Study on the Physiological Role of Carbohydrate Residues in Thyroxine Binding Globulin Using Introduction of its Gene by Transfection

甲状腺激素结合球蛋白(TbG)是血清中主要的甲状腺激素结合蛋白，在肝脏中合成。由于结构上的相似性，该蛋白属于α1-抗胰蛋白酶家族。本研究探讨了TBG中碳水化合物含量的生理意义。首先，我们使用了合成TBG和皮质类固醇结合球蛋白(CBG，也是α1-抗胰蛋白酶家族成员)的肝细胞系HepG2。当细胞用衣霉素(核心糖基化抑制剂)处理时，TBG永远不会分泌到培养液中。另一方面，CBG不需要糖基化就可以分泌到培养液中。然而，衣霉素处理后，TBG和CBG的细胞内降解率均增加。在衣霉素存在下合成的TBG与不与甲状腺激素结合的反变性TBG发生交叉反应。因此，TbG中的碳水化合物部分在T4结合、分泌和细胞内…中起重要作用。为了进一步研究碳水化合物残留物在TBG中的作用，对Murata等人在一名患者中发现的TBG-Gary进行了研究。TBG-Gary的特征是T4结合减少，等电聚焦和热不稳定性的注释移动。TbG基因组结构分析表明，外显子I的单点突变导致第96位氨基酸“异亮氨酸”替换为“天冬氨酸”。这种氨基酸取代导致了一个潜在的糖基化位点(天冬氨酸-半胱氨酸-丝氨酸)的产生。构建了编码正常TbG和TbG-Gary的完整cDNA，并将其克隆到表达载体pEUK-C1中。将所构建的重组真核表达载体分别导入Cos-1细胞，脉冲追逐实验表明，该重组蛋白确实含有额外的糖类残基，减少了T4结合，并与反变性的TBG发生交叉反应。此外，有迹象表明，TBG-Gary在细胞中的降解速度要快得多。通过定点突变，将突变的TBG-Gary中的天冬酰胺残基替换为天冬氨酸，突变的TBG表现出与正常TBG相似的行为。这些研究表明，TBG中的碳水化合物残基在激素结合、细胞转运和代谢中起重要作用。较少

Thyroxine-Binding Globulin (TBG) is a principal thyroxine-binding protein in serum and is synthesized in the liver. The protein belongs to alpha1-antitrypsin family because of its structural similarity. In this investigation, physiological significance of carbohydrate moiety in TBG was studied. At first, we employed hepatome cell line HepG2 which synthesizes TBG and corticosteroid-binding globulin (CBG, also a member of alpha1-antitrypsin family). When the cell was treated with tunicamycin (inhibitor of core glycosylation), TBG was never secreted into the medium. On the other hand CBG was secreted into the medium without glycosylation. However, intracellular degradation was increased in both TBG and CBG when treated with tunicamycin. It was also noted that TBG synthesized in the presence of tunicamycin cross-reacted with anti-denatured TBG which had no thyroxine binding. Thus, it is concluded that carbohydrate moiety in TBG play important roles in T4-binding, secretion and intracellula … More r degradation.To further investigate the role of carbohydrate residues in TBG, TBG-Gary found in a patient by Murata et al was studied. TBG-Gary was characterized by decreased T4-binding, annodal shift on isoelectric focusing and heat instability. The analysis of structure of TBG genome revealed that single point mutation in exon I causes amino acid substitution of 96th amino acid "isoleucine" to "asparagine". This amino acid substitution resulted in a creation of a potential glycosylation site (asparagine-cystine-serine). Complete cDNAs coding for normal TBG as well as TBG-Gary was constructed and inserted in expression vector pEUK-C1. Transfection of the respective plasmids into Cos 1 cells and subsequent analysis by pulse-chase experiment revealed that TBG-Gary was indeed has an additional carbohydrate residues, reduced T4-binding, and cross-reacted with anti-denatured TBG. Moreover, it was demons trated that TBG-Gary was much more rapidly degraded in the cells. When the mutant asparagine residue in TBG-Gary was substituted to aspartic acid by site-specific mutagenesis, the mutated TBG behaved like normal TBG.These studies demonstrate that carbohydrate residues in TBG play important role in hormone binding, cellular transport, and metabolism. Less