PROTECTIVE ANTIGEN-BINDING DOMAIN OF ANTHRAX LETHAL FACTOR MEDIATES TRANSLOCATION OF A HETEROLOGOUS PROTEIN FUSED TO ITS AMINO-TERMINUS OR CARBOXY-TERMINUS

The edema factor (EF) and lethal factor (LF) components of anthrax tox in require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosi s, PA facilitates the translocation of EF and LF across the membrane o f an acidic intracellular compartment To characterize the translocatio n process, we generated chimeric proteins composed of the PA recogniti on domain of LF (LF(N); residues 1-255) fused to either the amino-term inus or the carboxy-terminus of the catalytic chain of diphtheria toxi n (DTA), The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Bo th fusion proteins strongly inhibited protein synthesis in CHO-K1 cell s In the presence of PA, but not in its absence, and they showed simil ar levels of activity. This activity could be inhibited by adding LF o r the LF(N) fragment (which blocked the interaction of the fusion prot eins with PA), by adding inhibitors of endosome 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 resul ts demonstrate that LF(N) fused to either the amino-terminus or the ca rboxy-terminus of a heterologous protein retains its ability to comple ment PA in mediating translocation of the protein to the cytoplasm. Be sides its-importance in understanding translocation, this finding prov ides the basis for constructing a translocation vector that mediates e ntry of a variety of heterologous proteins, which may require a free a mino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.