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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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Effectof PA Phe clamp mutations, F427H and F427S, on the cytotoxicityof LFn-DTA and K12-DTA constructs in CHO-K1 cells.
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fig2: Effectof PA Phe clamp mutations, F427H and F427S, on the cytotoxicityof LFn-DTA and K12-DTA constructs in CHO-K1 cells.

Mentions: A key determinant of LFn-DTA translocationby PA is the Phe clamp, a structure formed by the F427 side chainswithin the lumen of the PA pore.34,37,38 We tested K12-DTA and DTA-K12 for cytotoxicity inthe presence of PA variants containing the F427H or F427S mutation,each of which blocks translocation of LFn-DTA without affecting poreformation. Both mutations completely blocked LFn-DTA translocation,as expected, but we found an unanticipated difference between F427Hand F427S in inhibiting translocation of the K12-tagged DTA constructs(Figure 2). Whereas F427H completely blockedtranslocation of K12-DTA and DTA-K12, F427S inhibited their translocation≤10-fold. These results support the notion that translocationpromoted by Lys tracts occurs via the pore lumen but also indicatedifferences in the mechanisms by which F427H and F427S inhibit translocation.


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

Sharma O, Collier RJ - Biochemistry (2014)

Effectof PA Phe clamp mutations, F427H and F427S, on the cytotoxicityof LFn-DTA and K12-DTA constructs in CHO-K1 cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Effectof PA Phe clamp mutations, F427H and F427S, on the cytotoxicityof LFn-DTA and K12-DTA constructs in CHO-K1 cells.
Mentions: A key determinant of LFn-DTA translocationby PA is the Phe clamp, a structure formed by the F427 side chainswithin the lumen of the PA pore.34,37,38 We tested K12-DTA and DTA-K12 for cytotoxicity inthe presence of PA variants containing the F427H or F427S mutation,each of which blocks translocation of LFn-DTA without affecting poreformation. Both mutations completely blocked LFn-DTA translocation,as expected, but we found an unanticipated difference between F427Hand F427S in inhibiting translocation of the K12-tagged DTA constructs(Figure 2). Whereas F427H completely blockedtranslocation of K12-DTA and DTA-K12, F427S inhibited their translocation≤10-fold. These results support the notion that translocationpromoted by Lys tracts occurs via the pore lumen but also indicatedifferences in the mechanisms by which F427H and F427S inhibit translocation.

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