title: Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.
abstract: For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.
authors.affiliation: Department of Oncology, Nagasaki University School of Medicine, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Haraguchi
authors.forename: S
authors.initials: S
authors.lastname: Yamashiro
authors.forename: A
authors.initials: A
authors.lastname: Yamamoto
authors.forename: K
authors.initials: K
authors.lastname: Furukawa
authors.forename: K
authors.initials: K
authors.lastname: Takamiya
authors.forename: K O
authors.initials: KO
authors.lastname: Lloyd
authors.forename: H
authors.initials: H
authors.lastname: Shiku
authors.forename: K
authors.initials: K
authors.lastname: Furukawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005732:Gangliosides / Q000276:immunology*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008546:Melanoma, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D005732:Gangliosides; D012333:RNA, Messenger; C019403:ganglioside, GD2; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase