The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity.
Anthrax toxin consists of three separate proteins produced by Bacillus anthracis: protective antigen (PA), lethal factor (LF), and edema factor (EF). Previous work showed that the process by which these proteins damage eukaryotic cells begins with binding of PA (83 kDa) to cell surface receptors. PA is then cleaved by a cell surface protease so as to expose a high-affinity binding site for LF or EF on the COOH-terminal, receptor-bound, 63-kilodalton fragment. In this report we more closely define a region of PA involved in receptor binding. The gene encoding PA was mutagenized so as to delete 3, 5, 7, 12, or 14 amino acids from the carboxyl terminus of the protein, and the truncated PA variants were purified from Bacillus subtilis or Escherichia coli. Deletion of 3, 5, or 7 amino acids reduced the binding of PA to cells and the subsequent toxicity of the PA.LF complex to J774A.1 cells and also the ability to cause EF binding to cells. Deletion of 12 or 14 amino acids completely eliminated all these activities. These results show that the carboxy terminus comprises or is part of the receptor-binding domain of PA.
Singh
Y
Y
Laboratory of Microbial Ecology, National Institute of Dental Research, Bethesda, Maryland 20892.
Klimpel
K R
KR
Quinn
C P
CP
Chaudhary
V K
VK
Leppla
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001412:Bacillus subtilis / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors