Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides.
Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.
Fukumoto
S
S
Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Nagoya 466-0065, Japan.
Miyazaki
H
H
Goto
G
G
Urano
T
T
Furukawa
K
K
Furukawa
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology; D005679:G(M3) Ganglioside / Q000096:biosynthesis; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012799:Sialyltransferases / Q000235:genetics*
D018076:DNA, Complementary; D005679:G(M3) Ganglioside; D012799:Sialyltransferases; C021343:haematoside synthetase