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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.

SUBMITTER: Rainey GJ

PROVIDER: S-EPMC1201603 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Receptor-specific requirements for anthrax toxin delivery into cells.

Rainey G Jonah A GJ Wigelsworth Darran J DJ Ryan Patricia L PL Scobie Heather M HM Collier R John RJ Young John A T JA

Proceedings of the National Academy of Sciences of the United States of America 20050901 37

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 ...[more]

PMID: 16141341

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Project description:UnlabelledThe actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.ImportanceBacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment. By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell. Our results show that the receptor specificity of the transport protein of anthrax toxin may be readily changed, raising the possibility that receptor-redirected forms of protective antigen (PA) and PA homologs may be useful for research and medical applications requiring modification or ablation of designated populations of cells.

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Receptor-specific requirements for anthrax toxin delivery into cells.

Publications

Receptor-specific requirements for anthrax toxin delivery into cells.

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