Purification and characterization of the Fas-ligand that induces apoptosis.
Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.
Suda
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T
Osaka Bioscience Institute, Japan.
Nagata
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S
D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis / Q000378:metabolism*; D017209:Apoptosis; D001483:Base Sequence; D002846:Chromatography, Affinity; D003208:Concanavalin A; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D019014:fas Receptor
D000954:Antigens, Surface; D017931:DNA Primers; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D003208:Concanavalin A