Disulfide bonds in the ectodomain of anthrax toxin receptor 2 are required for the receptor-bound protective-antigen pore to function

• Published in: PloS one Vol. 5; no. 5; p. e10553
• Main Authors: Sun, Jianjun, Collier, R John
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10-05-2010; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Analysis; Animals; Anthrax; Antigens; Antigens, Bacterial - chemistry; Antigens, Bacterial - metabolism; Bacterial Toxins - chemistry; Bacterial Toxins - metabolism; Binding sites; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell surface; Chemical bonds; CHO Cells; Conformation; Cricetinae; Cricetulus; Disulfide bonds; Disulfides - metabolism; E coli; Endocytosis; Escherichia coli - metabolism; Hydrogen-Ion Concentration - drug effects; Immunoglobulins; Infectious Diseases/Bacterial Infections; Kinases; Liposomes; Microbiology/Cellular Microbiology and Pathogenesis; Microbiology/Immunity to Infections; Molecular Sequence Data; Oxidation-Reduction - drug effects; pH effects; Pore formation; Potassium - metabolism; Protective antigen; Protein Binding - drug effects; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Transport - drug effects; Receptor mechanisms; Receptors; Receptors, Peptide - chemistry; Receptors, Peptide - metabolism; Recombinant Proteins - isolation & purification; Reducing Agents - pharmacology; Reduction; Self-assembly; Sodium Dodecyl Sulfate - pharmacology; Toxins; Translocation; Von Willebrand factor; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0010553

Cell-surface receptors play essential roles in anthrax toxin action by providing the toxin with a high-affinity anchor and self-assembly site on the plasma membrane, mediating the toxin entry into cells through endocytosis, and shifting the pH threshold for prepore-to-pore conversion of anthrax toxin protective antigen (PA) to a more acidic pH, thereby inhibiting premature pore formation. Each of the two known anthrax toxin receptors, ANTXR1 and ANTXR2, has an ectodomain comprised of an N-terminal von Willebrand factor A domain (VWA), which binds PA, and an uncharacterized immunoglobulin-like domain (Ig) that connects VWA to the membrane-spanning domain. Potential roles of the receptor Ig domain in anthrax toxin action have not been investigated heretofore.
We expressed and purified the ANTXR2 ectodomain (R2-VWA-Ig) in E. coli and showed that it contains three disulfide bonds: one in R2-VWA and two in R2-Ig. Reduction of the ectodomain inhibited functioning of the pore, as measured by K(+) release from liposomes or Chinese hamster ovary cells or by PA-mediated translocation of a model substrate across the plasma membrane. However, reduction did not affect binding of the ectodomain to PA or the transition of ectodomain-bound PA prepore to the pore conformation. The inhibitory effect depended specifically on reduction of the disulfides within R2-Ig.
We conclude that disulfide integrity within R2-Ig is essential for proper functioning of receptor-bound PA pore. This finding provides a novel venue to investigate the mechanism of anthrax toxin action and suggests new strategies for inhibiting toxin action.