forssman-glycolipid and Carcinoma--Embryonal

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [(14)C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Carbohydrate Sequence; Carcinoma, Embryonal; Cell Line, Tumor; Cell Membrane; Centrifugation, Density Gradient; Chromatography, Thin Layer; Clostridium perfringens; Collodion; Epitopes; Flow Cytometry; Galactose; Globosides; Glycosphingolipids; Glycosylation; Humans; Mass Spectrometry; Mice; Microscopy, Confocal; Molecular Sequence Data; Neuraminidase; Protein Binding; Protein Structure, Tertiary; Receptors, Laminin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stage-Specific Embryonic Antigens; Subcellular Fractions; Sucrose