Gesundheit N, Magner J A, Chen T, Weintraub B D
Endocrinology. 1986 Aug;119(2):455-63. doi: 10.1210/endo-119-2-455.
To determine whether sulfate and/or sialic acid are present on secreted mouse TSH, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [3H]methionine and [35S]sulfate, or [35S]methionine and [3H]N-acetylmannosamine. The metabolically labeled TSH and free alpha-subunits were then analyzed by gel electrophoresis. [3H]N-Acetylmannosamine was a specific precursor (greater than 80%) for the sialic acid [3H]N-acetylneuraminic acid, as established by HPLC characterization of tritium label released by acid hydrolysis. Each of the three secreted subunits (TSH alpha, TSH beta, and free alpha) incorporated both sulfate and sialic acid. The incorporation of these labels was confirmed by the release of [35S]sulfate by endoglycosidase F and of [3H]N-acetylneuraminic acid by neuraminidase. Differential labeling of newly synthesized secreted TSH subunits was observed. In secreted TSH dimer, TSH beta incorporated 1.3 times more [35S]sulfate (P less than 0.05) and 2.5 times more [3H] N-acetylmannosamine (P less than 0.02) per carbohydrate chain than did TSH alpha. Secreted free alpha-subunit incorporated more [3H]N-acetylmannosamine, but less [35S]sulfate, then did secreted TSH alpha. To investigate the effect of TRH on TSH sulfation and sialylation, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [35S]sulfate or [3H]N-acetylmannosamine, with or without 10(-7) M TRH; labeling was then normalized in each case to incorporation of [3H]mannose, a marker of the inner core sugars. TSH secreted in the presence of TRH had a lower sulfate to mannose ratio [28 +/- (+/- SE) 4% of control; P less than 0.05] and a lower sialic acid to mannose ratio (63 +/- 8% of control; P less than 0.05). TSH alpha and TSH beta were affected equally. No change was seen in the labeling of non-TSH secretory proteins. Differential glycoprotein sulfation and sialylation may, in part, explain the previously observed variability in isoelectric point, bioactivity, and MCR of TSH in different physiological states and may represent a point of regulation by TRH.
为了确定分泌型小鼠促甲状腺激素（TSH）上是否存在硫酸根和/或唾液酸，将促甲状腺肿瘤切碎组织和甲状腺功能减退的垂体与[3H]甲硫氨酸和[35S]硫酸根，或[35S]甲硫氨酸和[3H]N-乙酰甘露糖胺一起孵育。然后通过凝胶电泳分析代谢标记的TSH和游离α亚基。通过对酸水解释放的氚标记进行高效液相色谱（HPLC）表征确定，[3H]N-乙酰甘露糖胺是唾液酸[3H]N-乙酰神经氨酸的特异性前体（大于80%）。三种分泌亚基（TSHα、TSHβ和游离α）均掺入了硫酸根和唾液酸。糖苷内切酶F释放[35S]硫酸根以及神经氨酸酶释放[3H]N-乙酰神经氨酸证实了这些标记的掺入。观察到新合成的分泌型TSH亚基存在差异标记。在分泌型TSH二聚体中，每条碳水化合物链上TSHβ掺入的[35S]硫酸根比TSHα多1.3倍（P<0.05），掺入的[3H]N-乙酰甘露糖胺比TSHα多2.5倍（P<0.02）。分泌的游离α亚基掺入的[3H]N-乙酰甘露糖胺比分泌的TSHα多，但掺入的[35S]硫酸根比分泌的TSHα少。为了研究促甲状腺激素释放激素（TRH）对TSH硫酸化和唾液酸化的影响，将促甲状腺肿瘤切碎组织和甲状腺功能减退的垂体与[35S]硫酸根或[3H]N-乙酰甘露糖胺一起孵育，添加或不添加10-7M的TRH；然后在每种情况下将标记标准化为[3H]甘露糖的掺入量，[3H]甘露糖是内核糖的标志物。在TRH存在下分泌的TSH的硫酸根与甘露糖的比率较低[为对照的28±（±标准误）4%；P<0.05]，唾液酸与甘露糖的比率也较低（为对照的63±8%；P<0.05）。TSHα和TSHβ受到的影响相同。未观察到非TSH分泌蛋白的标记有变化。糖蛋白硫酸化和唾液酸化的差异可能部分解释了先前观察到的不同生理状态下TSH在等电点、生物活性和代谢清除率（MCR）方面的变异性，并且可能代表了TRH的一个调节点。
