Generation of novel chimeric LacdiNAcS by gene fusion of alpha-lactalbumin and beta1,4-galactosyltransferase 1

Abstract

Novel chimeric lacdiNAc (GalNAc(beta1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of alpha-lactalbumin (alpha-LA) and beta1,4-galactosyltransferase 1 (beta1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous alpha-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and beta1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine beta1,4-GalT1 and alpha-LA, indicating that each domain of alpha-LA and beta1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.