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.