Sulfation of lutropin oligosaccharides with a cell-free system.
Sulfation of lutropin oligosaccharides with a cell-free system.
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用无细胞系统硫酸化促黄体素寡糖。
DOI：
10.1073/pnas.81.17.5320
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发表时间：
1984
影响因子：
11.1
通讯作者：
Boime,I
中科院分区：
文献类型：
--
作者：
Green,ED;Gruenebaum,J;Bielinska,M;Baenziger,JU;Boime,I
硫酸盐与牛促黄体激素（促黄体激素; LH）α和β亚基上的寡糖共价连接，但不与人绒毛膜促性腺激素（hCG）上的寡糖共价连接。由于垂体和胎盘α亚基的氨基酸序列是同源的，比较它们的天冬酰胺连接的糖可以提供关于寡糖成熟的组织特异性的信息。为了表征这种翻译后修饰，我们开发了一种重组的无细胞硫酸化系统。在存在3 '-磷酸腺苷5'-磷酸[35 S]硫酸盐的情况下，硫酸盐通过Triton X-100裂解的高尔基体膜的磺基转移酶掺入外源添加的糖蛋白中，所述硫酸盐由克雷布斯腹水肿瘤细胞的无核糖体上清液从[35 S]硫酸盐产生。LH被垂体和肝膜硫酸化，但不被胎盘膜硫酸化。去唾液酸化hCG（AshCG）被胎盘和垂体的膜硫酸化，但不被肝脏硫酸化，而hCG不被任何这些膜硫酸化。糖苷内切酶F以单硫酸化寡糖和二硫酸化寡糖的异质混合物的形式从LH中释放所有掺入的硫酸盐。相比之下，添加到AshCG中的硫酸盐显然连接到肽而不是寡糖上。正如无细胞系统所发现的，通过垂体细胞代谢纳入LH的硫酸盐存在于单硫酸化和二硫酸化寡糖的异质群体中。因此，无细胞硫酸化系统准确地复制了体内过程。
Sulfate is covalently linked to the oligosaccharides on the alpha and beta subunits of bovine lutropin (luteinizing hormone; LH) but not to those on human chorionic gonadotropin (hCG). Since the amino acid sequences of the pituitary and placental alpha subunits are homologous, comparison of their asparagine-linked sugars can provide information regarding tissue specificity of oligosaccharide maturation. To characterize this post-translational modification, we have developed a reconstituted cell-free sulfation system. Sulfate is incorporated into exogenously added glycoproteins by sulfotransferases from Triton X-100-lysed Golgi membranes in the presence of 3'-phosphoadenosine 5'-phospho[35S]sulfate, which is generated from [35S]sulfate by a ribosome-free supernate from Krebs ascites tumor cells. LH is sulfated by pituitary and liver membranes but not by those from placenta. Desialylated hCG (AshCG) is sulfated by membranes from placenta and pituitary, but not liver, while hCG is not sulfated by any of these membranes. Endoglycosidase F releases all the incorporated sulfate from LH in the form of a heterogeneous mixture of mono- and disulfated oligosaccharides. In contrast, the sulfate added to AshCG is apparently attached to peptide rather than oligosaccharide. As found with the cell-free system, sulfate metabolically incorporated into LH by pituitary cells is present on a heterogeneous population of mono- and disulfated oligosaccharides. Thus the cell-free sulfation system accurately duplicates the in vivo process.