Type-II Ribosome Inactivating Proteins From Abrus Precatorius : Cytotoxicity And Mechanism Of Cell Death
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Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.
- Biochemistry (BC)