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). In our latest work, uptake of the toxin was shown to require only the extracellular domain of the recently-identified tumor endothelial marker 8 (TEM8) toxin receptor, and to involve partition of the PA-receptor complex into lipid rafts. Extensive studies showed that the lethal toxin, PA plus LF, caused a vascular collapse in several types of inbred mice, regardless of the sensitivity of their macrophages to lysis, and independent of a cytokine response. These finding may have relevance to supportive therapies for human anthrax patients. Retroviral mutagenesis of cultured CHO cells identified a small gene involved in synthesis of diphthamide, the target of ADP-ribosylating toxins such as diphtheria toxin. A PA variant protein activated by urokinase plasminogen activator was able to specifically target tumor cells in vitro and greatly reduced growth of three different types of tumors in mice. Specific targeting of tumor cells by requiring cell surface plasminogen activator action was extended to existing diphtheria toxin-based immunotoxins. Collaborative efforts related to anthrax vaccine development included studies that mapped an epitope in the receptor binding region of PA that is recognized by a neutralizing monoclonal antibody. Antibodies of higher affinity to this epitope were obtained by phage display of randomly mutated antibody libraries. PA was shown able to induce a mucosal immune response. Improved methods for PA and LF production were developed and the technologies transferred to academic and commercial groups working to develop improved anthrax vaccines. A conjugate of polyglutamate to PA was shown to induce antibodies to both PA and to the bacterial capsular material, so that both anti-toxin and anti-bacterial antibodies were provided by a single immunogen.
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