Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Abstract

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

Capillary morphogenesis gene 2 maintains gastric cancer stem-like cell phenotype by activating a Wnt/β-catenin pathway

“…CMG2 is originally identified as a gene upregulated in endothelial cells during capillary morphogenesis [ 8 ]. It has been implicated in tumor-related angiogenesis [ 28 ] and may be a target for anti-angiogenesis therapy for cancer [ 15 , 29 , 30 ]. However, the role of CMG2 in cancer has not been extensively investigated.…”

Tumor Targeting and Drug Delivery by Anthrax Toxin

“…Furthermore, PA R659S/M662R had enhanced specificity towards TEM8-expressing cells, making this PA variant another interesting tool for targeted therapies. In a recent publication, Chen et al described two further mutants of PA (PA I656Q and PA I656V) with decreased affinity towards TEM8 but with maintained high affinity for CMG2 [ 95 ].…”

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients

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