Prinetti A, Prioni S, Chigorno V, Karagogeos D, Tettamanti G, Sonnino S
Study Center for the Functional Biochemistry and Biotechnology of Sphingolipids, Department of Medical Chemistry and Biochemistry, University of Milano, Segrate, Milan, Italy.
J Neurochem. 2001 Sep;78(5):1162-7. doi: 10.1046/j.1471-4159.2001.00515.x.
Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.
用[1-(3)H]鞘氨醇喂养在培养中分化的大鼠小脑颗粒细胞，可实现所有细胞鞘脂和磷脂酰乙醇胺的代谢性放射性标记。通过蔗糖梯度离心从用[1-(3)H]鞘氨醇喂养的细胞中制备了一种去污剂不溶性富含鞘脂的膜组分，该组分含有约60%的细胞鞘脂，但仅含有痕量的磷脂酰乙醇胺。该组分富含Src家族蛋白酪氨酸激酶c-Src、Lyn和Fyn以及糖基磷脂酰肌醇锚定的神经元粘附分子TAG-1。在旨在保持结构域完整性的实验条件下，用抗GD3神经节苷脂单克隆抗体R24对细胞裂解物和富含鞘脂的膜组分进行免疫沉淀。从细胞裂解物和富含鞘脂的组分中获得的免疫沉淀物的放射性脂质组成非常相似，并且与整个富含鞘脂的膜组分的鞘脂组成非常相似。事实上，免疫沉淀物除了含有GD3神经节苷脂外，还含有所有细胞糖鞘脂和鞘磷脂，而不含有磷脂酰乙醇胺。此外，通过定性薄层层析分析和比色可视化在免疫沉淀物中检测到了胆固醇和磷脂酰胆碱。c-Src、Lyn、Fyn和TAG-1与抗GD3抗体免疫沉淀物相关。在与旨在保持结构域完整性的实验条件不同的条件下获得的免疫沉淀物中未检测到这些蛋白质。这些数据表明，含有胆固醇、磷脂酰胆碱、鞘脂和蛋白质的膜结构域可以通过抗GD3抗体免疫沉淀从总细胞膜中分离出来，并且c-Src、Fyn、Lyn和TAG-1与富含鞘脂的结构域的结合是由与复杂脂质环境的相互作用介导的，而不是由与单一鞘脂种类的特异性相互作用介导的。
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