Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the lumen of the Golgi complex. We demonstrate that ganglioside GD3 traffics from the trans-Golgi network (TGN) to plasma membrane by a Rab11-independent and Brefeldin A-insensitive exocytic pathway in CHO-K1 cells (JBC 279:47610, 2004). After arrival to the plasma membrane, gangliosides can undergo endocytosis, which has been examined by using fluorescent-labelled lipid analogues, toxins or radioactive lipids. In this work, we examined the endocytic transport of ganglioside GD3 in CHO-K1 cells by tracking the specific mouse monoclonal antibody anti-GD3, R24. CHO-K1 cells were incubated with R24 antibody (IgG3) at 4ºC to inhibit endocytosis. Then, cells were washed and incubated at 37°C for different times to allow endocytosis. By biochemical techniques, immunofluorescence and confocal microscopy we observed that GD3-R24 complex was properly internalized and accumulated in a perinuclear compartment. This compartment was positive for the expression of Rab11-GFP and internalized Alexa633-Transferrin, two recycling endosome markers; but not for GalNac-T, a TGN marker. After 60 min of R24 endocytosis, we could not detect the internalized antibody, unless it was continually present in the culture medium. NH4Cl and chloroquine, inhibitors of lysosomal degradation, could not prevent R24 depletion, indicating that the antibody was not targeted and degraded in lysosomes. Interestingly, after 60 min of the R24 endocytosis most of the antibody was recovered from the culture medium. In addition, we demonstrated that R24 exited the recycling compartment by a Brefeldin A and Monensin sensitive pathway. Taken together, these results indicate that GD3-R24 complex is rapidly endocyted in CHO-K1 cells, accumulated in recycling endosome and transported back to the plasma membrane.
