Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells.
Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.
D000116:Acetylgalactosamine / Q000378:metabolism; D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002999:Clone Cells; D003902:Detergents / Q000494:pharmacology; D005577:Forssman Antigen; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D005915:Globosides / Q000096:biosynthesis*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D006168:Guinea Pigs; D005779:Immunodiffusion; D007700:Kinetics; D017350:N-Acetylgalactosaminyltransferases; D014316:Tritium; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D000116:Acetylgalactosamine; D001564:Benzo(a)pyrene; D003902:Detergents; D005577:Forssman Antigen; D005700:Galactosyltransferases; D005915:Globosides; D006028:Glycosphingolipids; D017350:N-Acetylgalactosaminyltransferases; D014316:Tritium; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C010163:Forssman glycolipid; C023216:globoside alpha-N-acetylgalactosaminyltransferase