Expression cloning of mouse cDNA of CMP-NeuAc : lactosylceramide alpha 2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Expression cloning of a cDNA for the alpha 2,3-sialyltransferase (GM3 synth ase) (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 pdl302 7 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA c lone pM3T-7 predicted a type II membrane protein with 13 amino acids of cyt oplasmic domain, 17 amino acids of transmembrane region, and a large cataly tic domain with 329 amino acids. Introduction of the cDNA clone into L cell s resulted in the neo-synthesis of GM3 and high activity of alpha 2,3-sialy ltransferase. Among glycosphingolipids, only lactosylceramide showed signif icant activity as an acceptor, indicating that this gene product is a sialy ltransferase specific for the synthesis of GM3. An amino acid sequence dedu ced from the cloned cDNA showed the typical sialyl motif with common featur es among alpha 2,3-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.