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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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PA-mediated translocation of DTA constructs as measuredby proteinsynthesis inhibition in CHO K1 cells. (A and B) Cytotoxicity of N-terminallysine fusions in the presence and absence of PA, respectively. (Cand D) Cytotoxicity of C-terminal lysine fusions in the presence andabsence of PA, respectively. Solid lines show data with PA (20 nM)and dashed lines data without PA.
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fig1: PA-mediated translocation of DTA constructs as measuredby proteinsynthesis inhibition in CHO K1 cells. (A and B) Cytotoxicity of N-terminallysine fusions in the presence and absence of PA, respectively. (Cand D) Cytotoxicity of C-terminal lysine fusions in the presence andabsence of PA, respectively. Solid lines show data with PA (20 nM)and dashed lines data without PA.

Mentions: DTA constructs with tracts of 6, 9, 12, or 15Lys residues fused to the N- or C-terminus were tested for translocationinto CHO K1 cells in the presence or absence of PA (Figure 1). In the presence of PA, N-terminal tracts of 9or 12 Lys residues (K9-DTA or K12-DTA, respectively) were effectivepotentiators of DTA translocation, with half-maximal inhibition ofprotein synthesis occurring at concentrations of ∼20 pM (Figure 1A). Consistent with our earlier findings, LFn-DTAwas ∼10-fold more potent than the most active of these poly-Lys-taggedconstructs. In the absence of PA, constructs containing 9, 12, or15 N-terminal Lys residues blocked protein synthesis, but only at∼10000-fold higher concentrations (≥0.1 μM) thanin the presence (Figure 1B). Untagged DTA showedcytotoxicity at concentrations approaching 1 μM in the presenceof PA and was inactive at the highest concentration tested (1 μM)in the absence of PA.


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

Sharma O, Collier RJ - Biochemistry (2014)

PA-mediated translocation of DTA constructs as measuredby proteinsynthesis inhibition in CHO K1 cells. (A and B) Cytotoxicity of N-terminallysine fusions in the presence and absence of PA, respectively. (Cand D) Cytotoxicity of C-terminal lysine fusions in the presence andabsence of PA, respectively. Solid lines show data with PA (20 nM)and dashed lines data without PA.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: PA-mediated translocation of DTA constructs as measuredby proteinsynthesis inhibition in CHO K1 cells. (A and B) Cytotoxicity of N-terminallysine fusions in the presence and absence of PA, respectively. (Cand D) Cytotoxicity of C-terminal lysine fusions in the presence andabsence of PA, respectively. Solid lines show data with PA (20 nM)and dashed lines data without PA.
Mentions: DTA constructs with tracts of 6, 9, 12, or 15Lys residues fused to the N- or C-terminus were tested for translocationinto CHO K1 cells in the presence or absence of PA (Figure 1). In the presence of PA, N-terminal tracts of 9or 12 Lys residues (K9-DTA or K12-DTA, respectively) were effectivepotentiators of DTA translocation, with half-maximal inhibition ofprotein synthesis occurring at concentrations of ∼20 pM (Figure 1A). Consistent with our earlier findings, LFn-DTAwas ∼10-fold more potent than the most active of these poly-Lys-taggedconstructs. In the absence of PA, constructs containing 9, 12, or15 N-terminal Lys residues blocked protein synthesis, but only at∼10000-fold higher concentrations (≥0.1 μM) thanin the presence (Figure 1B). Untagged DTA showedcytotoxicity at concentrations approaching 1 μM in the presenceof PA and was inactive at the highest concentration tested (1 μM)in the absence of PA.

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