Anabolic sialosylation of gangliosides in situ in rat brain cortical slices.
Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)
Neuropsychiatric Institute, University of California, Los Angeles 90024.
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Durrie
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Rosenberg
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002540:Cerebral Cortex / Q000032:analysis* / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D046508:Culture Techniques; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D005732:Gangliosides / Q000032:analysis*; D006056:Golgi Apparatus / Q000201:enzymology; D006595:Hexosamines / Q000378:metabolism; D008985:Monensin / Q000378:metabolism; D019158:N-Acetylneuraminic Acid; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000032:analysis*; D012799:Sialyltransferases / Q000032:analysis
D005732:Gangliosides; D006595:Hexosamines; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D008985:Monensin; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine