STUDIES ON GLYCOPROTEINS PRODUCED BY WILD TYPE AND WHEAT GERM AGGLUTININ-RESISTANT B16 MOUSE MELANOMA CELLS.

摘要

Two variants of B16 mouse melanoma cells have been selected in serum-free medium for their resistance to toxic levels of wheat germ agglutinin isolation 1 (WGA). Chromosome analysis and characteristic melanin production showed that the variants are derived from the parent mouse melanoma cell lines. However, the two variants were less tumorigenic in mice compared to the parent B16 mouse melanoma cells. The variants showed a marked decrease in cell agglutination with WGA. Cell agglutination with ricin and peanut lectin was not different between the three cell lines, but the two variants showed a slight increase in agglutination with concanavalin A. The binding of ('125)I-labeled wheat germ agglutinin to the two variant cells was reduced compared to that of the parent cell.;Glycoproteins secreted or shed by the three lines were isolated after growth in serum-free medium in the presence of ('3)H glucosamine and bovine serum albumin (1%). These metabolically labeled products were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/ml). The WGA-Sepharose affinity chromatographic data suggested a decrease in WGA-binding glycoprotein(s) secreted to the medium by the two variants. The WGA-bound glycoproteins from the two variants upon SDS-PAGE revealed three bands of approximate molecular weights, 92,000, 56,000, and 42,000, none of which were present in the parent cell line (50,000 molecular weight). The glycoprotein contains both N- and O-linked saccharide chains. Neuraminidase treatment together with ricin-Sepharose affinity chromatography data suggested the presence of terminal NeuNAc-Gel sequences in most of the saccharide chains. Mild alkaline borohydride treatment together with high voltage paper electrophoresis and HPLC analysis showed that the O-linked structures are mainly tetrasaccharide. Hydrazinolysis followed by high voltage paper electrophoresis, HPLC and serial lectin affinity chromatography of the alkaline borohydride-resistant portion showed the presence of mainly trisialylated, tri-antennary N-linked saccharides with outer fucosylation. Polyclonal antibodies prepared against the 50K glycoprotein show unchanged reactivity with native, asialo or asialoagalacto forms. The glycoprotein can be detected in the circulation of tumor-bearing mice.