Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.
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.
Sjoberg
E R
ER
Department of Medicine, University of California, La Jolla 92093.
Manzi
A E
AE
Khoo
K H
KH
Dell
A
A
Varki
A
A
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D005732:Gangliosides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D008545:Melanoma / Q000201:enzymology*; D008745:Methylation; D008969:Molecular Sequence Data; D012794:Sialic Acids / Q000302:isolation & purification / Q000378:metabolism
D005732:Gangliosides; D012794:Sialic Acids; C019403:ganglioside, GD2; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C075983:ganglioside O-acetyltransferase