TY - JOUR
T1 - Lectin-deficient ricin toxin indicates cells bearing the D-mannose receptor
AU - Frankel, Arthur E.
AU - Fu, Tao
AU - Burbage, Christopher
AU - Tagge, Edward
AU - Harris, Billie
AU - Vesely, Joseph
AU - Willingham, Mark C.
N1 - Funding Information:
We thank James Nicholson for graphics analysis and Dr. H. Kirk Ziegler of Emory University for providing our peritoneal macrophage protocol. We thank Drs. Philip Stahl and Kurt Drickamer for mannose receptor-expressing cell lines. This work was supported by an institutional grant from the Medical University of South Carolina (AF).
PY - 1997/5/16
Y1 - 1997/5/16
N2 - Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor. Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors. We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin site domains (1α, 1β, and 2γ) yielding a 1000-fold reduced galactoside avidity. After reassociation with plant RTA, the recombinant heterpdimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor. Receptor expression was confirmed by immunofluorescence microscopy. Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting. Plant ricin A chain (RTA) and E. coli-derived RTA were also tested for cytotoxicity on J774E and KB cells. Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity. This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity. Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations. These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.
AB - Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor. Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors. We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin site domains (1α, 1β, and 2γ) yielding a 1000-fold reduced galactoside avidity. After reassociation with plant RTA, the recombinant heterpdimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor. Receptor expression was confirmed by immunofluorescence microscopy. Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting. Plant ricin A chain (RTA) and E. coli-derived RTA were also tested for cytotoxicity on J774E and KB cells. Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity. This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity. Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations. These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.
KW - Mannose receptor
KW - Ricin
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U2 - 10.1016/S0008-6215(97)00048-7
DO - 10.1016/S0008-6215(97)00048-7
M3 - Article
C2 - 9202409
AN - SCOPUS:0030959673
SN - 0008-6215
VL - 300
SP - 251
EP - 258
JO - Carbohydrate Research
JF - Carbohydrate Research
IS - 3
ER -