Kohls MD, Lappi DA (2003) Cholera toxin B-saporin cytotoxicity is correlated with the extent of GM1 expression on the cell surface. Neuroscience 2003 Abstracts 325.16. Society for Neuroscience, New Orleans, LA.
Summary: Cholera toxin is composed of five B-subunits that mediate binding to the cell surface monosialoganglioside GM1, and one A-subunit that irreversibly activates adenylate cyclase. GM1 is found in high concentrations on astrocytes and the myelin of oligodendrocytes, as well as on many other cell types. A targeted toxin was developed consisting of the ribosome-inactivating protein saporin coupled to the B-subunit of cholera toxin (CTB) for the purpose of eliminating cells that express GM1. This targeted toxin (CTB-SAP) binds to cell-surface GM1 and the entire molecule is then internalized. Saporin separates from CTB and is released from the endosome to inactivate ribosomes. The cytoxicity of CTB-SAP in a cell-based in vitro assay can be inhibited by the addition of free CTB, indicating binding specificity. CTB-SAP has been tested on a variety of cell lines and the effectiveness of the targeted toxin correlates with the extent of GM1 cell surface expression as demonstrated by fluorescence-activated cell sorting (FACS) analysis. For example, using FACS analysis, RBL-2H3 cells exhibit an 88% shift when labeled with CTB-FITC. The ED50 of CTB-SAP in a cytoxicity assay on these cells is 380 fM, which corresponds to 9150 CTB-SAP molecules per cell. In comparison, HS294T cells produce a 48% shift in FACS, and the ED50 in the cytotoxicity assay is 5.5 pM. The correlation of surface GM1 number with ED50 indicates that the number of saporin molecules internalized is of primary importance in the process of cytotoxicity. CTB-SAP has been used to demyelinate the lumbar spinal cord (Jasmin et al.), eliminate sympathetic preganglionic neurons, and eliminate facial motoneurons in the rat (Llewellyn-Smith et al.). CTB-SAP is an effective and specific tool for the in vitro and in vivo elimination of cells that express GM1 on the cell surface.
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