Expression of an NCA cDNA in NIH/3T3 cells yields a 110K glycoprotein, which is anchored into the membrane via glycosyl-phosphatidylinositol.
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.
Kolbinger
F
F
Institute of Immunobiology, University of Freiburg, FRG.
Schwarz
K
K
Brombacher
F
F
von Kleist
S
S
Grunert
F
F
D000818:Animals; D000941:Antigens / Q000235:genetics*; D000951:Antigens, Neoplasm; D015152:Blotting, Northern; D015139:Blotting, Southern; D002272:Carcinoembryonic Antigen / Q000235:genetics; D015815:Cell Adhesion Molecules; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005434:Flow Cytometry; D005796:Genes; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism*; D015183:Restriction Mapping
D000941:Antigens; D000951:Antigens, Neoplasm; D002272:Carcinoembryonic Antigen; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D004247:DNA