Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin.
Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required > 13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the nontoxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.
Klimpel
K R
KR
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Molloy
S S
SS
Thomas
G
G
Leppla
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects*; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D045683:Furin; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D009838:Oligodeoxyribonucleotides; C030325:anthrax toxin; D010450:Endopeptidases; D013381:Subtilisins; D045683:Furin