Biosynthesis in vitro of gangliosides containing Gg- and Lc-cores

On the basis of our previous and present studies with embryonic chicken brain system, we have proposed stepwise biosynthesis of GD1a (Gg-series) and LD1 (Lc-series) gangliosides, starting from ceramide (Fig. 4). At least three different galactosyltransferases GalT-2 (UDP-Gal:Glc-Cer), GalT-3(UDP-Gal:GM2) and GalT-4(UDP-Gal:LcOse3-Cer) and three different sialyltransferases SAT-1(CMP-NeuAc:Lac-Cer), SAT-2(CMP-NeuAc:GM3) and SAT-3(CMP-NeuAc:nLcOse4 Cer) are involved in the biosynthesis in vitro of these gangliosides. All six of these glycosyltransferases have been solubilized using nonionic detergents. Two forms of glycolipid:galactosyltransferases (GalT-3 and GalT-4) have been separated by DEAE sepharose CL-6B chromatography from solubilized supernatant of 11- to 13-day-old embryonic chicken brain. Using microisoelectric focusing (pH gradient 3 to 8) the galactosyltransferases (GalT-3 and GalT-4) have been separated from SAT-3. Two beta-N-acetylglucosaminyltransferases (GlcNAcT-2(UDP-GlcNAc:nLcOse4Cer(beta 1-3] and GlcNAcT-3(UDP-GlcNAc:nLcOse4Cer(beta 1-6] have also been solubilized from mouse T-lymphoma, P-1798, using Triton CF-54. These enzymes are involved in the synthesis of Ii-core gangliosides and 3H-products have been characterized by methylation studies. Further separation of these two GlcNAcT's are in progress.