Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.

Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin.

Cholera toxin (CT) is composed of a disulfide-linked A1/A2 heterodimer and a ring-like, cell-binding B homopentamer. The catalytic A1 subunit must dissociate from CTA2/CTB5 to manifest its cellular activity. Reduction of the A1/A2 disulfide bond is required for holotoxin disassembly, but reduced CTA1 does not spontaneously separate from CTA2/CTB5: protein disulfide isomerase (PDI) is responsible for displacing CTA1 from its non-covalent assembly in the CT holotoxin. Contact with PDI shifts CTA1 from a protease-resistant conformation to a protease-sensitive conformation, which is thought to represent the PDI-mediated unfolding of CTA1. Based solely on this finding, PDI is widely viewed as an 'unfoldase' that triggers toxin disassembly by unfolding the holotoxin-associated A1 subunit. In contrast with this unfoldase model of PDI function, we report the ability of PDI to render CTA1 protease-sensitive is unrelated to its role in toxin disassembly. Multiple conditions that promoted PDI-induced protease sensitivity in CTA1 did not support PDI-mediated disassembly of the CT holotoxin. Moreover, preventing the PDI-induced shift in CTA1 protease sensitivity did not affect PDI-mediated disassembly of the CT holotoxin. Denatured PDI could still convert CTA1 into a protease-sensitive state, and equal or excess molar fractions of PDI were required for both efficient conversion of CTA1 into a protease-sensitive state and efficient disassembly of the CT holotoxin. These observations indicate the 'unfoldase' property of PDI does not play a functional role in CT disassembly and does not represent an enzymatic activity.