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

甲状腺素结合球蛋白（TBG）是血清中主要的甲状腺素结合蛋白，在肝脏中合成。由于其结构相似，该蛋白属于α 1-抗胰蛋白酶家族。本研究对TBG中碳水化合物的生理意义进行了研究。首先，我们使用了合成TBG和皮质类固醇结合球蛋白（CBG，也是α 1-抗胰蛋白酶家族的成员）的肝细胞系HepG 2。当细胞用衣霉素（核心糖基化抑制剂）处理时，TBG从未分泌到培养基中。另一方面，CBG在无糖基化的情况下分泌到培养基中。然而，当用衣霉素处理时，TBG和CBG的细胞内降解增加。还注意到，在衣霉素存在下合成的TBG与不具有甲状腺素结合的抗变性TBG交叉反应。因此，TBG中的碳水化合物在T4结合、分泌和细胞内分泌中起重要作用 关于我们 为了进一步研究碳水化合物残基在TBG中的作用，研究了Murata等人在患者中发现的TBG-Gary。TBG-Gary的特征在于T4结合减少、等电聚焦的annodal移位和热不稳定性。TBG基因组结构分析表明，TBG基因第一外显子的单点突变导致第96位氨基酸“异亮氨酸”替换为“天冬酰胺”。该氨基酸取代导致产生潜在的糖基化位点（天冬酰胺-胱氨酸-丝氨酸）。构建了编码正常TBG和TBG-Gary的完整cDNA，并将其插入表达载体pEUK-C1中。将各质粒转染到Cos 1细胞中，随后通过脉冲追踪实验进行分析，结果显示TBG-Gary确实具有额外的碳水化合物残基，降低了T4结合，并与抗变性TBG交叉反应。此外，它是恶魔证明TBG-Gary在细胞中降解得更快。通过定点突变将TBG-Gary中的天冬酰胺残基突变为天冬氨酸后，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着重要作用。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