A subclass of cell surface carbohydrates revealed by a CHO mutant with two glycosylation mutations.
A novel lectin-resistance phenotype was displayed by a LEC10 Chinese hamster ovary (CHO) cell mutant that was selected for resistance to the erythroagglutinin, E-PHA. Biochemical and genetic analyses revealed that the phenotype results from the expression of two glycosylation mutations, LEC10 and lec8. The LEC10 mutation causes the appearance of N-acetylglucosaminyltransferase III (GlcNAc-TIII) activity and the production of N-linked carbohydrates with a bisecting GlcNAc residue. The lec8 mutation inhibits translocation of UDP-Gal into the Golgi lumen and thereby dramatically reduces galactosylation of all glycoconjugates. This reduction in galactose addition does not, however, cause Lec8 mutants to be very resistant to the galactose-binding lectin, ricin. By contrast, the double mutant LEC10.Lec8 behaved like a LEC10 mutant and was highly resistant to ricin. Based on structural studies of cellular glycopeptides as well as glycopeptides of the G glycoprotein of vesicular stomatitis virus grown in mutant cells, it appears that the ricin resistance of LEC10.Lec8 cells is due to the presence of a small number of Gal residues on branched, N-linked carbohydrates that also carry the bisecting GlcNAc residue. Labelling of N-linked cellular carbohydrates with [3H]galactose was found to occur at a low level for a wide spectrum of cellular glycoproteins in independent Lec8 mutants. Studies of the LEC10.Lec8 mutant have, therefore, led to the identification of a subset of structures that are acceptors for Gal when intra-Golgi UDP-Gal levels are limiting. This mutant also illustrates the potential for regulating cell surface recognition by carbohydrate-binding proteins by altering the expression of a single glycosyltransferase such as GlcNAc-TIII.
Stanley
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Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Sundaram
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Sallustio
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D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002250:Carbon Radioisotopes; D002846:Chromatography, Affinity; D006224:Cricetinae; D005690:Galactose / Q000378:metabolism; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics*; D006020:Glycopeptides / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases; D010641:Phenotype; D014316:Tritium; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D002250:Carbon Radioisotopes; D006020:Glycopeptides; D006023:Glycoproteins; D014316:Tritium; D014531:Uridine Diphosphate Galactose; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D005947:Glucose; D005690:Galactose