Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3.

Abstract

A glycolipid antigen, detected by a monoclonal antibody (ME 31 1) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized.Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164).Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography.On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Aca2+8Neu5Aca24 3Gal/31+4Glc/31+1-cer-amide) and specifically detected by monoclonal antibody R24.Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 'H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2a2+ 8Neu5Aca2+3Ga1/31~4Glc/3l+l-ceramide.This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D.