Protective antigen‐binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino‐ or carboxy‐terminus

Jill C. Milne, Steven R. Blanket, Philip C. Hanna, R. John Collier

Research output: Contribution to journalArticlepeer-review

Abstract

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor‐mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1–255) fused to either the amino‐terminus or the carboxy‐terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP‐ribosyltransferase activity and reacted with anti‐sera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO‐K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endo‐some acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP‐ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino‐terminus or the carboxy‐terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino‐ or carboxy‐terminus for biological activity, into the cytoplasm of mammalian cells.

Original languageEnglish (US)
Pages (from-to)661-666
Number of pages6
JournalMolecular Microbiology
Volume15
Issue number4
DOIs
StatePublished - Feb 1995

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Protective antigen‐binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino‐ or carboxy‐terminus'. Together they form a unique fingerprint.

Cite this