Type II Ribosome Inactivating Proteins (RIPs), commonly known as A/B toxins are heterodimers comprising of a catalytically active A chain, an RNA N-glycosidase which inhibits protein synthesis and a lectin-like B chain required for the binding of the toxin to the cell surface and internalization of the same. Abrin is a type II RIP obtained from the mature seeds of Abrus precatorius plant that is extremely toxic and has been shown to be 75 times more potent than its well studied sister toxin, ricin. The LD50 dose for abrin is only 2.8 µg/kg body weight of mice and its potential use in bio-warfare is a cause of major concern. Abrin has been classified as a select agent by the Centre for Disease Control and Prevention, U.S.A., because it is stable, effective at very low concentrations and easy to purify and disseminate in large amounts. In spite of abrin being a potential bio-warfare agent, there is no antidote or vaccine available against this toxin till date. The first and only neutralizing monoclonal antibody (mAb) against abrin, namely D6F10, was reported from our laboratory and has been shown to rescue toxicity of abrin in cells as well as in mice. The study reported in the thesis focuses on understanding the mechanism of neutralization of abrin by the mAb D6F10 and development of a potential vaccine candidate against the toxin. In order to map the epitope corresponding to the antibody, first, overlapping gene deletion constructs spanning the entire length, 251 amino acids, of ABA were generated and checked for binding to the mAb. Fragments shorter than 1-175 did not show immuoreactivity. Analysis of the crystal structure of abrin A chain revealed that a helix spanning the amino acids 148-167 was present at the core of the protein structure and truncation in this region of the protein possibly results in loss of conformation leading to abrogation of antibody binding. Therefore, a novel strategy of epitope mapping was adopted. Abrus precatorius agglutinin (APA) is a homologue of abrin obtained from the same plant source. The A chains of abrin and APA share 67% sequence identity and their crystal structures superimpose very well but unlike abrin the APA A chain does not bind the mAb D6F10. Chimeric constructs were generated within the region 1-175 of A chains of both ABA and APA and deletions and mutations of the ABA was then made on the APA as scaffold. It could be concluded that the amino acids of the region 75¬123 are involved in the formation of the epitope. Further, based on sequence alignment of ABA and APA A chain 13 residues in the chimera ABA1-123APA124-175 were mutated and it was found that the mutation of the residues Thr 112, Gly 114 and Arg 118 resulted in loss of binding to the antibody. Furthermore, the mAb D6F10 rescues inhibition of protein synthesis by abrin in HeLa cells by internalizing in cells along with abrin and possibly occluding the active site cleft of ABA. The antibody prevents cell attachment of abrin at higher concentrations. The observations provide novel insights into mechanisms of many known neutralizing antibodies against A/B toxins. The study also highlights that chimeric protein constructs could possibly be developed as potential vaccine candidates for neutralization of abrin intoxication.

Abrin is a potent toxin obtained from the seeds of Abrus precatorius. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a disulfide bond. The toxicity of the protein comes from the A subunit harboring RNA-N-glycosidase activity which cleaves the glycosidic bond between the ribose sugar and the adenine at position 4324 in 28S rRNA. The depurination of a specific adenine residue at position 4324 results in loss of conformation of the 28S rRNA at the α sarcin/ricin loop to which elongation factor-2 (EF-2) binds, during the transloction step of translation, leading to inhibition of protein synthesis. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the toxin to gain entry inside cells on binding to receptors with terminal galactose. After entering cells, a few molecules of abrin reach the endoplasmic reticulum (ER) via the retrograde transport, where the disulfide bond between the A and the B subunits gets cleaved. Then the A chain escapes into the cytosol where it binds to its target, the α-sarcin loop of the 28S ribosomal RNA and inhibits protein synthesis. Apart from inhibition of protein synthesis, exposure of cells to abrin leads to the loss of mitochondrial membrane potential (MMP) resulting in the activation of caspases and finally apoptosis. However, whether apoptosis is dependent on the inhibition of protein synthesis has not been elucidated. The major objectives of this study are therefore to delineate the signaling pathways involved in abrin-induced apoptosis. The thesis is divided into 4 Chapters: Chapter 1. provides a overview of the general properties of RIPs, with a brief history, classification, trafficking and biological activities of the toxins. This chapter also discusses their potential use in bio-warfare and the treatments available for management of toxicity. Chapter 2 and 3 discuss the results obtained on studies aimed at gaining insights into the signaling pathways involved in abrin-induced apoptosis. Chapter 4 focuses on the research carried out to understand the mechanisms of neutralization of abrin by the mAb D6F10. Towards the first objective, chapter 2 elucidates the role of endoplasmic reticulum (ER) stress signaling in abrin-induced apoptosis using the human T-cell line, Jurkat as a model system. It could be concluded that the inhibition of protein synthesis by the catalytic A subunit of abrin could result in accumulation of unfolded proteins in the ER leading to ER stress which triggers the unfolded protein response (UPR) pathway. The ER resident trans-membrane sensors IRE1 (Inositol-requiring enzyme 1), PERK (PKR-like ER kinase) and ATF6 (Activating transcription factor 6) are the important players of UPR in mammalian cells. These sensors inhibit translation and increase the levels of chaperones to restore protein homeostasis. However, if the ER stress is prolonged, apoptotic pathways get activated to remove severely damaged cells in which protein folding defects cannot be resolved. Recent studies have shown that endoplasmic reticulum (ER) stress induces apoptosis by activating initiater caspases such as caspase-2 and -8 which eventually trigger mitochondrial membrane potential loss and activation of downstream effector capases-9 and -3. Phosphorylation of eukaryotic initiation factor 2α and upregulation of CHOP [CAAT/enhancer binding protein (C/EBP) homologous protein], important players involved in ER stress signaling by abrin, suggested activation of ER stress in the cells. ER stress is also known to induce apoptosis via stress kinases such as p38 MAPK and JNK. Activation of both the pathways was observed upon abrin treatment and found to be upstream of the activation of caspases. However, abrin-induced apoptosis was found be dependent on p38 MAPK but not JNK. We also observed that abrin induced activation of caspase-2 and caspase-8 and triggered Bid cleavage leading to mitochondrial membrane potential loss and thus connecting the signaling events from ER stress to mitochondrial death machinery. Few toxins belonging to the family of ribosome inactivating proteins such as Shiga toxin have been observed to induce DNA damage in human endothelial cells and activate p53/ATM-dependent signaling pathway in mammalian cells. To further investigate the role of abrin on activation of DNA damage signaling pathway, we analysed the phosphorylation of H2AX and ATM, which are markers for double strand DNA breaks. We observed phosphorylation of H2AX and ATM upon abrin treatment but not when cells were pretreated with the broad spectrum pan caspase inhibitor. This study suggested that the DNA damage observed was an indirect effect of caspase-activated DNase. We concluded from the studies in chapter 2 that inhibition of protein synthesis by abrin can trigger endoplasmic reticulum stress leading to mitochondria-mediated apoptosis. Further studies were conducted to understand the dependence of ER stress on inhibition of protein synthesis and are presented in chapter 3. For this study, we have used an active site mutant of abrin A chain (R167L) which exhibits lower protein synthesis inhibitory activity than the wild type abrin A chain. Recombinant wild type and mutant abrin A chains were expressed in E.coli and purified. Since, abrin A chain requires the B chain for internalization into cells, both wild type and mutant abrin A chains were conjugated to native ricin B chain to generate a hybrid toxin. Next, we have compared the toxic effects of the two conjugates in cells. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild type ricin B-rABRA conjugate but it could trigger ER stress leading to mitochondrial mediated apoptosis in cells though delayed, suggesting that inhibition of protein synthesis is the major factor contributing to abrin-mediated apoptosis. Abrin is extremely lethal and considered as a potential agent for use in biological warfare. Currently, there are no antidotes or effective therapies available for abrin poisoning. Antibody based antitoxins function by either preventing toxin binding to cell surface receptors or by translocation. Antibodies against the B chain of RIPs function by inhibiting the binding of B chain of the toxin to cells, whereas the exact mechanism by which antibodies against A chain function is still not clear. The only known neutralizing monoclonal antibody against abrin A chain, namely, D6F10, was generated in our laboratory and was shown to rescue cells and mice from abrin intoxication. Earlier experiments with confocal microscopy suggested that mAb D6F10 could internalize in HeLa cells along with abrin, suggesting that the antibody can function intracellularly. Chapter 4 discusses the work carried out to delineate the mechanism of intracellular neutralization of abrin by the mAb D6F10. We observed significant reduction in binding and delay in abrin internalization in the presence of the neutralizing monoclonal antibody (mAb) D6F10. Considering that the majority of the abrin after internalization is removed by lysosomal degradation, we studied the fate of abrin in the presence of mAb D6F10. Confocal images did not show any difference in the distribution of abrin in the lysosomes in the absence or presence of antibody. However, the antibody remained persistently colocalized with abrin in the cells, suggesting that the antibody might inhibit enzymatic activity of abrin at its cellular site of action.