We have shown previously that Golgi-enriched vesicles from the human
melanoma cell line Melur can transfer [3H]
acetate from [acetyl-3H]
acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human
melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were
sialidase-sensitive and
alkali-labile, indicating incorporation as [3H]O-acetyl
esters on
sialic acids. Immunological reactivity,
sialidase sensitivity, chromatographic behavior, and the known
ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2.
Sialic acids released from this labeled molecule by
sialidase showed esterification with [3H]
acetate at both C7 and C9 hydroxyls.
Lipid extracts from cells metabolically labeled with [3H]
galactose showed a corresponding
ganglioside, which upon
alkali treatment yielded a species migrating with GD2. Analysis of purified
ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes,
periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1'
ceramide Total
gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with
monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated
gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated
ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated
gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated
gangliosides in
melanoma cells is much more complex than previously appreciated.