title: Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin.
abstract: Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Zdanovsky
authors.forename: M V
authors.initials: MV
authors.lastname: Zdanovskaia
authors.forename: D
authors.initials: D
authors.lastname: Strickland
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D005098:Exotoxins / Q000235:genetics / Q000633:toxicity*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011549:Pseudomonas / Q000235:genetics; D011971:Receptors, Immunologic / Q000187:drug effects* / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity; D037521:Virulence Factors; D000511:alpha-Macroglobulins / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002352:Carrier Proteins; D005098:Exotoxins; D006023:Glycoproteins; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins; D037521:Virulence Factors; D000511:alpha-Macroglobulins; D000097668:Pseudomonas aeruginosa Exotoxin A