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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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Experiments with biotinylated constructs. The PA63 preporewasadded to the cis compartment. Upon insertion of PApores, the DTA construct was added to the cis compartmentand occlusion of PA channels in the presence of cis 20 mV was monitored. (A and B) Interaction of biotin-C-K12-DTA (0.5μg) and DTA-K12-C-biotin (1 μg) constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa 5-fold (A) or 2.5-fold (B) molar excess of tetrameric streptavidinadded to the cis compartment. (C and D) Interactionof biotin-C-K12-DTA and DTA-K12-C-biotin constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa molar excess of tetrameric streptavidin added to the trans compartment.
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fig4: Experiments with biotinylated constructs. The PA63 preporewasadded to the cis compartment. Upon insertion of PApores, the DTA construct was added to the cis compartmentand occlusion of PA channels in the presence of cis 20 mV was monitored. (A and B) Interaction of biotin-C-K12-DTA (0.5μg) and DTA-K12-C-biotin (1 μg) constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa 5-fold (A) or 2.5-fold (B) molar excess of tetrameric streptavidinadded to the cis compartment. (C and D) Interactionof biotin-C-K12-DTA and DTA-K12-C-biotin constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa molar excess of tetrameric streptavidin added to the trans compartment.

Mentions: To probe the translocation of Lys-taggedconstructs across planarbilayers, we appended a Cys residue to the N-terminus of K12-DTA andto the C-terminus of DTA-K12 and labeled both constructs with a Cys-reactivebiotinylation reagent. The resulting biotin-C-K12-DTA and DTA-K12-C-biotinconstructs occluded PA channels with efficiencies comparable to thoseof the corresponding K12-DTA and DTA-K12 constructs. Addition of streptavidinto the cis compartment strongly hindered occlusionby both of the biotinylated constructs (Figure 4A,B) but had no effect on occlusion by DTA constructs lacking thebiotin label. Thus, the Lys tract initiated blockage of the PA poreirrespective of the terminus of DTA to which it was fused, and bindingof a bulky protein at the terminus adjacent to the Lys tract preventedthe blockage.


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

Sharma O, Collier RJ - Biochemistry (2014)

Experiments with biotinylated constructs. The PA63 preporewasadded to the cis compartment. Upon insertion of PApores, the DTA construct was added to the cis compartmentand occlusion of PA channels in the presence of cis 20 mV was monitored. (A and B) Interaction of biotin-C-K12-DTA (0.5μg) and DTA-K12-C-biotin (1 μg) constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa 5-fold (A) or 2.5-fold (B) molar excess of tetrameric streptavidinadded to the cis compartment. (C and D) Interactionof biotin-C-K12-DTA and DTA-K12-C-biotin constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa molar excess of tetrameric streptavidin added to the trans compartment.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Experiments with biotinylated constructs. The PA63 preporewasadded to the cis compartment. Upon insertion of PApores, the DTA construct was added to the cis compartmentand occlusion of PA channels in the presence of cis 20 mV was monitored. (A and B) Interaction of biotin-C-K12-DTA (0.5μg) and DTA-K12-C-biotin (1 μg) constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa 5-fold (A) or 2.5-fold (B) molar excess of tetrameric streptavidinadded to the cis compartment. (C and D) Interactionof biotin-C-K12-DTA and DTA-K12-C-biotin constructs, respectively,with PA pores incorporated into planar bilayers in the presence ofa molar excess of tetrameric streptavidin added to the trans compartment.
Mentions: To probe the translocation of Lys-taggedconstructs across planarbilayers, we appended a Cys residue to the N-terminus of K12-DTA andto the C-terminus of DTA-K12 and labeled both constructs with a Cys-reactivebiotinylation reagent. The resulting biotin-C-K12-DTA and DTA-K12-C-biotinconstructs occluded PA channels with efficiencies comparable to thoseof the corresponding K12-DTA and DTA-K12 constructs. Addition of streptavidinto the cis compartment strongly hindered occlusionby both of the biotinylated constructs (Figure 4A,B) but had no effect on occlusion by DTA constructs lacking thebiotin label. Thus, the Lys tract initiated blockage of the PA poreirrespective of the terminus of DTA to which it was fused, and bindingof a bulky protein at the terminus adjacent to the Lys tract preventedthe blockage.

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