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Changing the receptor specificity of anthrax toxin.

Mechaly A, McCluskey AJ, Collier RJ - MBio (2012)

Bottom Line: 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.Bacterial 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Unlabelled: The 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.

Importance: Bacterial 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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Related in: MedlinePlus

Composite representation of the heptameric prepore formed by PA63 (Protein Data Bank [PDB] accession no. 1TZO) with EGF (PDB accession no. 1JL9) linked to the C terminus. An axial view of the heptameric prepore is shown, with domains 1, 2, 3, and 4 in a single subunit of PA63 colored magenta, green, gold, and purple, respectively. EGF is in red. Broken lines represent an 8-amino-acid linker (SPGHKTQP) connecting the N terminus of EGF to the C terminus of PA63.
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fig1: Composite representation of the heptameric prepore formed by PA63 (Protein Data Bank [PDB] accession no. 1TZO) with EGF (PDB accession no. 1JL9) linked to the C terminus. An axial view of the heptameric prepore is shown, with domains 1, 2, 3, and 4 in a single subunit of PA63 colored magenta, green, gold, and purple, respectively. EGF is in red. Broken lines represent an 8-amino-acid linker (SPGHKTQP) connecting the N terminus of EGF to the C terminus of PA63.

Mentions: In this study, we ablated the receptor-binding activity of PA by mutating two residues within domain 4 and then fused each of two heterologous receptor-binding proteins, human epidermal growth factor (EGF) or the receptor-binding domain of diphtheria toxin (DTR), to the C terminus of the mutated protein (Fig. 1). Both of the resulting fusion proteins mediated the entry of effector enzymes, and entry was dependent in each case on the cellular receptor recognized by the receptor-binding polypeptide appended to PA.


Changing the receptor specificity of anthrax toxin.

Mechaly A, McCluskey AJ, Collier RJ - MBio (2012)

Composite representation of the heptameric prepore formed by PA63 (Protein Data Bank [PDB] accession no. 1TZO) with EGF (PDB accession no. 1JL9) linked to the C terminus. An axial view of the heptameric prepore is shown, with domains 1, 2, 3, and 4 in a single subunit of PA63 colored magenta, green, gold, and purple, respectively. EGF is in red. Broken lines represent an 8-amino-acid linker (SPGHKTQP) connecting the N terminus of EGF to the C terminus of PA63.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569862&req=5

fig1: Composite representation of the heptameric prepore formed by PA63 (Protein Data Bank [PDB] accession no. 1TZO) with EGF (PDB accession no. 1JL9) linked to the C terminus. An axial view of the heptameric prepore is shown, with domains 1, 2, 3, and 4 in a single subunit of PA63 colored magenta, green, gold, and purple, respectively. EGF is in red. Broken lines represent an 8-amino-acid linker (SPGHKTQP) connecting the N terminus of EGF to the C terminus of PA63.
Mentions: In this study, we ablated the receptor-binding activity of PA by mutating two residues within domain 4 and then fused each of two heterologous receptor-binding proteins, human epidermal growth factor (EGF) or the receptor-binding domain of diphtheria toxin (DTR), to the C terminus of the mutated protein (Fig. 1). Both of the resulting fusion proteins mediated the entry of effector enzymes, and entry was dependent in each case on the cellular receptor recognized by the receptor-binding polypeptide appended to PA.

Bottom Line: 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.Bacterial 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Unlabelled: The 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.

Importance: Bacterial 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.

Show MeSH
Related in: MedlinePlus