Expression of an NCA cDNA in NIH/3T3 cells yields a 110K glycoprotein, which is anchored into the membrane via glycosyl-phosphatidylinositol

• Published in: Biochemical and biophysical research communications Vol. 161; no. 3; pp. 1126 - 1134
• Main Authors: KOLBINGER, F, SCHWARZ, K, BROMBACHER, F, VON KLEIST, S, GRUNERT, F
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier 30.06.1989
• Subjects: Animals; Antigens - genetics; Antigens, Neoplasm; Biological and medical sciences; Blotting, Northern; Blotting, Southern; Carcinoembryonic Antigen - genetics; Cell Adhesion Molecules; Cell coat. Cell surface; Cell Membrane - metabolism; Cell structures and functions; Cells, Cultured; DNA - genetics; DNA - isolation & purification; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Genes; Genetic Vectors; Glycoproteins - biosynthesis; Glycoproteins - genetics; Membrane Lipids - metabolism; Mice; Molecular and cellular biology; Molecular Weight; Phosphatidylinositols - metabolism; Restriction Mapping; Cell culture; Flow cytometry; Membrane protein; Radiolabelling; Rodentia; 3T3-Cell line; Glycoproteins; Binding site; Gene expression; Immunological method; Northern blotting; Carcinoembryonic antigen; Vertebrata; Immunoprecipitation reaction; Mammalia; Cell line; Transfection; Complementary DNA; Cross reaction; Restriction map; Genetic engineering; ELISA assay
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(89)91359-4

The NCA cDNA, which represents a gene belonging to the CEA family, was inserted into an SV40 early promoter-driven expression vector and used for transfection of mouse NIH/3T3 cells. A cell line, NIH/3T3/KNCA IG7, was selected which expressed a molecule with an apparent molecular weight of 110,000. The mode of membrane attachment of this NCA, which we already proposed to be anchored via glycosyl-phosphatidylinositol, was investigated by treatment of NIH/3T3/KNCA IG7 cells with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Two independent methods, flow cytometry and immunoprecipitation of [3H]-labelled surface glycoproteins, clearly demonstrated that the NCA molecule expressed by NIH/3T3/KNCA IG7 cells is indeed anchored into the membrane via glycosyl-phosphatidylinositol. Furthermore, these results support our previous biochemical data on NCA-50, by unequivocally showing that the NCA cDNA used for transfection encodes an NCA molecule related to NCA-50 and NCA-90.