Carbohydrate chain analysis by lectin binding to electrophoretically separated glycoproteins from murine B16 melanoma sublines of various metastatic properties.

Cellular glycoprotein carbohydrate chains of B16 melanoma sublines of various metastatic colonization capacities were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and direct lectin staining, combined with chemical modification of carbohydrate chains in situ. For these studies, we utilized B16 sublines selected for low (B16-F1) or high lung (B16-F10), high brain (B16-B15b), or high ovary (B16-O13) colonization properties, or high tissue invasiveness in vitro (B16-BL6). The major B16 cell surface sialoglycoproteins were of Mr approximately 115,000, approximately 90,000, approximately 82,000, and 60,000 to 65,000, and were detectable by periodate NaB3H4 labeling and binding of 125I-wheat germ agglutinin (WGA). Terminal sialic acid residues in the carbohydrate chains were responsible for WGA binding, since chemical removal of sialic acid prevented WGA labeling of the glycoproteins. However, removal of sialic acid residues followed by Smith degradation resulted in reappearance of WGA-binding sites on these sialoglycoproteins, indicating that the carbohydrate chains possessed at least one branching point at an outer alpha-mannosyl residue. This structural feature was further indicated by the failure of 125I-Lens culinaris hemagglutinin to bind to these sialoglycoproteins. The fact that the carbohydrate residues of the Mr approximately 115,000, approximately 90,000, and approximately 82,000 sialoglycoproteins were of the complex type was confirmed by their reactivity with 125I-Ricinus communis agglutinin I, which preferentially binds to Gal leads to GlcNAc sequences after removal of sialic acid in situ. In contrast, 125I-peanut (Arachis hypogaea) agglutinin, specific for Gal leads to GalNAc sequences, failed to bind to the major WGA-reactive sialoglycoproteins, but strongly interacted after removal of sialic acid with Mr approximately 51,000 and approximately 56,000 glycoproteins from sublines B16-F1, -F10, and -BL6 and with a Mr approximately 63,000 glycoprotein from sublines B16-F10, -BL6, -O13, and -B15b. Thus, the small, mucin-type carbohydrate chains were expressed almost exclusively on these lower Mr sialoglycoproteins, and very little on the Mr approximately 82,000, approximately 90,000, and approximately 115,000 sialoglycoproteins. Differences in lectin binding to glycoproteins were observed with different sublines. These glycoproteins included: (a) a WGA-binding Mr 60,000 to 75,000 sialoglycoprotein prominent on B16-B15b cells.(ABSTRACT TRUNCATED AT 400 WORDS)