Role of N-terminal His6-Tags in binding and efficient translocation of polypeptides into cells using anthrax protective antigen (PA).
It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB(7/8)-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His(6)-tag to different proteins increased their binding affinity to the protective antigen (PA) PA(63)-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB(7/8)-family of toxins. His(6)-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to -70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA(63). Cell-based experiments demonstrated that addition of an N-terminal His(6)-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA(63). Our results revealed that addition of His(6)-tags to several factors increase their binding properties to PA(63) and enhance the property to intoxicate cells.
Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
Christoph
C
Beitzinger
Caroline
C
Stefani
Angelika
A
Kronhardt
Monica
M
Rolando
Gilles
G
Flatau
Emmanuel
E
Lemichez
Roland
R
Benz
D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004553:Electric Conductivity; D006639:Histidine; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D016062:Porosity; D011485:Protein Binding; D021381:Protein Transport; D014757:Viper Venoms / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D014757:Viper Venoms; C030325:anthrax toxin; C416405:edema factor; C067120:epidermal cell differentiation inhibitor, Staphylococcus aureus; D006639:Histidine