Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of sensitive cells, is cleaved by the cell surface protease furin, and only then captures either of the two other proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and pass to an acidic vesicle from which LF and EF escape to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase which causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease which cleaves mitogen-activated protein kinase kinase 1 (MEK1) both in vitro and within toxin-treated cells. Our work exploits unique features of these proteins to construct therapeutic agents. PA is the critical immunogen in the current and all proposed second generation human anthrax vaccines. Anthrax toxin LF and a catalytically inactive LF mutant were made in large amounts and supplied to a collaborator who used these proteins to solve the X-ray crystal structure. The three domains previously recognized by site specific mutagenesis are evident as distinct domains in the structure determined. Additional mutagenesis of the small loop of domain 4 of PA confirmed the importance of the small loop centered around residue N682 in interaction with the cellular receptor. Single amino acid substitutions were made in each residue in the small loop and the proteins were made and tested for toxicity. Several single amino acid substitutions caused more than 500-fold decreases in toxicity. Identification of PA mutants such as this which do not bind to receptor provides the basis for construction of fusion proteins targeted to new receptors. As an alternative method of achieving cell-type specificity, the furin site in PA was replaced with sequences cleaved by cell surface proteases which are enriched on tumor cells. These proteins were shown to be highly specific for cultured tumor cells while sparing normal cells in tissue culture systems. Methods for production of anthrax toxin components were further improved, so as to provide large amounts for use as improved anthrax vaccines.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Intramural Research (Z01)
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Dental & Craniofacial Research
United States
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