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 isolated from Chinese hamster ovary cells for resistance to pea lectin. Structural studies have shown that LEC18 cells synthesize complex N-glycans 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, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucosaminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to GlcNAcbeta1-O-pNP and to a GlcNAc-terminating, 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 acceptors, and transfers GlcNAc preferentially to a GlcNAc-terminating biantennary glycopeptide that contains a core fucose residue. The acceptor specificities and other biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previously characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of transferases.
Raju
T S
TS
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.
Stanley
P
P
D000117:Acetylglucosamine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006020:Glycopeptides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017351:N-Acetylglucosaminyltransferases / Q000145:classification / Q000235:genetics*; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D006020:Glycopeptides; D009844:Oligosaccharides; D011134:Polysaccharides; D008345:Manganese; D000255:Adenosine Triphosphate; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine