Enrichment and localization of ganglioside GD3 and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed GD3 expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of GD3 and caveolin-1, we used immunogold electron microscopy. This revealed localization of GD3 in small clusters in the plasma membrane as well as enrichment and localization of ganglioside GD3 and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both GD3 and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1. (C) 2000 Elsevier Science B.V. All rights reserved.