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Polylysine-mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore.

Sharma O, Collier RJ - Biochemistry (2014)

Bottom Line: Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation.In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro.Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School , 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.

ABSTRACT
The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with ∼12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10-100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.

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Kinetics of occlusionof PA pores in planar bilayers by LFn, DTA,and Lys-tagged DTA constructs. Up to 5 μg of PA63 prepore wasadded to the cis compartment. After insertion ofPA pores, 1 μg of a DTA construct was added to the cis compartment and the fraction of PA channels occluded in the presenceof cis 20 mV after 60 s was calculated.
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fig3: Kinetics of occlusionof PA pores in planar bilayers by LFn, DTA,and Lys-tagged DTA constructs. Up to 5 μg of PA63 prepore wasadded to the cis compartment. After insertion ofPA pores, 1 μg of a DTA construct was added to the cis compartment and the fraction of PA channels occluded in the presenceof cis 20 mV after 60 s was calculated.

Mentions: Binding of LFn to PApores in planar bilayers results in pore occlusion,as measured by ion conductance.39,40 We monitored occlusionof pores in DPhPC bilayers for 60 s following addition of variousconstructs (final concentration of 1 μg/mL) to the cis compartment (Figure 3). Free LFn blockedconductance rapidly (within 10 s) and almost completely (99%), whereasocclusion by Lys-tagged DTA constructs was slower and strongly dependenton the number of Lys residues in the tag. The greater the number ofLys residues, the more rapid and more complete the occlusion (at 60s). N-Terminally tagged constructs were more effective than the correspondingC-terminally tagged ones. Constructs with K9 tracts caused weak occlusion,and constructs with K6 caused almost none.


Polylysine-mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore.

Sharma O, Collier RJ - Biochemistry (2014)

Kinetics of occlusionof PA pores in planar bilayers by LFn, DTA,and Lys-tagged DTA constructs. Up to 5 μg of PA63 prepore wasadded to the cis compartment. After insertion ofPA pores, 1 μg of a DTA construct was added to the cis compartment and the fraction of PA channels occluded in the presenceof cis 20 mV after 60 s was calculated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230326&req=5

fig3: Kinetics of occlusionof PA pores in planar bilayers by LFn, DTA,and Lys-tagged DTA constructs. Up to 5 μg of PA63 prepore wasadded to the cis compartment. After insertion ofPA pores, 1 μg of a DTA construct was added to the cis compartment and the fraction of PA channels occluded in the presenceof cis 20 mV after 60 s was calculated.
Mentions: Binding of LFn to PApores in planar bilayers results in pore occlusion,as measured by ion conductance.39,40 We monitored occlusionof pores in DPhPC bilayers for 60 s following addition of variousconstructs (final concentration of 1 μg/mL) to the cis compartment (Figure 3). Free LFn blockedconductance rapidly (within 10 s) and almost completely (99%), whereasocclusion by Lys-tagged DTA constructs was slower and strongly dependenton the number of Lys residues in the tag. The greater the number ofLys residues, the more rapid and more complete the occlusion (at 60s). N-Terminally tagged constructs were more effective than the correspondingC-terminally tagged ones. Constructs with K9 tracts caused weak occlusion,and constructs with K6 caused almost none.

Bottom Line: Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation.In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro.Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School , 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.

ABSTRACT
The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with ∼12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10-100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.

Show MeSH
Related in: MedlinePlus