Oligosaccharide composition of the neurotoxin-responsive sodium channel of mouse neuroblastoma and requirement of sialic acid for biological activity.
A glycoprotein, M(r) 200,000, which has the biological activity of the neurotoxin-responsive Na+ channel, was isolated from a clonal line of mouse neuroblastoma cells, N-18. The glycoprotein was purified to homogeneity in 18% yield by methods used to purify glycoproteins, which included metabolic labeling of the cells with L-[3H]fucose and binding of the radioactive glycoproteins to WGA- and lentil-Sepharose, and DEAE-cellulose. The glycoprotein has biological activity of neurotoxin-responsive ion flux when reconstituted into artificial phospholipid vesicles. This activity was shown to depend on the presence of sialic acid since treatment of the purified, reconstituted glycoprotein with Vibrio cholerae neuraminidase abolished the response to neurotoxins of 86Rb flux. The [3H]fucose-containing glycopeptides derived by Pronase digestion of the glycoprotein were characterized by affinity to immobilized lectins and contained di-, tri-, and tetra-antennary oligosaccharides in a ratio of 2:4:3. Most of the glycopeptides were sialylated as shown by binding characteristics to immobilized serotonin-Sepharose with and without neuraminidase. The structure of the diantennary oligosaccharides was elucidated by 500-MHz 1H NMR spectroscopy. The Con A-bound fraction contains alpha-NeuNAc-(2-->6)-bound group on the GlcNAc5' antenna and an alpha-NeuNAc-(2-->3)-bound groups on the GlcNAc5 antenna. An alpha-L-fucosyl group is (1-->6)-bound to the Asn core GlcNAc1 residue.
Negishi
M
M
Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia.
van Kuik
J A
JA
Vliegenthart
J F
JF
Glick
M C
MC
D000818:Animals; D002240:Carbohydrate Sequence; D003208:Concanavalin A / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009447:Neuroblastoma / Q000737:chemistry*; D009498:Neurotoxins / Q000494:pharmacology*; D009844:Oligosaccharides / Q000032:analysis*; D012414:Rubidium Radioisotopes; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000494:pharmacology*; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects; D014407:Tumor Cells, Cultured
D006020:Glycopeptides; D006023:Glycoproteins; D009498:Neurotoxins; D009844:Oligosaccharides; D012414:Rubidium Radioisotopes; D012794:Sialic Acids; D015222:Sodium Channels; D003208:Concanavalin A; D019158:N-Acetylneuraminic Acid