Expression cloning of beta 1,4 N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides.
GM2 and GD2 gangliosides are sialic acid-containing glycosphingolipids expressed in some normal tissues such as brain and in various tumors such as neuroblastomas, astrocytomas, and malignant melanomas. We used a eukaryotic cell transient expression system to isolate cDNA clones that determine GM2 expression. We developed a new cell line from murine melanoma line B16 by transfecting with the polyoma T antigen gene that was suitable for this purpose. Two cDNA clones, both of which have a continuous open reading frame of 1683 base pairs, were isolated. Although the cloned cDNAs had no primary sequence similarity to reported glycosyltransferases, the deduced amino acid sequence predicted a type II transmembrane protein with an overall structure similar to other glycosyltransferases. The cDNA clones, when stably transfected, determined the expression of GM2 in B16 cells and GM2 and GD2 in the human melanoma line MeWo. Northern blot analysis revealed two transcripts in all cells that expressed either GM2 or GD2 or both. These findings indicate that the cDNAs catalyze the transfer of GalNAc onto GM3 and GD3 by a beta 1,4 linkage, resulting in the synthesis of GM2 and GD2, respectively. Namely they suggest that these cDNAs derive from the UDP-GalNAc: GM3/GD3 beta 1,4 N-acetylgalactosaminyltransferase (EC 2.4.1.92) gene.
Nagata
Y
Y
Department of Oncology, Nagasaki University School of Medicine, Japan.
Yamashiro
S
S
Yodoi
J
J
Lloyd
K O
KO
Shiku
H
H
Furukawa
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005434:Flow Cytometry; D005678:G(M2) Ganglioside / Q000235:genetics*; D005732:Gangliosides / Q000235:genetics*; D015870:Gene Expression; D006596:Hexosaminidases / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured; D048828:beta-N-Acetyl-Galactosaminidase
D005732:Gangliosides; D005678:G(M2) Ganglioside; C019403:ganglioside, GD2; D004247:DNA; D006596:Hexosaminidases; D048828:beta-N-Acetyl-Galactosaminidase