Isolation of GD3Synthase Gene by Expression Cloning of GM3α-2,8-Sialyltransferase cDNA Using Anti-GD2Monoclonal Antibody

For the isolation of ganglioside GD3synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2as well as GM3b...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1994-10, Vol.91 (22), p.10455-10459
Main Authors: Haraguchi, Masashi, Yamashiro, Shuji, Yamamoto, Akihito, Furukawa, Keiko, Takamiya, Kogo, Lloyd, Kenneth O., Shiku, Hiroshi, Furukawa, Koichi
Format: Article
Language:English
Subjects:
Amino acids
Cell lines
Complementary DNA
Gangliosides
Melanoma
Messenger RNA
Monoclonal antibodies
T lymphocytes
Transfection
Tumor cell line
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Summary:For the isolation of ganglioside GD3synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2as well as GM3but no GD3or GD2and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned β-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 GD3in KF3027 and B16 melanoma cells and GD3and GD2in 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.
ISSN:0027-8424