Role of N-terminal His6-Tags in binding and efficient translocation of polypeptides into cells using anthrax protective antigen (PA)

PLoS One. 2012;7(10):e46964. doi: 10.1371/journal.pone.0046964. Epub 2012 Oct 8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Bacterial / chemistry*
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Toxins / chemistry*
  • Bacterial Toxins / genetics
  • Bacterial Toxins / metabolism*
  • Electric Conductivity
  • Histidine*
  • Human Umbilical Vein Endothelial Cells / cytology
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Lipid Bilayers / metabolism
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism*
  • Porosity
  • Protein Binding
  • Protein Transport
  • Viper Venoms / metabolism

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • Bacterial Toxins
  • Lipid Bilayers
  • Peptide Fragments
  • Viper Venoms
  • anthrax toxin
  • edema factor
  • epidermal cell differentiation inhibitor, Staphylococcus aureus
  • Histidine

Grants and funding

This work was supported by the Deutsche Forschungsgemeinschaft (project A5 of the Sonderforschungsbereich 487 and area 2B of Graduate College 1141/1), by the Association pour la Recherche sur le Cancer (Grant ARC SFI20111203659 to EL and a fellowship to CS and MR) and by the ANR agency (11BSV3 004 01) to EL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.