Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin.
Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin.
复制标题
细胞表面新神经节蛋白的产生。
DOI：
10.1016/s0021-9258(19)39167-7
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
P. Fishman
中科院分区：
文献类型：
--
作者：
T. Pacuszka;P. Fishman
以神经节苷脂为原料，经臭氧分解和碱性裂解法制备GM1 (II3Neu5Ac-GgOse4Cer)-低聚糖，经还原胺化，偶联到异双功能交联剂丁二酰亚胺基4-（n -马来酰亚胺甲基）环己烷-1-羧酸盐。在缺乏gm1的大鼠胶质瘤C6细胞上，所得到的衍生物与游离巯基反应并容易交联到细胞表面成分。gm1寡糖衍生物的附着是时间和浓度依赖的，通过125i霍乱毒素与细胞的结合增加来监测。先前用二硫苏糖醇处理的细胞通过产生额外的游离巯基来增强附着。霍乱毒素对GM1寡糖衍生物或GM1处理过的细胞的亲和力相似。Western blotting检测新生成的毒素受体的性质。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离衍生细胞的膜，并将分离的组分电泳转移到硝化纤维素片上，在硝化纤维素片上覆盖125i -霍乱毒素。这种毒素与多种膜蛋白结合，其中大多数是胰蛋白酶敏感的。对照细胞的膜未观察到这种结合。尽管在大鼠胶质瘤C6细胞表面新生成的gm1 -新神经节蛋白很容易结合霍乱毒素，但通过环AMP的增加或腺苷酸环化酶的激活来测量，细胞对霍乱毒素的反应并没有增强。相比之下，暴露于GM1的细胞对毒素产生了高度反应。因此，细胞表面的新神经节蛋白表现为霍乱毒素的无功能受体。
GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.