Role of N-Terminal His(6)-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.