Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells.
To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed. Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N. Arora and S. H. Leppla, J. Biol. Chem. 268:3334-3341, 1993). In this study, it was found that fusion proteins containing the ADP-ribosylation domain of diphtheria toxin fused at either the amino end or the carboxyl end of LF1-254 are highly toxic to Chinese hamster ovary (CHO) cells, indicating that translocation does not strictly require that the amino terminus of LF be free. A fusion protein containing the ribosome-inactivating A1 subunit of Shiga toxin fused to the carboxyl terminus of LF1-254 was also highly toxic for CHO cells. All fusion proteins were toxic only when administered with the anthrax toxin protective antigen component. The data show that the combination of protective antigen and LF fusion proteins can efficiently import polypeptides from diverse bacterial sources to the cytosol of eukaryotic cells and that LF fusion proteins may have the passenger polypeptides fused at either the amino terminus or the carboxyl terminus of LF1-254. These LF fusion proteins could potentially be used as components of a therapeutic agent when the destruction of certain types of cells is desired (e.g., in treating cancer).
Arora
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Laboratory of Microbial Ecology, National Institute of Dental Research, Bethesda, Maryland 20892.
Leppla
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SH
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000737:chemistry* / Q000633:toxicity*; D001692:Biological Transport; D016466:CHO Cells / Q000187:drug effects; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000633:toxicity; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; D013329:Structure-Activity Relationship
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; C030325:anthrax toxin