The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated.
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.
Fabbrini
M S
MS
Dibit-Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy. fabbris@dibit.hsr.it
Carpani
D
D
Bello-Rivero
I
I
Soria
M R
MR
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D000922:Immunotoxins / Q000493:pharmacokinetics* / Q000633:toxicity; D009699:N-Glycosyl Hydrolases; D010446:Peptide Fragments / Q000493:pharmacokinetics* / Q000633:toxicity; D010940:Plant Proteins / Q000493:pharmacokinetics* / Q000633:toxicity; D011500:Protein Synthesis Inhibitors / Q000493:pharmacokinetics / Q000633:toxicity; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000502:physiology; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects; D000076985:Saporins; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000493:pharmacokinetics*
D000922:Immunotoxins; C524898:PLAUR protein, human; D010446:Peptide Fragments; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C109077:urokinase-ATF-SAP-3, recombinant; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D014568:Urokinase-Type Plasminogen Activator