Crespo Pilar Maria, Zurita Adolfo Ramón, Daniotti Jose Luis
Centro de Investigaciones en Química Biológica de Córdoba, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba 5000, Argentina.
J Biol Chem. 2002 Nov 22;277(47):44731-9. doi: 10.1074/jbc.M204604200. Epub 2002 Sep 16.
Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.
糖基磷脂酰肌醇（GPI）锚定蛋白主要聚集在质膜的鞘脂 - 胆固醇微结构域中。研究了具有不同糖脂组成的中国仓鼠卵巢（CHO）-K1细胞膜中GPI锚定的融合黄色荧光蛋白（GPI-YFP）的分布。通过抑制葡糖神经酰胺合酶活性耗尽鞘糖脂的细胞、通过稳定转染适当的神经节苷脂糖基转移酶表达不同神经节苷脂的细胞系或暴露于外源性GM1的细胞，用GPI-YFP cDNA进行转染。使用膜不可渗透的交联剂双（磺基琥珀酰亚胺基）辛二酸酯研究了质膜上表达的GPI-YFP融合蛋白的分布。结果表明，GPI-YFP在表达GM3的细胞表面、耗尽糖脂的细胞、主要表达GD3和GT3或GM1和GD1a或大多表达GM2或高表达GM1的转染细胞中形成簇。然而，在所研究的细胞系膜提供的不同糖脂环境中，未检测到GPI-YFP膜微结构域的显著变化。另一方面，在与双（磺基琥珀酰亚胺基）辛二酸酯交联之前暴露于100微摩尔GM1的野生型CHO-K1细胞显示GPI-YFP簇的数量显著减少。这些发现清楚地表明，仅通过改变细胞的神经节苷脂生物合成活性来操纵细胞膜的糖脂含量，不会显著影响CHO-K1细胞表面上GPI-YFP的缔合。外源性GM1神经节苷脂对GPI-YFP质膜分布的影响可能是质膜中达到的神经节苷脂水平和/或导致膜结构和/或动态修饰的特定神经节苷脂种类（微团）的作用的结果。
