Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide.
Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.
Clark
G F
GF
Department of Biochemistry and Nutrition, Virginia Tech., Blacksburg, Virginia 24061.
Gorbea
C M
CM
Cummings
R D
RD
Mattox
S
S
Smith
D F
DF
D000818:Animals; D002240:Carbohydrate Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002846:Chromatography, Affinity; D005577:Forssman Antigen / Q000096:biosynthesis / Q000737:chemistry; D005915:Globosides / Q000096:biosynthesis* / Q000737:chemistry; D037102:Lectins; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D013724:Teratoma / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D005915:Globosides; C038095:Helix lectin; D037102:Lectins; D009844:Oligosaccharides; D014212:Tretinoin; C010163:Forssman glycolipid; D005577:Forssman Antigen