Abstract
In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Line
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Cell Survival / drug effects
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Cloning, Molecular
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Escherichia coli
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Humans
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Immunotoxins / pharmacokinetics*
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Immunotoxins / toxicity
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N-Glycosyl Hydrolases*
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Peptide Fragments / pharmacokinetics*
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Peptide Fragments / toxicity
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Plant Proteins / pharmacokinetics*
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Plant Proteins / toxicity
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Protein Synthesis Inhibitors / pharmacokinetics
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Protein Synthesis Inhibitors / toxicity
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Receptors, Cell Surface / biosynthesis
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Receptors, Cell Surface / physiology
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Receptors, Urokinase Plasminogen Activator
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Recombinant Fusion Proteins*
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Recombinant Proteins / pharmacokinetics
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Ribosome Inactivating Proteins, Type 1
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Ribosomes / drug effects
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Saporins
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Tumor Cells, Cultured
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Urokinase-Type Plasminogen Activator / pharmacokinetics*
Substances
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Immunotoxins
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PLAUR protein, human
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Peptide Fragments
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Plant Proteins
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Protein Synthesis Inhibitors
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Receptors, Cell Surface
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Receptors, Urokinase Plasminogen Activator
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Recombinant Fusion Proteins
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Recombinant Proteins
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Ribosome Inactivating Proteins, Type 1
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urokinase-ATF-SAP-3, recombinant
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N-Glycosyl Hydrolases
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Saporins
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Urokinase-Type Plasminogen Activator