BIOSYNTHESIS AND GLYCOSYLATION OF THYROTROPIN

围产期和成熟期大鼠分泌型TSH的寡糖结构 与甲状腺功能正常的对照组相比， 阴离子交换高效液相色谱法。与对照TSH相比， 甲状腺功能减退的TSH表现为唾液酸相对增多， 硫酸盐，以及更复杂的结构与3个或更多的负 电荷，可能代表多触角结构。TSH来源于 慢性甲状腺功能减退动物与产生的重组TSH相似 在将α和TSH-β基因转染到中国仓鼠卵巢中后， 细胞这样的重组TSH被完全唾液酸化，并显示没有 Ga 1 Nac或末端硫酸盐部分的证据。重组激素 在各种体外生物测定中的效力低3至8倍，但显示出 当注射到甲状腺功能正常的大鼠中时，半衰期显著延长。 甲状腺功能减退以及重组TSH将有助于确定 唾液酸与硫酸盐在人促甲状腺激素作用中的生理作用。在 此外，该重组激素将在临床上用于 甲状腺癌的诊断和治疗。 使用定点突变，我们目前正在确定 人TSH的结构-功能关系。我们已经变异了 各种氨基酸以及糖基化位点，并确定重要的 不同密码子在β亚基中的功能作用。我们也 产生几个稳定的细胞系产生这些TSH突变体， 可以对生物合成、降解和分泌进行详细的研究， 测定最后，我们使用了各种寡糖抑制剂 处理以确定碳水化合物结构对分泌的作用 从分散的啮齿动物垂体细胞中提取TSH。两者的抑制剂 葡萄糖醛酸酶以及甘露糖醛酸酶损害了 TSH和含有葡萄糖的高甘露糖形式特别是 易受细胞内降解的影响。几种稳定的突变细胞系 产生具有改变的碳水化合物链的TSH分子 以扩大这些研究。

The oligosaccharide structure of secreted TSH in perinatal and mature rats with hypothyroidism was compared to euthyroid controls using newly developed methods of anion exchange HPLC. Compared to control TSH, hypothyroid TSH showed relatively more sialic acid and less terminal sulfate, as well as more complicated structure with 3 or more negative charges, probably representing multiantennary structures. TSH derived from chronically hypothyroid animals was similar to recombinant TSH produced after transfection of alpha and TSH-beta genes into Chinese hamster ovary cells. Such recombinant TSH was completely sialylated and showed no evidence of Ga1Nac or terminal sulfate moieties. The recombinant hormone was 3- to 8-fold less potent in various in vitro bioassays, but showed a considerably longer half-life when injected into euthyroid rats. Hypothyroid as well as recombinant TSH will be useful in determining the physiologic roles of sialic acid vs. sulfate in the action of human TSH. In addition, the recombinant hormone will be of clinical utility in the diagnosis and treatment of patients with thyroid cancer. Using site-directed mutagenesis, we are currently determining the structure-function relationships of human TSH. We have already mutated various amino acids as well as glycosylation sites and determined important functional roles of various codons in the beta subunit. We are also generating several stable cell lines producing these TSH mutants so that detailed studies of biosynthesis, degradation and secretion can be determined. Finally, we have used various inhibitors of oligosaccharide processing to determine the role of carbohydrate structure on the secretion of TSH from dispersed rodent pituitary cells. Inhibitors of both glucosidases as well as mannosidases impair the assembly and secretion of TSH and the high mannose forms containing glucose are particularly susceptible to intracellular degradation. Several stable mutant cell lines producing TSH molecules with altered carbohydrate chains are being established to extend these studies.