Isolation and characterization of a wheat germ agglutinin-binding glycoprotein from B16 mouse melanoma cells.
Cells of B16 mouse melanoma grown in serum-free medium in the presence of [3H]glucosamine secreted or shed labeled glycoproteins. A wheat germ agglutinin-binding glycoprotein was isolated that accounted for 37% of the total [3H]glucosamine incorporated; it had a molecular weight of approximately 50,000 and was absent in less-tumorgenic wheat germ agglutinin (isolectin I)-resistant variants of the cells. The glycoprotein contained approximately 25% of serine and threonine plus equimolar amounts of glucosamine and galactosamine, indicating both N- and O-linked oligosaccharide chains. Neuraminidase treatment released approximately 60% of the glycoprotein's 3H radioactivity as N-acetylneuraminic acid. The sialoglycoprotein did not, but the desialylated species did, bind (97%) to ricin-Sepharose, suggesting the presence of terminal sialic acid and penultimate galactose residues in most of the saccharide units. Alkaline borohydride released 61% of the glycoprotein's radioactivity as oligosaccharide alcohols that were mainly tetrasaccharides (75%) with some branched trisaccharides (10%) from the O-linked structures. Hydrazinolysis and analysis of the alkaline borohydride-resistant portion of the glycoprotein indicated the presence of mainly triantennary, complex-type structures (N-linked) containing three sialic acids residues plus L-fucose. Serial lectin-affinity chromatography of the hydrazine-released saccharides with concanavalin A-agarose, pea lectin-agarose, L-PHA-agarose, and E-PHA-agarose, indicated the absence of high-mannose or hybrid-type structures and further confirmed the presence of triantennary, complex-type units.
D000818:Animals; D002460:Cell Line; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000032:analysis; D011975:Receptors, Mitogen / Q000302:isolation & purification* / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D014316:Tritium; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; D011975:Receptors, Mitogen; D012794:Sialic Acids; D014909:Wheat Germ Agglutinins; C027503:wheat germ agglutinin receptor; D014316:Tritium; D019158:N-Acetylneuraminic Acid; D005944:Glucosamine