Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids.

ABSTRACT

The assembly of most of the ceramide-linked glycolipids (GSLs) in eukaryotic cells occurs in Golgi bodies. At least 18 different glycolipidglycosyltransferases (GSLGLTs) have been characterized, 10 of which have been solubilized. These GLTs can be classified into 2 distinct groups 1) GLTs dedicated to either Dol-P-P-sugar(s) or ceramide-linked sugar(s); and 2) GLTs with dual loyalties (i.e., they compete with glycolipid- and glycoprotein-bound oligosaccharides). Studies with solubilized and purified GalNAcT-1 and GalNAcT-2 from embryonic chicken brains prove that GalNAcT-1 (UDP-GalNAcGM3 beta 1-4GalNAcT) is specific for GSL, whereas GalNAcT-2 (UDP-GalNAcGb3 beta 1-3GalNAcT) can transfer to an oligosaccharide containing the alpha-linked terminal galactose. Similarly, GalT-3 (UDP-GalGM2 beta 1-3GalT) is more specific for ganglio-oligosaccharide and GalT-4 (UDP-GalLc3 beta 1-4GalT) can transfer galactose to N-acetylglucosamine linked to p-nitrophenol, glycolipid or glycoprotein. Both GalT-3 and GalT-4 have been separated and purified from embryonic chicken brains. Studies with solubilized SAT-4 and SAT-3, from bovine spleen and embryonic chicken brains, respectively, suggest the existence of 2 different gene-expressed alpha 2-3SATs. The newly discovered FucT-3 (GDP-FucNeuGc-iLc6-alpha 1-3FucT) from human colon carcinoma (Colo-205) has also been solubilized and separated from other GSLGLTs. Using a new activity gel-Western blot combined technique, the molecular mass of this FucT-3 was determined to be 105 kDa.