Yoshiharu Murata

Regulation of Glycosaminoglycan Synthesis by Thyroid Hormone in Vitro

Human skin fibroblasts synthesize and accumulate glycosaminoglycans (GAG). Recently, we reported that fibroblasts incubated in thyroid hormone-deficient media accumulate more GAG than do cultures incubated in the same media enriched with 0.1 muM triiodothyronine (T(3)) (1981. Endocrinology. 108: 2397). The current study characterizes that enhanced accumulation. Confluent cultures were maintained in thyroid hormone-deficient media without or with added T(3), labeled with [(3)H]acetate and analyzed for total [(3)H]GAG and [(3)H]hyaluronic acid content. Addition of T(3) to thyroid hormone-depleted media consistently inhibited the incorporation of [(3)H]acetate into GAG by 28-60% in fibroblast cultures from four different normal human donors. Maximal inhibitory effect was observed within 3 d after hormone addition at concentrations > 1 nM. 73% of the maximal inhibitory effect was observed in the presence of physiologic concentrations of T(3) (0.16 nM total T(3) or 1.4 pM free T(3)). The following observations indicated that T(3) inhibition of [(3)H]GAG accumulation is most likely due to a decrease in GAG synthesis rather than to changes in the acetate pool or GAG degradation: (a) Addition of 0, 100, 500, and 2,500 muM unlabeled acetate progressively decreased [(3)H]acetate incorporation into GAG, up to 80%, without altering the further inhibitory effect of T(3) (35-40%); (b). A similar effect of T(3) on GAG (32% inhibition) was observed using [(3)H]glucosamine as substrate; (c) T(3) decreased hyaluronate synthetase activity by 32%; and (d) There was no effect of T(3) on GAG degradation in a pulse-chase experiment. The effect of T(3) on [(3)H]GAG accumulation appears to be quite specific, since the hormone had no effect on the incorporation of [(3)H]leucine into trichloroacetic acid-precipitable material.Thus, thyroid hormone inhibits GAG accumulation in a dose-, time-dependent, and reversible manner. This inhibition is apparently due to specific effects on the rate of macromolecular synthesis.

Variant thyroxine-binding globulin in serum of Australian Aborigines: its physical, chemical and biological properties

Low serum total thyroxine (TT4) and triiodothyronine (TT3) is found in approximately 40% of Australian Aborigines. Studies were carried out to characterize the properties of thyroxine-binding globulin (TBG) in these Australian Aborigines to explain the observed reduction of thyroid hormone concentration in their serum. TBG from Aborigines with low serum TT4 concentrations was compared to TBG fram Aborigines with normal TT4 concentration and Caucasians and American Blacks with normal or reduced serum TBG levels due to familial partial TBG deficiency. TBG from Aborigines with low serum TT4 concentrations had a reduced affinity for thyroid hormone (Ka). The Ka for T4 was 54% and for T3 30% of the Ka values for TBG from Aborigines with normal TT4 concentration or non-Aborigines. Maximal binding values were in agreement with TBG measurements by RIA for Aborigines with low or normal serum TT4 and for non-Aborigines. An increase in the rate of heat denaturation of TBG at temperatures from 54 to 60 C was also observed in sera from Aborigines with low TT4. The heat lability was lowered by 2 C. The low concentration of TT4 in serum of these Aborigines could not explain this higher heat lability of TBG since only addition of greater than 80-fold the physiologic T4 concentration obliterated the difference of heat inactivation by denaturation. Nevertheless, decreased T4 occupancy of this TBG with lower affinity for thyroid hormone may explain reduced stability at high temperatures. There were no differences in the microheterogeneity by isoelectric focusing between TBGs from Aborigines with low serum TT4 concentration and those with normal TT4 or non-Aborigines. From data on maximal binding capacity and TBG measurement by radioimmu-noassy it could be determined that TBG in these Aborigines as in non-Aborigines has a single thyroid hormone binding site. These results indicate that euthyroid Aborigines with low serum TT4 and TT3 concentrations have a variant TBG with reduced affinity for these hormones. The difference between this variant TBG as compared to the more common type of TBG in non-Aborigines appears to reside in the polypeptide chain rather than in the carbohydrate moiety. It fully accounts for the reduced serum total thyroid hormone concentration in the presence of clinical euthyroidism with normal serum free T4 and thyrotropin levels.

Thyroxine-Binding Globulin: Organization of the Gene and Variants

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.

Variant thyroxine-binding globulin in serum of Australian Aborigines: a comparison with familial TBG deficiency in Caucasians and American Blacks

About 40% of clinically euthyroid Australian Aborigines have low concentrations of total thyroxine (TT4) and triiodothyronine (TT3) in serum. While the finding of normal concentrations of serum thyrotropin (TSH) in such individuals is compatible with their eumetabolic state, the reason for the finding of a low free T4 index (FT4I) has been unclear. A genetic variant of T4-binding globulin (TBG) with reduced affinity for T4 has been suggested but decrease in the absolute concentration of TBG has also been reported. In this study, we measured various parameters of thyroid function in 20 serum samples from euthyroid Australian Aborigines selected for their low TT4 levels. Results were compared to those obtained in serum samples from Caucasians and American Blacks with inherited partial TBG deficiency, 15 of which were matched to the Aborigines by their TBG and 20 by their TT4 concentrations. Results were also compared with those from another group of 20 samples from Caucasians and American Blacks with normal TBG concentration, matched to the Aborigines by their serum TT4 concentration. TBG in serum from these Australian Aborigines was immunologically identical to that in Caucasians and American Blacks in terms of parallelism of serially diluted samples in the TBG radioimmunoassay (RIA). Comparison of measurements of TBG concentration by RIA and by T4-binding capacity (CAP) gave identical results indicating that, in Aborigines as in non-Aborigines, TBG molecules have a single T4-binding site. Serum TBG concentration in this group of Aborigines with lowTT4was 1.1 ± 0.3 mg/dl (mean ± SD) and significantly lower (p< 0.001) than that in non-Aborigines of 1.6 ± 0.2 mg/dl. When the Aboriginal samples were matched to euthyroid Caucasians and American Blacks by their TBG concentration, their mean TT4 concentration was significantly lower (4.1 ± 0.8µg/dl vs. 5.6 ± 1.1 µg/dl, p < 0.001), as was their mean TT3 concentration (88 ± 17 ng/dl vs. 139 ± 27 ng/dl, p < 0.001). In contrast when matched by their TT4 concentration, the mean serum TBG level was significantly higher (1.1 ± 0.3 mg/dl vs. 0.7 ± 0.3 mg/dl, p < 0.001). TSH concentration was normal and not significantly different in the Aborigines and the two non-Aboriginal groups with familial partial TBG deficiency. All serum samples from non-Aborigines with normal TBG which were matched to the Aborigines by their TT4 concentration had high serum TSH values and thus belonged to patients with primary hypothyroidism. The mean FT4I value was significantly (p < 0.001) lower in the Aborigines (4.9 ± 1.0) as compared to the two euthyroid non-Aboriginal groups matched by their TBG or TT4 (7.4 ± 0.8 and 7.5 ± 1.3, respectively). In contrast, the free T4 (FT4) values measured by equilibrium dialysis in 5 Aboriginal samples spanned over the same normal range as that of 8 euthyroid non-Aborigines. There was an excellent correlation between the FT4I and FT4 in both Aborigines and non-Aborigines (r = 0.957 and 0.918 respectively). However, the slopes of the two regression lines were significantly different (p < 0.001). These results indicate that the degree of reduction of TT4 and TT3 concentration in euthyroid Australian Aborigines cannot be explained solely on the basis of diminished serum TBG level. They support the hypothesis that such individuals have a variant TBG with reduced affinity for T4 and T3.

A New Form of X-Chromosome Linked TBG Abnormality with No Demonstrable T4-Binding Activity

Diagnostic work-up for short stature uncovered TBG deficiency in an 11 year old girl (propositus) with XO Turner’s syndrome. Studies on members of the family revealed that the two hemizygous affected subjects (propositus and father) had, compared to the mean normal levels, 1.5% native (n) TBG and 9-fold to 10-fold increased denatured (dn) TBG, both measured by specific RIAs. The heterozygous sister had 30% nTBG and 5.6-fold increased dnTBG, while the mother had normal levels (Table 1).

Direct application of radioiodinated aminoacyl tRNA for radiolabeling nascent proteins

A two-step procedure to incorporate 125I-iodotyrosine into protein synthesized in a reticulocyte lysate is described. In the first step, the iodination of tyrosyl tRNA was catalyzed by a solid-state glycouril compound. More than one-third of 200 microCi of radioiodine became bound to 70 micrograms of aminoacyl tRNA after 15 min at 0 degrees C. The isotope was distributed in a three-to-one ratio of monoiodotyrosine to di-iodotyrosine. In the second step, the soluble product of the radioiodination was transferred directly into a nuclease-treated reticulocyte lysate coded with RNA isolated from the human hepatoma cell line Hep G2. Fractional recovery of radioiodine in nascent protein was maximally 7.6%. Reaction of the product of translation with antibody against alpha-antitrypsin separated an 125I-containing protein having a molecular weight estimated as 47,000. The synthesis of unprocessed alpha-antitrypsin was confirmed by cleavage of the labeled protein with leader peptidase and by its displacement from immunocomplex formation with purified alpha-antitrypsin. The amount of 125I incorporated into alpha-antitrypsin was proportionate to iodinated tRNA additions up to a concentration of 70 micrograms/ml. The synthesis of alpha-antitrypsin as detected in radioautograms after gel electrophoresis was more than twice as sensitive using radioiodinated aminoacyl tRNA as compared with [35S]methionine. Iodine labeling of thyroxine-binding globulin was also demonstrated in the translation product of Hep G2 RNA. Since the specific activity of the radioiodine is high and the means for detection of the isotope efficient, the method described can facilitate the demonstration of quantitatively minor translation products.