Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen.
The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid sequence of APO-1 showed sequence identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.
Oehm
A
A
Institute for Immunology and Genetics, German Cancer Research Center, Heidelberg.
Behrmann
I
I
Falk
W
W
Pawlita
M
M
Maier
G
G
Klas
C
C
Li-Weber
M
M
Richards
S
S
Dhein
J
J
Trauth
B C
BC
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D016923:Cell Death; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009414:Nerve Growth Factors / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D012689:Sequence Homology, Nucleic Acid; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019014:fas Receptor
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D004247:DNA