GAIN-OF-FUNCTION CHINESE-HAMSTER OVARY MUTANTS LEC18 AND LEC14 EACH EXPRESS A NOVEL N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY

LEC18 and LEC14 cells are gain-of-function glycosylation mutants isola ted from Chinese hamster ovary cells for resistance to pea lectin. Str uctural studies have shown that LEC18 cells synthesize complex N-glyca ns with a GlcNAc residue linked at the O-6 position of the core GlcNAc (Raju, T. S.,, Ray, M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-2 position of the core beta-linked Man residue (Raju, T. S., and Stanley, P., (1996) J. Biol. Chem. 271, 748 4-7493). Both modifications are novel and have not been reported in gl ycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucos aminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcN Ac-TVIII, transfers GlcNAc to GlcNAc beta 1-O-pNP and to a GlcNAc-term inating, biantennary, complex N-glycan, with or without a core fucose. By contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have significant activity with simple accepters, and transfers GlcNAc pref erentially to a GlcNAc-terminating biantennary glycopeptide that conta ins a core fucose residue. The acceptor specificities and other bioche mical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previousl y characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of tra nsferases.