Cell Surface Tumor Endothelium Marker 8 Cytoplasmic Tail-independent Anthrax Toxin Binding, Proteolytic Processing, Oligomer Formation, and Internalization

Abstract

The interaction of anthrax toxin protective antigen (PA) and target cells was assessed, and the importance of the cytosolic domain of tumor endothelium marker 8 (TEM8) in its function as a cellular receptor for PA was evaluated. PA binding and proteolytic processing on the Chinese hamster ovary cell surface occurred rapidly, with both processes nearly reaching steady state in 5 min. Remarkably, the resulting PA63 fragment was present on the cell surface only as an oligomer, and furthermore, the oligomer was the only PA species internalized, suggesting that oligomerization of PA63 triggers receptor-mediated endocytosis. Following internalization, the PA63 oligomer was rapidly and irreversibly transformed to an SDS/heat-resistant form, in a process requiring an acidic compartment. This conformational change was functionally correlated with membrane insertion, channel formation, and translocation of lethal factor into the cytosol. To explore the role of the TEM8 cytosolic tail, a series of truncated TEM8 mutants was transfected into a PA receptor-deficient Chinese hamster ovary cell line. Interestingly, all of the cytosolic tail truncated TEM8 mutants functioned as PA receptors, as determined by PA binding, processing, oligomer formation, and translocation of an lethal factor fusion toxin into the cytosol. Moreover, cells transfected with a TEM8 construct truncated before the predicted transmembrane domain failed to bind PA, demonstrating that residues 321-343 are needed for cell surface anchoring. Further evidence that the cytosolic domain plays no essential role in anthrax toxin action was obtained by showing that TEM8 anchored by a glycosylphosphatidylinositol tail also functioned as a PA receptor.Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PA, 1 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa) (1, 2). These three proteins are individually non-toxic. To intoxicate mammalian cells, PA binds to a ubiquitously expressed, recently identified cellular receptor, tumor endothelium marker 8 (TEM8) variant 2 (3), and is cleaved at the sequence RKKR 167 on the cell surface by furin or furin-like proteases (4, 5). Proteolysis yields the amino-terminal 20-kDa fragment (PA20), which is released into the medium, and the carboxyl-terminal 63-kDa fragment (PA63), which remains bound to the receptor and self-associates to form a ring-shaped heptamer (6, 7). The heptamer binds up to 3 molecules of LF or EF (8, 9). The resulting oligomeric complex is then internalized into endosomes, where the decreased pH causes the PA63 heptamer to insert into the endosomal membrane and produce a channel through which LF and EF translocate to the cytosol (10). Therefore, PA is the central part of anthrax toxin, serving as the delivery vehicle for binding and translocation of LF and EF into the cytosol of the cells. The combination of PA plus LF kills animals (11, 12) and certain cells, including mouse macrophages (13,14). LF is a zinc-dependent metalloprot...

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

“…This finding is consistent with an interaction between the carboxyl group of Asp 683 and the metal coordination site of the anthrax receptors, TEM8 and CMG2. Recent studies in our laboratory have shown that the extracellular domain and a transmembrane domain or membrane anchor are necessary for PA activity, whereas the cytoplasmic tail is not essential (6). The extracellular portions of the receptors contain a von Willebrand factor type A domain with a metal ion-dependent adhesion site (MIDAS) motif.…”

“…RAW264.7, a mouse macrophage cell line, was grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 10 mM HEPES, 2 mM Glutamax I, and 50 g/ml gentamycin. CHO cell clone 6 (CHO-CL6) and a furin-deficient cell line, CHO FD11, were previously described (6,31) and were grown in ␣-minimal essential medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM GlutaMax I, and 50 g/ml gentamycin.…”

“…One of the molecular reactions it would be beneficial to understand in molecular detail would be that between anthrax toxin and its cellular receptor. As noted above, much progress has been made in the study of PA and recently also with its cellular receptors, TEM8 and capillary morphogenesis protein 2, but studies of the interaction between the toxin with receptor have to date been limited to deletions or multiple substitutions in the proteins (6,25). One goal of this study was to determine which individual amino acids in the previously identified region of PA were important for interaction with its receptor.…”

Receptor-specific requirements for anthrax toxin delivery into cells

“…PA is synthesized as an 83-kDa protein (PA 83 ) for which two cell surface receptors have been identified: tumor endothelial marker 8 (TEM8) (2,3) and capillary morphogenesis protein 2 (CMG2) (4). Two splice variant mRNAs derived from the TEM8 gene (sv1 and sv2) encode functional anthrax toxin receptors (2,5). TEM8 expression has been documented in epithelium of the lung, intestine, and skin, the three routes of entry in anthrax infection (6).…”

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

“…Cells and Culture Media-Parental wild type CHO cells (CHO WTP4) and the PA receptor-expressing CHO CMG2-C4 and CHO TEM8-T4 cells, which overexpress CMG2 and TEM8, respectively, are as described previously (26,30). CHO cells were grown in minimum essential medium ␣ with 5% FBS, 2 mM glutamine, 5 mM HEPES, pH 7.4, and 50 g/ml gentamicin, with or without 300 g/ml hygromycin B. HeLa cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS, 2 mM glutamine, and 50 g/ml gentamicin.…”