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


RESULTS
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.


CONCLUSIONS
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.
Sun
Jianjun
J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America. jsun@utep.edu
Collier
R John
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D019163:Reducing Agents / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004220:Disulfides; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D019163:Reducing Agents; C030325:anthrax toxin; C070309:anthrax toxin receptors; D012967:Sodium Dodecyl Sulfate; D011188:Potassium