Isolation of a cDNA encoding a murine UDP galactose: beta -D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha -1,3-galactosyltransferase: Expression cloning by gene transfer

The authors have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose: beta -D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha -1,3-galactosyltransferase (( alpha 1-3)GT: EC 2.4.1.151). This library was transfected into COS-1 cells, which lack expression of ( alpha 1-3)GT. Transfected cells containing functional ( alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the ( alpha 1-3)GT enzyme. One cloned ( alpha 1-3)GT cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide.