Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen
| Autor: | Leon Eidels, Jacqueline G. Brown, Brian D. Almond, Joseph G. Naglich |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Recombinant Fusion Proteins
Receptor expression Molecular Sequence Data Enzyme-Linked Immunosorbent Assay Receptors Cell Surface Biology Transfection Polymerase Chain Reaction Tetraspanin 29 L Cells (Cell Line) Mice L Cells Antigen Antigens CD Animals Humans Diphtheria Toxin Receptors Cholinergic Cloning Molecular Receptor Vero Cells Diphtheria toxin Membrane Glycoproteins Multidisciplinary Base Sequence Molecular biology Kinetics Phenotype Oligodeoxyribonucleotides Cell culture Vero cell Intercellular Signaling Peptides and Proteins Heparin-binding EGF-like Growth Factor Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 90:8184-8188 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.90.17.8184 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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