A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects.The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16,17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21,22). Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of...

This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol ( Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol ( Arora et al. 1992 ; Milne et al. 1995 ). This work used the catalytic domains of other toxins which are normally translocated across membranes ( Arora & Leppla 1994) . Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non‐catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV‐1. Administration of this recombinant protein along with PA to antigen‐presenting cells sensitized them to cytolysis by cytotoxic T‐cells specific to gp120 peptides ( Goletz et al. 1997 ). Further exploitation of the anthrax toxin system as a cell‐targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure ( Petosa et al. 1997 ) allows rational engineering to modify or replace the receptor‐binding domain with specific ligand structures. A model system was produced by fusing a c‐Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non‐toxic mutants of PA ( Varughese et al. 1998 ).