Enrichment and localization of ganglioside G(D3) 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 shed ding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified hum an ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immuno staining 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 a n active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell mem branes also express caveolin-1, a characteristic protein marker for caveola e, which was detected by flow cytometric analysis and by Western blotting i n both the cell membranes and the isolated membrane vesicles. To further de fine the expression of GD3 and caveolin-1, we used immunogold electron micr oscopy. This revealed localization of GD3 in small clusters in the plasma m embrane as well as enrichment and localization of ganglioside GD3 and caveo lin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both GD3 and caveolin-1. Together, these results suggest that membrane vesicle shedd ing originates in plasma membrane domains enriched in gangliosides and cave olin-1. (C) 2000 Elsevier Science B.V. All rights reserved.