Swainsonine对胰岛素受体功能影响的研究

Studies on the Effects of Swainsonine on the Function of Insulin Receptor

以Ｓｗａｉｓｏｎｉｎｅ（Ｓｗ）作为高尔基体付糖链加工酶系中α－甘露糖苷酶Ⅱ的特异抑制剂，研究Ｎ－糖链结构和胰岛素受体（Ｉｎｓ－Ｒ）功能的关系．发现Ｓｗ不影响细胞生长和３Ｈ－亮氨酸参入ＳＭＭＣ７７２１细胞，但明显促进３Ｈ－甘露糖参入细胞总糖蛋白和表面糖蛋白，并使后者的ＣｏｎＡ强结合组分显著增加，提示Ｓｗ使Ｉｎｓ－Ｒ的Ｎ－糖链变成杂合型及高甘露糖型。胰岛素结合试验后作Ｓｃａｔｃｈａｒｄ分析：发现Ｓｗ不改变Ｉｎｓ－Ｒ的结合容量和每个细胞表面的结合位点数，也不改变结合动力学。再用部分纯化的Ｉｎｓ－Ｒ研究自身磷酸化和对外源底物的酪氨酸蛋白激酶活力，也未发现Ｓｗ处理和对照细胞间的明显区别，表示Ｓｗ也不影响Ｉｓｎ－Ｒ的跨膜信息传递，结合已报道的衣霉素使细胞表面Ｉｎｓ－Ｒ减少的结果，提示Ｉｎｓ－Ｒ运送至细胞膜需要Ｎ－糖链存在。

Using swainsonine (Sw)as a specific inhibitor of a-mannosidase Ⅱ of the N-glycan processing enzyme system in Golgi body, the relationship between the structure and function of Nglycan on insulin receptor (Ins-R) was investigated,It was found that Sw did not affect the cell growth and incorporation of 3H-leucine into SMMC-7721 cells, but significantly stimulated the incorporation of 3H-mannose into total cell and surface glycoproteins, also obviously increased the Con A strongly binding fraction of the surface glycoprotein, suggesting that the N-glycans of InsR were changed to hybrid and high mannose type by Sw. When insulin binding assay was analyzed by Scatchard plot, it showed that Sw did not change the binding capacity of insR and the binding sites on each cell, as well as the binding kinetics of the receptor. The autophosphorylation and the tyrosine protein kinase activity to exogeneous substrate of the partially purified Ins-R were also studied, and no obvious difference between Sw treated and control cells was found,suggesting that Sw also did not affect the transmembranous signal transduction of Ins-R.Considering that tunicamycin decreased the number of ins-R on cell surface in our previous report, it can be concluded that the presence of Nglycan on ins-R is necessary for its transfer to cell membrane,but the structure of N-glycan may not be important for receptor metabolism and binding kinetics.