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 LF and EF translocate 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). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2(CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action have not been well understood. During the current period, we approached this important question by generating mice conditionally deleted for each of the two receptors. Surprisingly, the knockout mice lacking each receptor were viable and showed only subtle phenotypic changes. These mice and cells obtained from them were tested for sensitivity to the two anthrax toxins. The most striking result was that the mice lacking TEM8 were almost indistinguishable from normal mice in response to toxin, arguing that the TEM8 receptor plays a very small role in facilitating toxin action in mice. In contrast, mice lacking CMG2 were highly resistant to challenge with PA and either LF or EF, showing the dominant role played by CMG2. In these mice, death occurred only much later and with higher doses of toxin, again suggesting that the TEM8 receptor (remaining present in these mice) plays a limited and qualitatively different role in toxin action. By breeding the two knockout mice, progeny were obtained lacking both receptors. These mice were completely resistant to doses of toxin that were highly lethal to normal mice. Analysis of the knockout mice was extended to bacterial challenges. The CMG2 mice were highly resistant to infection with the Sterne strain of B. anthracis. This proves that the uptake of anthrax toxin through the CMG2 receptor plays a key role in facilitating anthrax infections, and validates this protein as a therapeutic target. To explain the greater role of CMG2, we isolated and cultured primary cells from each of the knockout mice. These were used in competitive binding studies (e.g., Schild analysis) that showed PA binds with 10-fold higher affinity to CMG2 than to TEM8. This difference may explain at least in part the greater role of CMG2 in toxin action in mice. In this period we also extended use of a fusion of the N-terminal portion of LF (LFn, amino acids 1-254) with beta-lactamase (BLA). The availability of the fluorescent beta-lactase substrate CCF2/AM makes the LFnBLA fusion protein a sensitive reporter on the process of LF internalization into cultured cells and animal tissues. We improved the potency of this reporter by restoring the N-terminus of the recombinant protein to the native AGG.. sequence that was recently shown to enhance the activity of full-size, native LF. In addition, we developed methods for producing this protein in avirulent strains of B. anthracis, which potentially can yield more protein than the previously used E. coli expression system. The LFnBLA protein is being used in high throughput chemical genomic screens to identify chemicals that block steps in the action of anthrax toxin or the cancer therapeutic agents based on this toxin. The results of one such screen were recently published, as is reported under a parallel project.
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