disulfide bonds in the ectodomain of anthrax toxin receptor 2 are required for the receptor-bound protective-antigen pore to function二硫键的ectodomain炭疽毒素受体2需要receptor-bound保护抗原孔功能.pdfVIP

Disulfide Bonds in the Ectodomain of Anthrax Toxin Receptor 2 Are Required for the Receptor-Bound Protective-Antigen Pore to Function ¤ Jianjun Sun* , R. John Collier Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America Abstract 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. Methodology/Principal Findings: 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/Significance: We conclude that disulfide integrity within R2-Ig is essential for proper functioning of receptor