A chimeric saporin-transferrin conjugate compared to ricin toxin: role of the carrier in intracellular transport and toxicity.
Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.
Ippoliti
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Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche, Università di Roma La Sapienza, Italy.
Lendaro
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D'Agostino
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Fiani
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Guidarini
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Vestri
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Benedetti
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Brunori
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D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D001692:Biological Transport; D016923:Cell Death; D002461:Cell Line, Transformed / Q000187:drug effects; D004337:Drug Carriers; D006801:Humans; D000922:Immunotoxins; D018715:Microscopy, Video; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D000076985:Saporins; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D000972:Antineoplastic Agents, Phytogenic; D004337:Drug Carriers; D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014168:Transferrin; D012276:Ricin; D009699:N-Glycosyl Hydrolases; D000076985:Saporins