Synthesis and mass spectrometric characterization of digoxigenin and biotin labeled ganglioside GM1 and their uptake by and metabolism in cultured cells.

ABSTRACT

Selective acylation of mono-deacetyl lyso-GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl -(1-->4)-(alpha-D-neuraminyl-(2-->3))-beta-D-galactopyranosyl- (1-->4)-beta-D-glucopyranosyl-(1-->1)-(2S,3R,4E)-2-amino-4-octa decen-1,3-diol, with N-succinimidyl-[1-14C]stearate afforded labeled mono-deacetyl GM1, i.e. beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyr ano syl- (1-->4)-(alpha-D-neuraminyl-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta -D- glucopyranosyl-(1-->1)-(2S,3R,4E)-2-[1-14C]octadecanamido-4- octadecen-1, 3-diol, in good yield. Its condensation with either N-succinimidyl-digoxigenyl-3-O-methyl carbonyl-epsilon-amino caproate or N-succinimidyl-D-biotinyl-epsilon-aminocaproate led to radioactive GM1 derivatives carrying a tag for immuno-electron microscopy at the sialic acid residue. These GM1 derivatives could be hydrolyzed to the corresponding GM3 derivatives by treatment with GM1-beta-galactosidase and beta-hexosaminidases. There was no further degradation by sialidases due to the bulky tag in the sialic acid residue. The uptake of biotin labeled GM1 by human skin fibroblasts, rat neuroblastoma cells B104 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, significantly higher (3.1, 6.8, and 20.0 nmol lipid/mg cellular protein, respectively). Both the biotin and digoxigenin labeled GM1 analogs were catabolized to the corresponding GM2 and GM3 derivatives in lysosomes of cultured cells. This demonstrates that these synthetic analogues are suitable for studying, by immuno-electron microscopy, their endocytosis and distribution in intralysosomal membranes.