Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies.

David J Vance,Amanda Y Poon,Nicholas J Mantis

doi:10.1371/journal.pone.0236538

Abstract

Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.

Highlights

The plant toxin, ricin, is classified by military and public health officials as a biothreat agent because of its extreme potency following inhalation, coupled with the ease by which the toxin can be procured from castor beans (Ricinus communis) [1]
The relative epitope locations of the 10 Ricin toxin’s B subunit (RTB)-specific toxin-neutralizing and non-neutralizing monoclonal antibodies (mAbs) in our collection are not known, because the collection as a whole has never been subject to cross-competition assays
We performed competitive sandwich ELISAs in which capture mAbs were immobilized on microtiter plates and probed with biotinylated ricin in the presence of molar excess competitor mAb (Table 1; Fig 2A)

Summary

Introduction

The plant toxin, ricin, is classified by military and public health officials as a biothreat agent because of its extreme potency following inhalation, coupled with the ease by which the toxin can be procured from castor beans (Ricinus communis) [1]. Ricin’s A and B subunits each contribute to toxicity. The A subunit (RTA) is a ribosome-inactivating protein (RIP) that functions by depurination of a conserved adenine residue within the sarcin-ricin loop (SRL) of 28S rRNA [2, 3]. The B subunit (RTB) is a galactose (Gal)- and N-acetylgalactosamine (GalNAc)-. Revised B cell epitope map of RTB

Methods

Results

Conclusion