Expression of a differentiation antigen and poly-N-acetyllactosaminyl O-glycans directed by a cloned core 2 beta-1,6-N-acetylglucosaminyltransferase

Abstract

Chinese hamster ovary (CHO) cells do not contain detectable amounts of core 2 beta-1,6-N-acetylglucosaminyltransferase, C2GnT, and thus lack various modifications in their branched O-linked oligosaccharides. In the present study, the O-linked oligosaccharides and the occurrence of a differentiation antigen were analyzed in CHO cells stably transfected with cDNA encoding human leukosialin alone (CHO-leu) or with cDNAs encoding both leukosialin and C2GnT (CHO-leu.C2GnT). The analysis of O-glycans, released from [3H]glucosamine-labeled cells, revealed that CHO-leu cells synthesize O-glycans with a Gal beta 1-->3GalNAc backbone, whereas CHO-leu.C2GnT cells synthesize in addition O-glycans with a Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc backbone. Moreover, CHO-leu.C2GnT cells express poly-N-acetyllactosaminyl extensions from the GlcNAc beta 1-->6 branch in O-glycans, while CHO-leu cells express no detectable amount of poly-N-acetyllactosaminyl O-glycans. It was also demonstrated that leukosialin in CHO-leu.C2GnT cells is recognized by the T305 monoclonal antibody, while the same antibody did not react at all with CHO-leu cells. In addition, the transient expression cloning scheme using the T305 monoclonal antibody as a selectin marker and COS-1 cells, which endogenously express C2GnT as recipient cells, resulted in the isolation of cDNA encoding leukosialin. These results indicate that C2GnT determines the expression of poly-N-acetyllactosamines in O-glycans and together with leukosialin, an onco-differentiation antigen recognized by the T305 antibody.