SYNTHESIS AND MASS-SPECTROMETRIC CHARACTERIZATION OF DIGOXIGENIN AND BIOTIN-LABELED GANGLIOSIDE GM1 AND THEIR UPTAKE BY AND METABOLISM IN CULTURED-CELLS

Selective acylation of mono-deacetyl lyso-GM1, i.e. beta-D-galactopyra nosyl-(1 --> 3)-2-acetamido-2-deoxy-beta-D-galactopyranosyl-(1 --> 4)- (alpha-D-neuraminyl-(2 --> 3))-beta-D-galactopyranosyl-(1 --> 4)-beta- D-glucopyranosyl-(1 --> 1)-(2S,3R,4E)-2-amino-4-octadecen-1,3-diol, wi th N-succinimidyl-[1-C-14]stearate afforded labeled mono-deacetyl GM1, i.e. beta-D-galactopyranosyl-(1 --> 3)-2-acetamido-2-deoxy-beta-D-gal actopyranosyl-(1 --> 4)-(alpha-D-neuraminyl-(2 --> 3))-beta-D-galactop yranosyl-(1 --> 4)-beta-D-glucopyranosyl-(1 --> 4E)-2-[1-C-14]octadeca namido-4-octadecen-1,3-diol, in good yield. Its condensation with eith er N-succinimidyl-digoxigenyl-3-O-methyl carbonyl-epsilon-amino caproa te or N-succinimidyl-D-biotinyl-epsilon-aminocaproate led to radioacti ve GM1 derivatives carrying a tag for immune-electron microscopy at th e sialic acid residue. These GM1 derivatives could be hydrolyzed to th e corresponding GM3 derivatives by treatment with GM1-beta-galactosida se and beta-hexosaminidases. There was no further degradation by siali dases due to the bulky tag in the sialic acid residue. The uptake of b iotin labeled GM1 by human skin fibroblasts, rat neuroblastoma cells B 104 and human neuroblastoma cells SHSY5Y was 0.85, 0.58 and 1.62 nmol lipid/mg cellular protein, respectively, after an incubation for 66 h at 37 degrees C and was similar to that of untagged GM1. The uptake of digoxigenin labeled GM1 by these cell types was, however, significant ly higher (3.1, 6.8, and 20.0 nmol lipid/mg cellular protein, respecti vely). Both the biotin and digoxigenin labeled GM1 analogs were catabo lized to the corresponding GM2 and GM3 derivatives in lysosomes of cul tured cells. This demonstrates that these synthetic analogues are suit able for studying, by immune-electron microscopy, their endocytosis an d distribution in intralysosomal membranes. (C) 1997 Elsevier Science Ireland Ltd.