GD3 Ganglioside, Expression, Modification and its Biological Function

免疫共沉淀实验检测神经节苷脂GD3与糖基化磷脂酰肌醇锚定的神经细胞黏附分子Tag-1的相互作用。以前，我们已经证明，抗神经节苷脂GD3抗体(R24)免疫沉淀大鼠小脑中的Src家族激酶Lyn，R24处理原代小脑培养物诱导Lyn激活和80 kDa蛋白(P80)的快速酪氨酸磷酸化。我们现在报告R24从原代小脑培养物中免疫共沉淀出135 kDa的蛋白(P135)。用磷脂酰肌醇专一性磷脂酶C处理p135，发现p135是糖基磷脂酰肌醇-锚定在膜上。用抗TAG-1抗体进行序贯免疫沉淀，鉴定为TAG-1。抗体介导的Tag-1交联物诱导Lyn活化和p80的快速酪氨酸磷酸化。Src家族的选择性抑制剂降低了p80的酪氨酸磷酸化水平。蔗糖密度梯度分析显示，小脑培养物中的TAG-1和酪氨酸磷酸化的p80存在于脂筏部分。这些数据表明，Tag-1通过Lyn将信号传递到小脑脂筏中的p80。此外，细胞表面糖鞘糖脂被内糖神经酰胺酶降解，导致OptiPrep梯度上Tag-1分布的改变，并降低了Tag-1介导的Lyn激活和p80的酪氨酸磷酸化。这些观察结果表明，鞘糖脂参与了脂筏中Tag-1介导的信号传递。

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specitic phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.