Abstract
Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.
Highlights
Abrin, a type II ribosome inactivating protein of plant origin comprises of catalytically active A chain harboring the RNA Nglycosidase activity and a lectin-like B chain responsible for binding and trafficking of the toxin in cells [1]
Mapping the Epitope of the monoclonal antibodies (mAbs) D6F10 In order to map the epitope corresponding to the neutralizing mAb D6F10, overlapping truncated constructs spanning the entire length of the 251 amino acids long abrin A chain (ABA) were generated (Figure 1A)
When probed with the mAb D6F10, a faintly stained band observed with the truncated protein ABA 76–175 suggested that the amino acids within the stretch 76–175 of ABA form a part of the epitope (Figure 1B)
Summary
A type II ribosome inactivating protein of plant origin comprises of catalytically active A chain harboring the RNA Nglycosidase activity and a lectin-like B chain responsible for binding and trafficking of the toxin in cells [1]. Abrin is known to be 75 times more potent than ricin with an LD50 of 2.8 mg/kg body weight in mice [2]. Despite the understanding of the molecular details of inhibition of protein synthesis by ricin and abrin [3,4,5,6], the development of effective antidotes against these lethal potential bio-terror agents has been elusive. The antibody was shown to rescue cells and importantly mice challenged with a lethal dose of abrin. Delineating epitopes of neutralizing antibodies is important to understand mechanisms involved in the immunoneutralization of toxins. Such studies would provide better rationale for the design of vaccines against these lethal toxins [15]
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