Functional analysis of the anthrax toxin receptor CMG2 by alanine scanning mutagenesis




Manam, Srikanth

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The causative agent of anthrax is Bacillus anthracis, a large rod-shaped Gram-positive bacterium. The hallmarks of the bacterium's virulence are its capsule and anthrax toxin. The toxin is a collection of three secreted proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). EF and LF are the catalytic moieties that intoxicate cells. PA, itself nontoxic, delivers EF and LF inside cells, and it does this by receptor-mediated endocytosis. The toxin employs two mammalian cell surface proteins as the receptors: tumor endothelial marker 8 (TEM8; antxr1) capillary morphogenesis gene 2 protein (CMG2; antxr2). Both are closely related single-pass integral membrane proteins that have a von Willebrand Factor A (vWA) domain in their extracellular portions. Within the vWA domain, both receptors have a metal ion dependent adhesion site (MIDAS). The MIDAS motif and the vWA domain are indispensable for PA binding to both receptors. The receptors also have a membrane proximal segment that lies between the vWA domain and the transmembrane helix. While ample evidence has accumulated that the vWA domain is crucial for toxin binding and entry, so far the role of the membrane proximal segment has not been elucidated. Further, the mutational analysis of even the vWA domain has not been done to an appreciable degree, and such analysis of the membrane proximal region has not been done at all. Thus, identification of specific residues crucial for these proteins as anthrax toxin receptors remains to be done.

To identify specific residues important for receptor function, we have carried out extensive mutational analysis of the vWA domain of CMG2-488, one of four splice variants of this protein. We also carried out limited mutational analysis of the membrane proximal portion of this receptor. Our overall strategy was to individually replace certain acidic, basic, polar, and aromatic residues with alanine, but we also targeted some hydrophobic residues for analysis. To assess the mutant receptors' capacity to support anthrax toxin entry, we expressed the plasmid-encoded receptors in JCR65, a functionally receptor negative hamster cell line. The results show that the receptor loses function when the MIDAS residues are replaced with alanine, consistent with previous findings. Of the acidic residues, D180A and E233A mutations rendered the receptor nonfunctional. Likewise, the basic residue mutations K101A, R111A, and R242A proved equally disruptive. Of the polar residues, only S87A mutation killed receptor function. Among the aromatic residues, F85A was the only mutation that proved detrimental to receptor function. A hydrophobic residue mutation, V115A, completely blocked intoxication of cells. The E233A, E289A, R242A mutations, which abolish receptor function, are in the membrane proximal segment of the receptor. None of these mutations has been described before. Indeed the three mutations constitute the first experimental evidence that the membrane proximal portion is important for toxin entry. A number of mutations did not abolish receptor function, but nonetheless proved significantly disruptive. Many of the mutations had little or no effect on receptor function.

While much more analysis remains to be done, we think the current study represents a significant advance in understanding what particular residues in CMG2 are important for receptor function. Devising strategies to intercept anthrax toxin during late stages of infection is an imperative. Clearly a thorough understanding of how the toxin employs its receptors for entry and what particular receptor residues and subregions are crucial to support toxin entry would help devise such strategies.


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Integrative Biology