Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro

Abstract

The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin’s enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin’s disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…Unfortunately, our current report does not address the mechanism(s) by which the supercluster II antibodies neutralize ricin. We can only speculate that they interfere with a region of the toxin that is required for retrograde transport from the plasma membrane to the ER and/or retrotranslocation from the ER to the cytoplasm (45)(46)(47)(48).…”

A monoclonal antibody to an abrin chimera recognizing a unique epitope on abrin A chain confers protection from abrin-induced lethality

“…Antibodies to ricin, a sister protein of abrin, have been demonstrated to exert neutralization by hindering cell surface attachment of ricin 19 or inhibiting the catalytic activity of the A chain 20 or interfering with the reduction of the holotoxin to A and B chains. 21 It is less likely that mAb A7C4 acts via the last mechanism as the epitopic region is present away from the interface between the A and B chains. All the observations mentioned above strongly suggest an alternate mechanism of neutralization of abrin by the mAb A7C4.…”

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…Legler and colleagues recently demonstrated, using V H Hs, a correlation between an antibody's impact on RTA unfolding dynamics and in vitro toxin-neutralizing activity, possibly due to events associated with ricin endocytosis or retrograde transport (39). Other reports have shown that anti-RTA MAbs reduced intracellular transport of ricin from the plasma membrane to the endoplasmic reticulum (ER) and even prevented protein-disulfide isomerase (PDI)-mediated dissociation of RTA from RTB in a cell-free assay (62)(63)(64). Whether these in vitro activities are relevant in vivo The strongly protected regions for each cluster are summed over all MAbs belonging to that cluster and shown mapped to the crystal structure of the ricin holotoxin (PDB ID: 2AAI) (14), with each cluster colored as indicated.…”