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

Kuang-Hua Chen,Shihui Liu,Clinton E Leysath,Sharmina Miller-Randolph,Yi Zhang,Rasem Fattah,Thomas H Bugge,Stephen H Leppla

doi:10.1074/jbc.m116.753301

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.

Highlights

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
7.4 ϫ 102 7.0 ϫ 104 5.6 ϫ 103 a Binding of bacteriophage to Dynabeads without the addition of an extracellular domain. b 100 ng of PA was mixed with bacteriophage lysate before adding to the residue Ile-656 is required for strong binding to tumor endothelial marker 8 (TEM8) and that substitution of Ile-656 with certain amino acids, such as Gln, produces PA variants that have high affinity for capillary morphogenesis protein 2 (CMG2) but little to no affinity for TEM8
PA variants were selected using plates coated with mouse monoclonal antibody 14B7, which mimics the receptors by binding to the same PA domain 4 (PAD4) surface

Summary

Results

Selection of PAD4 Mutants That Bind to CMG2—Our previous study [17] identified PAD4 mutants that bind differentially to TEM8 and CMG2 This was accomplished by bacteriophage display of a library of randomly mutated PAD4 variants. Among the resulting total of 43,680 bp examined, a total of 616 changes were found, for a mutation rate of 1.4% This value and the subsequent analyses are for the phage library after selection for binding to CMG2. There were multiple amino acid mutations in nearly every clone from the CMG2-selected library (supplemental File 2), strikingly, we found that mutation of Ile656 to Val, in the region between 4␤6 and 4␤7 (amino acids 656 – 665), occurred in most of the clones, indicating that residue Ile-656 is critical for binding of PA to the CMG2 receptor. PA (native) PA I656Q PA I656S PA I656N PA I656H PA I656E PA I656A PA I656G PA I656K

Ͼ1667 Ͼ1190 Ͼ694 Ͼ490 Ͼ490 Ͼ320 Ͼ287 Ͼ269

Discussion

Experimental Procedures