NATURAL FORMS OF SHED TUMOR GANGLIOSIDES

Gangliosides shed by tumor cells are immunosuppressive molecules, but the mechanisms of shedding are poorly understood. We therefore conduct ed a comprehensive study of shedding to identify the natural forms of shed gangliosides. By chemical detection and mass spectrometric analys is of the gangliosides of YAC-1 murine lymphoma cells, we first confir med that all major ganglioside species are released. Then, by the comb ination of metabolic and cell surface radiolabeling, we further demons trated that gangliosides are released directly from the cell plasma me mbrane, i.e. by shedding. Ultracentrifugation separated the conditione d medium of metabolically radiolabeled cells cultured in either serum- free or serum-containing medium into: (1) a pellet of 100-200 nm membr ane vesicles (visualized by electron microscopy) containing nearly one -third of total shed gangliosides; and (2) the supernatant, which cont ained soluble gangliosides (two-thirds of the total shed gangliosides) . Although the ganglioside concentration in the conditioned medium (6- 14 x 10(-8) M) was above the critical micelle concentration of purifie d YAC-1 gangliosides (< 1 x 10(-8) M), by gel filtration > 90% of the soluble gangliosides were found in monomeric form (MW < 2 kDa) and onl y < 10% in micelles (130 kDa). Ultrafiltration of fresh conditioned me dium likewise showed the existence of monomers, and the findings were confirmed in human Daoy medulloblastoma and mouse MEB4 melanoma cells. Thus, in their natural states, shed tumor cell gangliosides exist in three forms: membrane vesicles, micelles, and monomers. (C) 1998 Publi shed by Elsevier Science B.V. All rights reserved.