Biosynthesis of glycolipids GA2, GA1, GM1b, and GD1c was studied in Golgi vesicles isolated from rat liver. Sequential addition of N-acetylgalactosamine, galactose and two sialic acid residues to lactosylceramide led to the endproduct GD1c. Activities of the corresponding glycosyltransferases were shown to be present in isolated Golgi vesicles and their respective kinetic data were determined. The products of each reaction were characterized by their mobility on thin-layer chromatography, by enzymic degradation to their respective precursors, and in case of GM1b by FAB mass spectrometry.

Glycolipids (GSL) are characteristic components of the outer leaflet of plasma membranes (PM) where they form cell-specific patterns which change with differentiation. They are synthesized by membrane-bound transferases mainly in the Golgi compartment and are degraded in the lysosomes (for rev. 1). In order to gain some insight into factors involved in the maintainance of the profiles of acidic GSL (gangliosides) on cellular surfaces we synthesized spin-, radio-, fluorescent- (2) and biotin-labeled ganglioside derivatives and studied their uptake, localisation and metabolism in cultured cells. ESR spectra obtained from cultured cells after feeding of spin-labeled gangliosides show that they are slowly incorporated into the PM where they obtain a position similar to that of endogenous gangliosides (3, 4). Gangliosides inserted into the PM are subjected to endocytosis and metabolism. They are transported into the lysosomal as well as into the Golgi compartment (5). This is evidenced by the distribution of biotin-labeled ganglioside within the cells as well as by metabolic products obtained from radio-labeled gangliosides and ganglioside derivatives in normal and mutant cells with deficiencies in different steps of glycolipid catabolism (6,7).

Competition experiments using lactosylceramide, ganglioside GM3 and ganglioside GD3 as substrates, as well as mutual inhibitors for ganglioside N-acetylgalactosaminyltransferase, in Golgi vesicles derived from rat liver suggested that N-acetylgalactosamine transfer to these three respective compounds, leading to gangliosides GA2, GM2, and GD2, respectively, is catalyzed by one enzyme. Analogous studies with gangliosides GA1, GM1, and GD1b as glycolipid acceptors in sialyltransferase assays indicated GM1b, GD1a, and GT1b synthases to be identical. These results are incorporated into a model for ganglioside biosynthesis and its regulation.

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside GM3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside GM3 as well as for ganglioside GD3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside GM3. Based on this idea we used a variety of GM3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for GD3 synthase (of rat liver Golgi vesicles). This study revealed that those GM3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (GD3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.

Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.

Metabolism of [3H]ganglioside derivatives GM3-amide and GM2-amide has been investigated in normal human skin fibroblasts. In a cell-free system the ganglioside analogues have been shown to enter biosynthetic pathways, their degradation, however, was curtailed at an early stage, as GM3-amide could not be hydrolysed by sialidase action. GM2-amide was susceptible to beta-hexosaminidase degradation yielding GM3-amide. When incorporated into fibroblasts [3H]GM2-amide was degraded to [3H]GM3-amide presumably in the lysosomes, and at the same time glycosylation to [3H]GD1a-monoamide took place most likely in the Golgi apparatus. [3H]GM3-amide, however, did not seem to reach the glycosylation sites in the Golgi apparatus. It could be detected in the lysosomes, where it was not degraded due to its sialidase resistance. From these results we conclude that in cells exogenously administered [3H]GM3-amide and [3H]GM2-amide both are directed to the lysosomes and that [3H]GM2-amide also reaches the Golgi apparatus. The synthesis of higher [3H]ganglioside-amides from incorporated [3H]GM2-amide can occur by direct glycosylation. [3H]GM3-amide, however, even if it reaches the Golgi compartment, does not enter the biosynthetic pathway.