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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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Disruption of lipid rafts blocks formation of the PA channel and intracellular delivery of LF. (A) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with 500 ng/ml trypsin-nicked PA83, and were transferred to 37°C for different periods of time. Aliquots of 80 μg total cell extract proteins were loaded on a 7.5% SDS-gel and probed for PA by Western blotting. (B) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with a mixture of 1 μg/ml trypsin-nicked PA83 and 1 μg/ml LF, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting (12.5% SDS-gel) for the presence of LF, total MEK1 (anti-COOH–terminal antibody), LF-processed MEK1 (anti-NH2–terminal antibody), and PA (using 7.5% SDS-gels for the latter).
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fig4: Disruption of lipid rafts blocks formation of the PA channel and intracellular delivery of LF. (A) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with 500 ng/ml trypsin-nicked PA83, and were transferred to 37°C for different periods of time. Aliquots of 80 μg total cell extract proteins were loaded on a 7.5% SDS-gel and probed for PA by Western blotting. (B) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with a mixture of 1 μg/ml trypsin-nicked PA83 and 1 μg/ml LF, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting (12.5% SDS-gel) for the presence of LF, total MEK1 (anti-COOH–terminal antibody), LF-processed MEK1 (anti-NH2–terminal antibody), and PA (using 7.5% SDS-gels for the latter).

Mentions: To evaluate the physiological relevance of the role of rafts in the delivery of PA to endosomes, we measured the effect of cholesterol depletion on the appearance of the SDS-resistant PA63 heptamer because this form only appears in an acidic environment. As shown in Fig. 4 A, formation of the membrane-inserted SDS-resistant heptamer was strongly delayed on β-MCD treatment.


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)

Disruption of lipid rafts blocks formation of the PA channel and intracellular delivery of LF. (A) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with 500 ng/ml trypsin-nicked PA83, and were transferred to 37°C for different periods of time. Aliquots of 80 μg total cell extract proteins were loaded on a 7.5% SDS-gel and probed for PA by Western blotting. (B) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with a mixture of 1 μg/ml trypsin-nicked PA83 and 1 μg/ml LF, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting (12.5% SDS-gel) for the presence of LF, total MEK1 (anti-COOH–terminal antibody), LF-processed MEK1 (anti-NH2–terminal antibody), and PA (using 7.5% SDS-gels for the latter).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172673&req=5

fig4: Disruption of lipid rafts blocks formation of the PA channel and intracellular delivery of LF. (A) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with 500 ng/ml trypsin-nicked PA83, and were transferred to 37°C for different periods of time. Aliquots of 80 μg total cell extract proteins were loaded on a 7.5% SDS-gel and probed for PA by Western blotting. (B) CHO cells were treated or not with β-MCD, incubated for 1 h at 4°C with a mixture of 1 μg/ml trypsin-nicked PA83 and 1 μg/ml LF, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting (12.5% SDS-gel) for the presence of LF, total MEK1 (anti-COOH–terminal antibody), LF-processed MEK1 (anti-NH2–terminal antibody), and PA (using 7.5% SDS-gels for the latter).
Mentions: To evaluate the physiological relevance of the role of rafts in the delivery of PA to endosomes, we measured the effect of cholesterol depletion on the appearance of the SDS-resistant PA63 heptamer because this form only appears in an acidic environment. As shown in Fig. 4 A, formation of the membrane-inserted SDS-resistant heptamer was strongly delayed on β-MCD treatment.

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