Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

Zhu, Yuwei; Dai, Jing; Zhang, Tiancheng; Xu, Li; Fang, Pengfei; Wang, Huajing; Jiang, Yong-Liang; Yu, Xiaojie; Tian, Xia; Niu, Liwen; Guo, Yajun; Teng, Maikun

doi:10.1074/jbc.m113.480830

Cited by 12 publications

(15 citation statements)

References 38 publications

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“…In addition, CDR3 generates six hydrogen bonds to RTA with one crucial hydrogen bond between CDR3 residue Ser 98 and RTAs Thr 116 in β-strand H and the most prominent interaction occurring between CDR3 and α-helix B (residues 97–107) that forms two of the six hydrogen bonds. This interaction results in the burial of 472 Å 2 and, importantly, involves hydrogen bonding between CDR3 residue Arg104 and RTA residue Thr105 (Figure 4A), a residue within α-helix B that we have predicted is a common contact point among a number of very potent ricin toxin-neutralizing murine mAbs 15; 26 .…”

Section: Resultsmentioning

confidence: 99%

“…Based on differential peptide reactivity, we postulate that R70 and PB10 contact residues Q98, E99, E102, T105 and H106 (Table S2; Figure S3). Another R70/PB10-like murine mAb, 6C2, was recently described 12 and the X-ray crystal structure solved of its Fab fragments in complex with RTA [ PDB: 4KUC ] 26 . The crystal structure revealed that 6C2 is virtually identical to R70/PB10 in that it makes key contacts with RTA at residues Gln98, Glu99, Glu102, and Thr105.…”

Section: Discussionmentioning

confidence: 99%

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Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Rudolph

Vance

Cheung

et al. 2014

Journal of Molecular Biology

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Ricin is a Select Agent Toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins (RIPs). In this study, we determined x-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA’s RNA N-glycosidase activity or induced notable allosteric changes in the toxin’s subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Rudolph

Vance

Cheung

et al. 2014

Journal of Molecular Biology

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“…Interestingly, the ricin-neutralizing antibody 6C2 recognizes an epitope distinct from the catalytic active site of RTA and is more potent in its neutralizing ability relative to antibodies that bind the active site (Dai et al , 2011). These researchers further proposed that this antibody may inhibit binding of RTA to the ribosome (Zhu et al , 2013). We show here for the first time that targeting specific arginine residues of RTA can reduce its toxicity in mammalian cells by inhibiting its interactions with the ribosome while leaving the active site intact.…”

Section: Discussionmentioning

confidence: 99%

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

Jetzt

Xiao-ping

Tumer

et al. 2016

Toxicology and Applied Pharmacology

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Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication.

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“…High resolution epitope mapping studies using a phage displayed peptide library demonstrated that PB10 and R70 recognize slightly different epitopes, even though they are focused on the same α-helix and bind the same peptide [101]. Recent X-ray crystallographic studies of toxin-antibody complexes have enabled atomic level resolution of epitopes within Cluster I. Zhu et al solved the structure of RTA in complex with Fab fragments from a PB10-like mAb called 6C2 [102], which revealed antibody contact primarily with α-helix B (residues 97–107).…”

Section: G Establishing Surrogate Markers Of Immunity To Ricinmentioning

confidence: 99%

“…In addition, a non-human primate study with RiVax demonstrated the vaccine’s potential to elicit immunity to aerosolized ricin challenge [47]. Finally, the first atomic level insights into the nature of neutralizing B cell epitopes on RTA emerged [97,102]. …”

Section: H Expert Commentary and Five-year Viewmentioning

confidence: 99%

Progress and challenges associated with the development of ricin toxin subunit vaccines

Vance

Mantis

2016

Expert Review of Vaccines

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Summary The past several years have seen major advances in the development of a safe and efficacious ricin toxin vaccine, including the completion of two Phase I clinical trials with two different recombinant A subunit (RTA)-based vaccines: RiVax™ and RVEc™ adsorbed to aluminum salt adjuvant, as well as a non-human primate study demonstrating that parenteral immunization with RiVax elicits a serum antibody response that was sufficient to protect against a lethal dose aerosolized ricin exposure. One of the major obstacles moving forward is assessing vaccine efficacy in humans, when neither ricin-specific serum IgG endpoint titers nor toxin-neutralizing antibody levels are accepted as definitive predictors of protective immunity. In this review we summarize ongoing efforts to leverage recent advances in our understanding of RTA-antibody interactions at the structural level to develop novel assays to predict vaccine efficacy in humans.

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Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

Cited by 12 publications

References 38 publications

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

Progress and challenges associated with the development of ricin toxin subunit vaccines

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