Sub-Domains of Ricin’s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

Anastasiya Yermakova,Nicholas J Mantis,David J Vance,Michel R Popoff

doi:10.1371/journal.pone.0044317

Abstract

The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

Highlights

Ricin, a natural product of the castor bean plant (Ricinus communis), is one of the most lethal protein toxins known [1,2]
surface plasmon resonance (SPR) analysis using a Biacore instrument revealed that JB4, C/M A2 and B/J F9 each bound ricin holotoxin with affinities roughly equal to other previously described RTBspecific neutralizing monoclonal antibodies (mAbs), including SylH3 and 24B11 (Table 1)
We found that when tested in series JB4, C/M A2 and B/J F9 were largely unaffected in their abilities to bind ricin holotoxin when the Biacore chips were first saturated with one or both of the other mAbs

Summary

Introduction

A natural product of the castor bean plant (Ricinus communis), is one of the most lethal protein toxins known [1,2]. In its mature form, ricin consists of two distinct subunits, RTA and RTB, joined by a single disulfide bond. RTA (32 kDa) is an RNA N-glycosidase that irreversibly inactivates eukaryotic ribosomes through hydrolytic cleavage of a conserved adenosine residue within in the sarcin-ricin loop (SRL) of the 28S rRNA [3,4]. RTB (34 kDa) is a galactose- and N-acetylgalactosamine (Gal/GalNac)specific lectin that mediates attachment, endocytosis, and trafficking of RTA from the plasma membrane to the trans-Golgi network (TGN) and the endoplasmic reticulum (ER)[5]. Once in the ER, RTA is transported via a process known as retrotranslocation, across the ER membrane and into the cytoplasm where it refolds into its enzymatically active conformation and initiates ribosome depurination [6]. Ricin’s potency is due in large part to RTB’s ability to adhere to and be internalized by virtually all mammalian cell types [7]

Methods

Results

Conclusion