Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin 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 that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Our work exploits unique features of these proteins to construct therapeutic agents. Mutagenesis of the small loop of domain 4 of PA confirmed that residues surrounding N682 are critical for interaction with the cellular receptor. Single amino acid substitutions were made in each residue in the small loop and several substitutions were identified that decrease toxicity by more than 100-fold. Identification of PA mutants such as this provides the basis for construction of fusion proteins targeted to new receptors. PA proteins were constructed that are redirected to the GMCSF and IL-2 receptors. As an alternative method of achieving cell-type specificity, the furin site in PA was replaced by sequences cleaved by cell surface plasminogen activators. These altered PA proteins were shown to be highly specific for cultured tumor cells while sparing normal cells in tissue culture systems. Studies in knockout mice confirmed that all components of the plasminogen activator system are needed to activate these proteins in vivo. These constructs have potent anti-tumor activity in mouse tumor models. PA is the critical immunogen in the current and all proposed second-generation human anthrax vaccines. Methods for production of anthrax toxin components were further improved, so as to provide large amounts for use as improved anthrax vaccines. A collaborator used purified LF to solve the X-ray crystal structure. This structure is being used to aid in the design of protease inhibitors that may act to treat or prevent anthrax infections.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Intramural Research (Z01)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Dental & Craniofacial Research
United States
Zip Code
Ramirez, D M; Leppla, S H; Schneerson, R et al. (2002) Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis. J Ind Microbiol Biotechnol 28:232-8
Koo, Han-Mo; VanBrocklin, Matt; McWilliams, Mary Jane et al. (2002) Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proc Natl Acad Sci U S A 99:3052-7
Leppla, Stephen H; Robbins, John B; Schneerson, Rachel et al. (2002) Development of an improved vaccine for anthrax. J Clin Invest 110:141-4
Frankel, Arthur E; Bugge, Thomas H; Liu, Shihui et al. (2002) Peptide toxins directed at the matrix dissolution systems of cancer cells. Protein Pept Lett 9:1-14
Rosovitz, M J; Leppla, Stephen H (2002) Virus deals anthrax a killer blow. Nature 418:825-6
Chaudry, G Jilani; Moayeri, Mahtab; Liu, Shihui et al. (2002) Quickening the pace of anthrax research: three advances point towards possible therapies. Trends Microbiol 10:58-62
Maynard, Jennifer A; Maassen, Catharina B M; Leppla, Stephen H et al. (2002) Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat Biotechnol 20:597-601
Engelholm, L H; Nielsen, B S; Netzel-Arnett, S et al. (2001) The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis. Lab Invest 81:1403-14
Duesbery, N S; Resau, J; Webb, C P et al. (2001) Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways. Proc Natl Acad Sci U S A 98:4089-94
Price, B M; Liner, A L; Park, S et al. (2001) Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect Immun 69:4509-15

Showing the most recent 10 out of 15 publications