A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.
Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.
Ray
M K
MK
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Yang
J
J
Sundaram
S
S
Stanley
P
P
D017485:1-Deoxynojirimycin; D000818:Animals; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A / Q000378:metabolism; D006224:Cricetinae; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000378:metabolism; D000520:alpha-Glucosidases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D003208:Concanavalin A; D017485:1-Deoxynojirimycin; C042059:glucosidase I; D000520:alpha-Glucosidases; D005947:Glucose; D005944:Glucosamine