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Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

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Related in: MedlinePlus

Antibody cross-linking promotes raft association of PA83 in a cholesterol-dependent manner. (A) CHO cells were incubated at 4°C with 500 ng/ml PA83 for 20 min. Surface PA was then clustered by an antibody sandwich at 4°C. DRMs were prepared and fractions were probed for PA by Western blotting. (B) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA83, and then were either fixed with PFA followed by incubation with primary and secondary antibodies to detect PA (Fix+Xlink), or incubated for 30 min at 4°C with rabbit polyclonal anti-PA, or for 30 min at 4°C with the secondary antibodies and then fixed (Xlink+Fix). Bar, 10 μm. (C) CHO cells were treated or not with β-MCD or filipin, then treated as in A. DRMs were prepared and fractions were probed for the presence of PA as in A. (D) ATR-HA–expressing CHO cells were incubated or not with 500 ng/ml PA83 for 20 min and submitted to antibody (polyclonal) clustering as in A. DRMs were prepared and fractions were probed for the presence of ATR-HA by HA antibodies. (E) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA (either native PA83, trypsin nicked PA83, or PASNKE) and 500 ng/ml of an inactive aerolysin mutant (ASSP), a monovalent probe for GPI-anchored proteins. Cells were then successively incubated for 30 min at 4°C with rabbit polyclonal anti-PA and chicken polyclonal anti-aerolysin antibodies for 30 min at 4°C with corresponding fluorescent secondary antibodies. Cells were fixed and visualized. For ease of analysis, only a small region of the plasma membrane is shown for each condition. Bar, 2.5 μm. (F) CHO cells were treated as in B (Xlink+Fix), and were then permeabilized with saponin and double labeled for the presence of caveolin-1. Bar, 5 μm. (G) Schematic behavior of ATR at the cell surface. ATR and the ATR–PA83 complex are present in the glycerophospholipid area of the plasma membrane. On clustering of ATR either via heptamerization of PA63 or by anti-body cross-linking, the transmembrane protein is stabilized within lipid rafts.
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fig2: Antibody cross-linking promotes raft association of PA83 in a cholesterol-dependent manner. (A) CHO cells were incubated at 4°C with 500 ng/ml PA83 for 20 min. Surface PA was then clustered by an antibody sandwich at 4°C. DRMs were prepared and fractions were probed for PA by Western blotting. (B) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA83, and then were either fixed with PFA followed by incubation with primary and secondary antibodies to detect PA (Fix+Xlink), or incubated for 30 min at 4°C with rabbit polyclonal anti-PA, or for 30 min at 4°C with the secondary antibodies and then fixed (Xlink+Fix). Bar, 10 μm. (C) CHO cells were treated or not with β-MCD or filipin, then treated as in A. DRMs were prepared and fractions were probed for the presence of PA as in A. (D) ATR-HA–expressing CHO cells were incubated or not with 500 ng/ml PA83 for 20 min and submitted to antibody (polyclonal) clustering as in A. DRMs were prepared and fractions were probed for the presence of ATR-HA by HA antibodies. (E) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA (either native PA83, trypsin nicked PA83, or PASNKE) and 500 ng/ml of an inactive aerolysin mutant (ASSP), a monovalent probe for GPI-anchored proteins. Cells were then successively incubated for 30 min at 4°C with rabbit polyclonal anti-PA and chicken polyclonal anti-aerolysin antibodies for 30 min at 4°C with corresponding fluorescent secondary antibodies. Cells were fixed and visualized. For ease of analysis, only a small region of the plasma membrane is shown for each condition. Bar, 2.5 μm. (F) CHO cells were treated as in B (Xlink+Fix), and were then permeabilized with saponin and double labeled for the presence of caveolin-1. Bar, 5 μm. (G) Schematic behavior of ATR at the cell surface. ATR and the ATR–PA83 complex are present in the glycerophospholipid area of the plasma membrane. On clustering of ATR either via heptamerization of PA63 or by anti-body cross-linking, the transmembrane protein is stabilized within lipid rafts.

Mentions: Our hypothesis also predicts that clustering ATR by means other than PA heptamerization, for example by cross-linking ATR using a sandwich of primary and secondary antibodies (Harder et al., 1998), should lead to raft association. Not having anti-ATR antibodies, ATR was labeled with PA83, which cannot heptamerize unless processed, and thus clustering at 4°C was induced with antibodies against PA. Whether a monoclonal or a polyclonal antibody was used, the clustered PA83 was predominantly present in DRMs, in contrast to the nonclustered PA83 (Fig. 2 A). That antibody clustering modified the surface distribution of ATR was confirmed by fluorescence microscopy. As shown in Fig. 2 B, a very punctate and intense signal was observed on cross-linking, whereas on cells that had been fixed before the addition of antibodies, the staining was weaker and more diffuse (note that some punctate structures can be observed that could well be due to cross-linking after PFA fixation as previously observed for GPI-anchored proteins; Maxfield and Mayor, 1997). The antibody-induced DRM association was cholesterol-dependent, as shown by the sensitivity to filipin and β-MCD (Fig. 2 C), strengthening the notion that lipid rafts control this process. Importantly, clustering of PA83 also led to the redistribution of a population of ATR-HA to DRMs (Fig. 2 D ; the majority of ATR-HA remains in detergent soluble fractions because of the vast excess of receptor with respect to PA, and because intracellular receptors are unavailable for PA63 binding). We further confirmed the raft association of clustered PA83 by cellular localization studies. As a typical raft marker, we used antibody-clustered GPI-anchored proteins (Harder et al., 1998) that were labeled using an inactive mutant of aerolysin, a specific probe for this class of proteins (Fivaz et al., 2002). As shown in Fig. 2 E (arrows), we found strong colocalization with GPI-anchored proteins not only for PA63, as expected, but also for clustered wild-type PA83 and PASNKE, consistent with our biochemical analysis. However, we could not see any significant colocalization at the cell surface of clustered PA83 with caveolin-1 (Fig. 2 F), strengthening the notion that PA associates with noncaveolar rafts (Fig. 1). The above experiments show that clustering of ATR, either by PA63 heptamerization or by antibody cross-linking, promotes its partitioning into rafts (Fig. 2 G).


Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

Antibody cross-linking promotes raft association of PA83 in a cholesterol-dependent manner. (A) CHO cells were incubated at 4°C with 500 ng/ml PA83 for 20 min. Surface PA was then clustered by an antibody sandwich at 4°C. DRMs were prepared and fractions were probed for PA by Western blotting. (B) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA83, and then were either fixed with PFA followed by incubation with primary and secondary antibodies to detect PA (Fix+Xlink), or incubated for 30 min at 4°C with rabbit polyclonal anti-PA, or for 30 min at 4°C with the secondary antibodies and then fixed (Xlink+Fix). Bar, 10 μm. (C) CHO cells were treated or not with β-MCD or filipin, then treated as in A. DRMs were prepared and fractions were probed for the presence of PA as in A. (D) ATR-HA–expressing CHO cells were incubated or not with 500 ng/ml PA83 for 20 min and submitted to antibody (polyclonal) clustering as in A. DRMs were prepared and fractions were probed for the presence of ATR-HA by HA antibodies. (E) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA (either native PA83, trypsin nicked PA83, or PASNKE) and 500 ng/ml of an inactive aerolysin mutant (ASSP), a monovalent probe for GPI-anchored proteins. Cells were then successively incubated for 30 min at 4°C with rabbit polyclonal anti-PA and chicken polyclonal anti-aerolysin antibodies for 30 min at 4°C with corresponding fluorescent secondary antibodies. Cells were fixed and visualized. For ease of analysis, only a small region of the plasma membrane is shown for each condition. Bar, 2.5 μm. (F) CHO cells were treated as in B (Xlink+Fix), and were then permeabilized with saponin and double labeled for the presence of caveolin-1. Bar, 5 μm. (G) Schematic behavior of ATR at the cell surface. ATR and the ATR–PA83 complex are present in the glycerophospholipid area of the plasma membrane. On clustering of ATR either via heptamerization of PA63 or by anti-body cross-linking, the transmembrane protein is stabilized within lipid rafts.
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Related In: Results  -  Collection

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fig2: Antibody cross-linking promotes raft association of PA83 in a cholesterol-dependent manner. (A) CHO cells were incubated at 4°C with 500 ng/ml PA83 for 20 min. Surface PA was then clustered by an antibody sandwich at 4°C. DRMs were prepared and fractions were probed for PA by Western blotting. (B) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA83, and then were either fixed with PFA followed by incubation with primary and secondary antibodies to detect PA (Fix+Xlink), or incubated for 30 min at 4°C with rabbit polyclonal anti-PA, or for 30 min at 4°C with the secondary antibodies and then fixed (Xlink+Fix). Bar, 10 μm. (C) CHO cells were treated or not with β-MCD or filipin, then treated as in A. DRMs were prepared and fractions were probed for the presence of PA as in A. (D) ATR-HA–expressing CHO cells were incubated or not with 500 ng/ml PA83 for 20 min and submitted to antibody (polyclonal) clustering as in A. DRMs were prepared and fractions were probed for the presence of ATR-HA by HA antibodies. (E) CHO cells were treated for 1 h at 4°C with 500 ng/ml PA (either native PA83, trypsin nicked PA83, or PASNKE) and 500 ng/ml of an inactive aerolysin mutant (ASSP), a monovalent probe for GPI-anchored proteins. Cells were then successively incubated for 30 min at 4°C with rabbit polyclonal anti-PA and chicken polyclonal anti-aerolysin antibodies for 30 min at 4°C with corresponding fluorescent secondary antibodies. Cells were fixed and visualized. For ease of analysis, only a small region of the plasma membrane is shown for each condition. Bar, 2.5 μm. (F) CHO cells were treated as in B (Xlink+Fix), and were then permeabilized with saponin and double labeled for the presence of caveolin-1. Bar, 5 μm. (G) Schematic behavior of ATR at the cell surface. ATR and the ATR–PA83 complex are present in the glycerophospholipid area of the plasma membrane. On clustering of ATR either via heptamerization of PA63 or by anti-body cross-linking, the transmembrane protein is stabilized within lipid rafts.
Mentions: Our hypothesis also predicts that clustering ATR by means other than PA heptamerization, for example by cross-linking ATR using a sandwich of primary and secondary antibodies (Harder et al., 1998), should lead to raft association. Not having anti-ATR antibodies, ATR was labeled with PA83, which cannot heptamerize unless processed, and thus clustering at 4°C was induced with antibodies against PA. Whether a monoclonal or a polyclonal antibody was used, the clustered PA83 was predominantly present in DRMs, in contrast to the nonclustered PA83 (Fig. 2 A). That antibody clustering modified the surface distribution of ATR was confirmed by fluorescence microscopy. As shown in Fig. 2 B, a very punctate and intense signal was observed on cross-linking, whereas on cells that had been fixed before the addition of antibodies, the staining was weaker and more diffuse (note that some punctate structures can be observed that could well be due to cross-linking after PFA fixation as previously observed for GPI-anchored proteins; Maxfield and Mayor, 1997). The antibody-induced DRM association was cholesterol-dependent, as shown by the sensitivity to filipin and β-MCD (Fig. 2 C), strengthening the notion that lipid rafts control this process. Importantly, clustering of PA83 also led to the redistribution of a population of ATR-HA to DRMs (Fig. 2 D ; the majority of ATR-HA remains in detergent soluble fractions because of the vast excess of receptor with respect to PA, and because intracellular receptors are unavailable for PA63 binding). We further confirmed the raft association of clustered PA83 by cellular localization studies. As a typical raft marker, we used antibody-clustered GPI-anchored proteins (Harder et al., 1998) that were labeled using an inactive mutant of aerolysin, a specific probe for this class of proteins (Fivaz et al., 2002). As shown in Fig. 2 E (arrows), we found strong colocalization with GPI-anchored proteins not only for PA63, as expected, but also for clustered wild-type PA83 and PASNKE, consistent with our biochemical analysis. However, we could not see any significant colocalization at the cell surface of clustered PA83 with caveolin-1 (Fig. 2 F), strengthening the notion that PA associates with noncaveolar rafts (Fig. 1). The above experiments show that clustering of ATR, either by PA63 heptamerization or by antibody cross-linking, promotes its partitioning into rafts (Fig. 2 G).

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

Show MeSH
Related in: MedlinePlus