Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-β- d-galactopyranosyl group (1→3)-linked and forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Abstract

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:GbOse 3Cer β- N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of GalNAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 ([6- 3H]Galα1→4Galβ1→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 GbOse 4Cer ([ 3H]GalNAc→Galα1→4Galβ1→4Glc→Cer) was treated with purified clam β-hexosaminidase. The present results establish the formation of a β- d-Gal pNAc-(1→3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse 4Cer α-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[ 14C]- or -[ 3H]GalNAc to GbOse 4Cer (GalNAcβ1→3Galα1→4Galβ1→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.