Mutational analysis of tumor endothelial marker 8, an anthrax toxin receptor

Anthrax is caused by Bacillus anthracis, a rod-shaped, Gram-positive, aerobic, spore-forming soil bacterium. Two attributes that make it a potent pathogen: the capsule and anthrax toxin. The toxin is the major lethality factor. It comprises three secreted proteins-protective antigen (PA), edema factor (EF), and lethal factor (LF). Individually, the proteins have no toxicity, but PA+EF and PA+LF are highly toxic. EF is an adenylyl cyclase that harms cells by raising cAMP. LF is a Zn-dependent metalloprotease that cuts MAP kinase kinases, thus inactivating them. PA itself has no toxicity, but it is nonetheless crucial because it delivers EF and LF inside the cells by receptor-mediated endocytosis.

PA uses two similar cell surface proteins as the receptors, tumor endothelial marker 8 (TEM8) and capillary morphogenesis gene 2 protein (CMG2), both single-pass integral membrane receptors. The ectopic portion of each receptor has a vWA domain and within this domain a metal ion dependent adhesion site (MIDAS). The vWA domain and the MIDAS motif are required for PA binding. TEM8 and CMG2 have multiple splice variants, some secreted and some membrane bound. Only the membrane bound forms support toxin entry.

This work reports mutational analysis of TEM8, employing both random and site-directed mutagenesis. For this analysis, we used TEM8-var4, one of three membrane bound forms of the receptor. Var4 was amplified by nested PCR from human fetal brain cDNA and cloned into pIREShyg3, a mammalian expression vector. Over a hundred clones were sequenced to identify the wild-type, as well as the random mutations. All clones had more than one mutation, and no wild-type receptor was found. Thus, the wild-type var4 was constructed by recombining the wild-type segments of two mutants. The normal receptor and its mutant forms were expressed in a functionally receptor negative host cell line. The host cells expressing various receptors were then subjected to cytotoxicity assays with a modified form of anthrax toxin, PA+FP59, a potent cytocidal toxin.

Receptor mutants derived by random mutagenesis had multiple mutations, many with combination of silent, conservative, and nonconservative changes. The effects of these mutations on receptor function varied dramatically; some had no effect and some others killed the receptor function. To segregate their effects, many mutations were individually made in the wild-type receptor by site-directed mutagenesis. The receptor functions were quantitatively assessed by comparing the IC50 values, defined as the toxin concentrations that resulted in 50% cell death. The IC50 for the wild-type receptor was 3-4 ng/mL of toxin. The individual mutations that resulted in the most drastic loss of receptor function were Q101L and R126W (IC50 values > 2000 ng/mL), both in the vWA domain. The mutations that did not kill receptor function but nonetheless resulted in considerable disruption were P37L, L69S, S143N, and G172C (IC50 values 30-80 ng/mL). An unexpected result was the markedly reduced receptor function when Q110L and C347R mutations were present simultaneously (IC50 ∼ 200 ng/mL), but little effect when they were present individually (IC50 ∼ 10 ng/mL). How Q110L mutation, which is in the vWA domain (outside the cell) and C347R mutation, which is in the cytoplasmic portion, together disrupt receptor function is unclear.