Receptor-specific requirements for anthrax toxin delivery into cells

Abstract

The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.capillary morphogenesis protein 2 ͉ tumor endothelial marker 8 ͉ toxin entry B acillus anthracis, the causative agent of anthrax, secretes a toxin that is believed to be instrumental in causing anthrax disease symptoms leading to death. Anthrax toxin consists of three proteins, protective antigen (PA), which is a receptorbinding and pore-forming subunit; lethal factor (LF), which is a protease that cleaves mitogen-activated protein kinase kinase family members; and edema factor (EF), which is an adenylate cyclase that raises cAMP levels in cells (1). 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). The CMG2 gene, which has been shown to be broadly expressed in different tissues (4), encodes three protein isoforms, two of which, CMG2 488 and CMG2 489 , are anthrax toxin receptors (4) (H.M.S., unpublished data).Receptor-bound PA 83 is cleaved by a cellular protease to generate a 20-kDa PA 20 subunit and a 63-kDa subunit (PA 63 ). The larger subunit assembles into the heptameric (PA 63 ) 7 prepore in lipid rafts (7-9). EF and LF bind to the prepore, and the toxin-receptor complexes are internalized by clathrindependent endocytosis and by other endocytic mechanisms (9, 10). These complexes are then exp...

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

“…15,16 For this event to occur in the presence of the receptor, domain 2 must partially dissociate from the receptor and lose potential stabilizing interactions with domains 3 and 4. Indeed, recent studies using immunoprecipitation have shown that both ATR/TEM8 and CMG2 dissociate from the prepore at pH values required for pore formation, 10 the latter corroborated with data from NMR studies. 17 Although not wellunderstood, we hypothesized that the large structural change from the prepore to the pore is likely to induce receptor dissociation, through a pH-induced unfolding of domain 4.…”

“…In vitro studies have shown that at this same pH, the receptor dissociates. 10,17 This implied to us that domain 4, in addition to domain 2, may also undergo a conformational change (unfolding) that results in the release the receptor. The crystal structure of domain 4 revealed a structure with amazing adherence to the topology of domain 4 within the context of the full-length PA protein.…”

“…9,10 When bound to the von Willebrand factor A (vWA) domain of ATR/TEM8, the pH threshold of pore formation is pH 6, whereas when bound to the vWA domain of CMG2 the pH threshold for pore formation is lowered to pH 5. 10 Each of the receptors utilize a conserved metal-ion-dependant-adhesion (MIDAS) site within the vWA domain for binding to domain 4 of PA, which along with residues in the receptor, coordinates a magnesium ion that is critically important for binding. 2 Domain 4, comprising residues 595-735, was shown as an isolated domain fused to glutathione S-transferase (GST) to be sufficient for binding to the vWA domain of CMG2, although interactions from domain 2 are also important.…”

Anthrax protective antigen is a calcium-dependent serine protease

“…the interaction between PA and its receptor, is critical for the development of effective treatment strategies against B. anthracis related infections. In previous research, it has been shown that the interaction between PA 83 and its receptors resembles an pH-dependent α-integrin/ligand binding interaction [9,10]. Additionally, Bradley and co-workers showed that EDTA inhibits the binding of PA 83 to its receptor, suggesting a role of divalent cations in receptor binding [3].…”

Polyvalent inhibitors of anthrax toxin that target host receptors

“…Internalization depends on a coreceptor, low-density lipoprotein (LDL) receptor-related protein 6, that binds ANTXR1͞2 directly (17). Upon reaching an acidic compartment, [PA 63 ] 7 dissociates from its receptors and inserts flexible loops into the membrane to form a ␤-barrel pore (18). Insertion of [PA 63 ] 7 that has been internalized by ANTXR1 occurs at a higher pH found in early endosomes compared with ANTXR2-internalized [PA 63 ] 7 , which inserts only at a lower pH found in late endosomes.…”