci (QTL) for testis size, plasma FSH in boars, and body composition (backfat) have been identified near the centromere on the X chromosome in a Meishan-White Composite resource population. Since thyroid function affects Sertoli cell development and adult testis size in rodents, and thyroxine-binding globulin (TBG) maps to this region on the porcine X chromosome, TBG was a positional candidate gene for testis size. We discovered a polymorphism in exon 2 of the porcine TBG gene that results in an amino acid change of the consensus histidine to an asparagine. This single nucleotide polymorphism (SNP) resides in the ligand-binding domain of the mature polypeptide, and the Meishan allele is the conserved allele found in human, bovine, sheep, and rodent TBG. Binding studies indicate altered binding characteristics of the allelic variants of TBG with the asparagine (White Composite) isoform having significantly greater affinity for thyroxine than the histidine (Meishan) isoform. Alternate alleles in boars from the resource population are also significantly associated with testis weight. Therefore, this polymorphism in TBG is a candidate for the causative variation affecting testis size in boars.     

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.     

Thyroxine-binding globulin variant (TBG-Kumamoto): identification of a point mutation and genotype analysis of its family.

Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial or complete TBG deficiencies have been shown to be caused by either one or two nucleotide substitutions, or one nucleotide deletion in the coding regions of the TBG gene. In this report, a Japanese female patient (proband) with hyperthyroid state, whose lower TBG levels did not return to normal under the euthyroid state after treatment was examined. Genomic DNA samples from the proband with thyroxine-binding globulin deficiency (termed TBG-Kumamoto) and her family were subjected to the polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide substitution in the codon for the amino acid 363 of native TBG molecule (CCT to CTT) was found, resulting in the replacement of proline by leucine. It was revealed that the proband was a heterozygote and her father was a hemizygote. The mutation was confirmed by the allele-specific amplification of genomic DNAs from the proband and her father using oligonucleotide primers of normal or mutant residues at the 3' position in the polymerase chain reaction. These results indicate that the abnormality of TBG-Kumamoto is the consequence of this mutation. Genetically, this point mutation observed in TBG-Kumamoto might be classified as a new type of TBG deficiency.     

Comparative characteristics of molecular forms of human thyroxine-binding globulin

Two molecular variants, of thyroxin-binding globulin (TBG), TBG-1 and TBG-2, were obtained from human retroplacental blood by fractionation of pure TBG on concanavalin A-Sepharose. It was found that both variants are immunologically identical, have similar molecular weights, amino acid composition and spectral properties, and possess the same affinity for thyroid hormones. However, TBG-1 and TBG-2 differed in charge upon isoelectrofocusing and had different monosaccharide composition. The existence of two molecular variants of TBG in pregnancy is probably due to the peculiarities of the polypeptide chain glycosylation during TBG biosynthesis.     

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.