Crespo Pilar M, von Muhlinen Natalia, Iglesias-Bartolomé Ramiro, Daniotti Jose L
Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
FEBS J. 2008 Dec;275(23):6043-56. doi: 10.1111/j.1742-4658.2008.06732.x.
Gangliosides are glycosphingolipids mainly present at the outer leaflet of the plasma membrane of eukaryotic cells, where they participate in recognition and signalling activities. The synthesis of gangliosides is carried out in the lumen of the Golgi apparatus by a complex system of glycosyltransferases. After synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analysed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized by Madin-Darby canine kidney (MDCK) cell lines genetically modified to express appropriate ganglioside glycosyltransferases. Using biochemical techniques and confocal laser scanning microscopy analysis, we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, were transported and accumulated mainly at the plasma membrane of nonpolarized MDCK cell lines. More interestingly, both complex gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that, after arrival at the plasma membrane, GD3 and GM1 gangliosides were endocytosed using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, was accumulated in endosomal compartments and transported back to the plasma membrane. In contrast, endocytosed GM1, in association with cholera toxin, was transported to endosomal compartments en route to the Golgi apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells, and that GD3 and GM1 gangliosides are internalized by clathrin-independent endocytosis to follow different intracellular destinations.
神经节苷脂是糖鞘脂，主要存在于真核细胞膜的外小叶，在那里它们参与识别和信号传导活动。神经节苷脂的合成是在高尔基体腔内由一个复杂的糖基转移酶系统进行的。合成后，神经节苷脂通过运输小泡的腔表面离开高尔基体，这些运输小泡的目的地是质膜。在本研究中，我们分析了由基因改造以表达适当神经节苷脂糖基转移酶的Madin-Darby犬肾（MDCK）细胞系内源性合成的GD3和GM1神经节苷脂的合成及膜分布。使用生化技术和共聚焦激光扫描显微镜分析，我们证明GD3和GM1在高尔基体合成后，主要运输并积累在非极化MDCK细胞系的质膜上。更有趣的是，当这些细胞系被诱导极化时，发现这两种复合神经节苷脂主要富集在顶端区域。此外，我们证明，到达质膜后，GD3和GM1神经节苷脂通过一种不依赖网格蛋白的途径被内吞。然后，内化的GD3与一种特异性单克隆抗体结合，积累在内体区室并运回质膜。相比之下，内化的GM1与霍乱毒素结合，被运输到内体区室，然后前往高尔基体。总之，我们的结果表明，复合神经节苷脂在极化的MDCK细胞中进行顶端分选，并且GD3和GM1神经节苷脂通过不依赖网格蛋白的内吞作用被内化，以遵循不同的细胞内目的地。
