Identification of a Novel Functional Domain of Ricin Responsible for Its Potent Toxicity

Abstract

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved ␣-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an ␣-helix comprising the residues 99 -106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the ␣-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the ␣-helix were rationally designed. Our data clearly demonstrated that the flexibility of the ␣-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved ␣-helix might be considered as a potential target for the prevention and treatment of RIP poisoning. Ribosome-inactivating proteins (RIPs)3 are depurinating rRNA N-glycosidases (E.C. 3.2.2.22) that cleave a single bond between a specific adenine and ribose of rRNA in eukaryotes (1). They are generally divided into two classes (1-3). Type I RIPs such as trichosanthin and gelonin are monomeric enzymes of ϳ30 kDa. Type II RIPs are heterodimeric proteins with an approximate molecular mass of 60 kDa, in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain). The B-chain is able to bind to a galactose-containing receptor on the surface of sensitive cells and mediate transport of the A-chain through the secretory pathways into the cytoplasm.Ricin (a type II RIP), which has emerged as a powerful catalyst for mammalian ribosomes, is a good prototype to investigate the N-glycosidase mechanism of RIPs. Ricin A-chain (RTA) is the catalytic subunit of ricin, which catalyzes the depurination of an invariant adenosine residue, A 4324 , within the GA 4324 GA tetraloop motif of the highly conserved sarcinricin loop of eukaryotic 28 S rRNA (4). Most previous studies have focused on the role of the active-site residues that are crucial for catalytic activity of RIPs. Day et al. (5) have presented the crystal structure of ricin, indicating that RTA has a prominent cleft able to recognize the target rRNA stem-loop. Sitedirected mutagenesis, as well as analysis by systematic deletion ...

Characterization and Epitope Mapping of the Polyclonal Antibody Repertoire Elicited by Ricin Holotoxin-Based Vaccination

“…The observation that epitope 1 has a role in ricin neutralization is in agreement with a previous report that demonstrated that this epitope is the target of an anti-ricin monoclonal antibody (32). In line with this, epitopes 2 and 3, which were not as effective in neutralizing ricin, are located in the so-called ricin-immunodominant regions V and VI, respectively, which are the targets of nonneutralizing antibodies (24).…”

“…4). Interestingly, this epitope was shown previously to be the exact target of a ricin-neutralizing monoclonal antibody (32).…”

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

“…The first hot spot encompasses RTA's ␣-helix B and ␣-helix D. The importance of ␣-helix B as a target of neutralizing antibodies has been recognized for more than 2 decades (22, 49), although it is only recently that the critical holdfasts have been identified (16,18,21,26,32). The second hot spot (as noted above) is located at the RTA-RTB interface, defined by RTA's ␣-helix F and the F-G loop and (possibly) RTB's subdomains 2␣ and/or 2␥ (16).…”

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

“…B cell epitope mapping studies of RTA have been conducted using peptide arrays (pepscan), peptide affinity enrichment by phage display, site-directed mutagenesis, differential reactivity with Ricinus communis agglutinin 1 (RCA-1), competition ELISAs, and surface plasmon resonance (SPR) analysis (28,30,34,(36)(37)(38). X-ray crystallography is also being used to define the structural B cell epitopes on RTA, although these studies have been restricted (with one exception) to the use of single-domain camelid antibodies (37,(39)(40)(41)(42).…”