An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion.
An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion.
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DOI：
10.1210/mend.6.3.1584218
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发表时间：
1992-03
影响因子：
--
通讯作者：
F. Kambe;H. Seo;Y. Mori;Y. Murata;O. Janssen;S. Refetoff;N. Matsui
中科院分区：
文献类型：
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作者：
F. Kambe;H. Seo;Y. Mori;Y. Murata;O. Janssen;S. Refetoff;N. Matsui
T4结合球蛋白-Gary（TBG-G）变体严重损害了T4结合，在37 ℃下不稳定，并且当进行等电聚焦时呈现出所有同种型的明显阳极位移。这种异常TBG的遗传导致血清中天然TBG水平显著降低，其变性形式发生相互变化，导致甲状腺激素浓度与完全TBG缺乏症一样低。TBG-G基因具有用Asn（AAC）替换正常IIe 96（ATC）的单核苷酸取代，从而产生用于N-连接糖基化的新位点。为了确定TBG-G是否含有额外的碳水化合物链，如由等电聚焦结果间接表明的，将含有正常TBG（TBG-N）和TBG-G的cDNA插入适当的载体中，以允许它们在哺乳动物细胞（COS-1）和两栖动物（非洲爪蟾）卵母细胞中表达。在这两个系统中，表达TBG-G产生了一个更大的分子比TBG-N在变性条件下通过聚丙烯酰胺凝胶电泳分析时。然而，当在衣霉素存在下在COS-1细胞中合成时，或当在爪蟾卵母细胞中合成后去糖基化时，两者的大小相同。脉冲追踪实验显示TBG-G相对于TBG-N的分泌受损和过度的总体细胞内降解。正如对受影响受试者血清的研究所预期的那样，体外表达的TBG-G对T4的亲和力低10倍。这些研究证明TBG-G中点突变产生的潜在糖基化的新位点确实是糖基化的。（250字处删节）
The T4-binding globulin-Gary (TBG-G) variant has severely impaired T4 binding, is unstable at 37 C, and presents an apparent anodal shift of all isoforms when submitted to isoelectric focusing. Inheritance of this abnormal TBG produces a profound decrease in the serum levels of native TBG with reciprocal changes in its denatured form, causing thyroid hormone concentrations to be as low as those found in complete TBG deficiency. The TBG-G gene possesses a single nucleotide substitution replacing the normal IIe96 (ATC) with Asn (AAC), thus creating a new site for N-linked glycosylation. In order to determine whether TBG-G contains an additional carbohydrate chain as indirectly suggested by the isoelectric focusing results, cDNAs containing the normal TBG (TBG-N), and TBG-G were inserted in the appropriate vectors to allow their expression in mammalian cells (COS-1) and in amphibian (Xenopus) oocytes. In both systems, expression of TBG-G yielded a larger molecule than TBG-N when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. However, both were identical in size when synthesized in COS-1 cells in the presence of tunicamycin or when deglycosylated after their synthesis in Xenopus oocytes. Pulse chase experiments revealed impaired secretion and excessive overall intracellular degradation of TBG-G relative to TBG-N. As expected from studies on serum from affected subjects, in vitro expressed TBG-G had a 10-fold lower affinity for T4. These studies prove that the new site for potential glycosylation created by the point mutation in TBG-G is indeed glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)