Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen.
DTS-II is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTS-II cells are as toxin-sensitive as Vero cells, have approximately 3-fold more receptors than Vero cells, and have approximately 10-fold lower affinity for DT than Vero cells. We now cotransfected DTS-II cells with a plasmid containing the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensitive colony was isolated and purified. The purified DT-hypersensitive cells, DTS-III, (i) are approximately 10-fold more toxin-sensitive than DTS-II and Vero cells and (ii) bear approximately 10(6) DT receptors per cell (i.e., approximately 20-fold and approximately 60-fold more receptors than DTS-II and Vero cells, respectively), but their receptor affinity is still approximately 10-fold lower than that of Vero cells. Cross-linking experiments employing 125I-labeled DT demonstrated that DTS-II and DTS-III cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTS-III cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTS-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).
Brown
J G
JG
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Almond
B D
BD
Naglich
J G
JG
Eidels
L
L
D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000378:metabolism / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D007739:L Cells; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000096:biosynthesis* / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects; D060245:Tetraspanin 29; D014162:Transfection; D014709:Vero Cells
D015703:Antigens, CD; C554869:CD9 protein, human; C554870:Cd9 protein, mouse; D004167:Diphtheria Toxin; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D060245:Tetraspanin 29