Generation and characterization of alanine scanning mutations in TEM8/ANTXR1 vWA domain

Cowsert, Brandon L.
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Anthrax is a potentially fatal disease caused by the bacterium Bacillus anthracis. The toxins produced by the bacterium are lethal to animals. The three secreted proteins that make anthrax toxin are protective antigen (PA), lethal factor (LF), and edema factor (EF). Separately, each protein is nontoxic. But LF combined with PA forms an active anthrax toxin, termed lethal toxin. EF combined with PA forms the second active toxin, termed edema toxin. EF is an adenylate cyclase that raises cAMP to potentially deadly levels. LF is a zinc-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPKKs), resulting in lethal consequences for the host. PA has no lethal activity per se, but is essential for delivery of EF and LF inside cells via cell surface receptors. EF and LF have no receptor-binding property, which explains why the combination of two proteins does not result in an active toxin.

PA receptors are tumor endothelial marker 8 (TEM8) and capillary morphogenesis gene 2 (CMG2), also designated ANTXR1 and ANTXR2, respectively. Both receptors are single-pass integral membrane proteins. Each receptor has a von Willebrand Factor A domain (vWA), which carries a metal ion dependent adhesion site (MIDAS). The vWA domain and MIDAS are required for PA binding. After binding PA goes through a series of events that the end with EF and LF entry to cytosol. The normal cellular functions of the receptors are not known with certainty, but evidence has accumulated that both are important for neo-angiogenesis. It is also known that CMG2 and TEM8 have splice variants that encode both the membrane-bound and secreted forms.

The focus of this work was to carry out mutational analysis of TEM8. To do that, HA-tagged cloned variant 4 (V4) of the receptor was used as the template for in vitro site-directed mutagenesis. A number of acidic residues in the vWA domain of the receptor were picked as the targets. The overall strategy was to separately replace each residue with alanine, express the mutant receptors in a receptor-negative host, and assess the effect of each mutation on the receptor's capacity to support anthrax toxin entry. Alanine-scanning mutagenesis is a suitable approach because alanine is nonpolar and has moderate hydrophobicity. JCR65, a Chinese hamster lung fibroblast derivative, was used as the host cell to express the mutant receptors. JCR65 is functionally receptor-negative, and consequently it does not support anthrax toxin entry. However, upon expression of a normal receptor it becomes sensitive to anthrax toxin.

The results show that E100A, E108A, and E122A mutations considerably disrupted receptor function; the toxin IC50 values were greater than those for the wild-type receptor. But the most disruptive mutation was E129A; the receptor harboring this change did not support anthrax toxin entry at all. Remarkably, the mutant receptor appeared to bind PA as well as the wild-type receptor, suggesting the E129A lesion disrupts a step in intoxication after initial toxin binding. Several other mutations had no effect on receptor function.

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Integrative Biology