Ribosome Inactivating Toxins Julien Barbier and Daniel Gillet www.mdpi.com/journal/toxins Edited by Printed Edition of the Special Issue Published in Toxins Ribosome Inactivating Toxins Ribosome Inactivating Toxins Special Issue Editors Julien Barbier Daniel Gillet MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Special Issue Editors Julien Barbier CEA—Universit ́ e Paris-Saclay France Daniel Gillet CEA—Universite ́ Paris-Saclay France Editorial Office MDPI St. Alban-Anlage 66 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Toxins (ISSN 2072-6651) from 2016 to 2018 (available at: http://www.mdpi.com/journal/toxins/special issues/ribosome) For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. 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Contents Preface to ”Ribosome Inactivating Toxins” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Julien Barbier and Daniel Gillet Ribosome Inactivating Proteins: From Plant Defense to Treatments against Human Misuse or Diseases Reprinted from: Toxins 2018 , 10 , 160, doi: 10.3390/toxins10040160 . . . . . . . . . . . . . . . . . . 1 Maria Serena Fabbrini, Miku Katayama, Ikuhiko Nakase and Riccardo Vago Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions) Reprinted from: Toxins 2017 , 9 , 314, doi: 10.3390/toxins9100314 . . . . . . . . . . . . . . . . . . . 5 Jeroen De Zaeytijd and Els J. M. Van Damme Extensive Evolution of Cereal Ribosome-Inactivating Proteins Translates into Unique Structural Features, Activation Mechanisms, and Physiological Roles Reprinted from: Toxins 2017 , 9 , 123, doi: 10.3390/toxins9040123 . . . . . . . . . . . . . . . . . . . 38 Ludger Johannes Shiga Toxin—A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis Reprinted from: Toxins 2017 , 9 , 340, doi: 10.3390/toxins9110340 . . . . . . . . . . . . . . . . . . . 64 Bj ̈ orn Becker, Tina Schn ̈ oder and Manfred J. Schmitt Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking Reprinted from: Toxins 2016 , 8 , 366, doi: 10.3390/toxins8120366 . . . . . . . . . . . . . . . . . . . 75 Xiao-Ping Li and Nilgun E. Tumer Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins Reprinted from: Toxins 2017 , 9 , 133, doi: 10.3390/toxins9040133 . . . . . . . . . . . . . . . . . . . 89 Wei-Wei Shi, Yun-Sang Tang, See-Yuen Sze, Zhen-Ning Zhu, Kam-Bo Wong and Pang-Chui Shaw Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2 Reprinted from: Toxins 2016 , 8 , 296, doi: 10.3390/toxins8100296 . . . . . . . . . . . . . . . . . . . 101 Annie Villysson, Ashmita Tontanahal and Diana Karpman Microvesicle Involvement in Shiga Toxin-Associated Infection Reprinted from: Toxins 2017 , 9 , 376, doi: 10.3390/toxins9110376 . . . . . . . . . . . . . . . . . . . 113 Jun-Young Park, Yu-Jin Jeong, Sung-Kyun Park, Sung-Jin Yoon, Song Choi, Dae Gwin Jeong, Su Wol Chung, Byung Joo Lee, Jeong Hun Kim, Vernon L. Tesh, Moo-Seung Lee and Young-Jun Park Shiga Toxins Induce Apoptosis and ER Stress in Human Retinal Pigment Epithelial Cells Reprinted from: Toxins 2017 , 9 , 319, doi: 10.3390/toxins9100319 . . . . . . . . . . . . . . . . . . . 135 Christina C. Tam, Thomas D. Henderson II, Larry H. Stanker, Xiaohua He and Luisa W. Cheng Abrin Toxicity and Bioavailability after Temperature and pH Treatment Reprinted from: Toxins 2017 , 9 , 320, doi: 10.3390/toxins9100320 . . . . . . . . . . . . . . . . . . . 155 v Christina C. Tam, Luisa W. Cheng, Xiaohua He, Paul Merrill, David Hodge and Larry H. Stanker A Monoclonal–Monoclonal Antibody Based Capture ELISA for Abrin Reprinted from: Toxins 2017 , 9 , 328, doi: 10.3390/toxins9100328 . . . . . . . . . . . . . . . . . . . 167 Yoav Gal, Ohad Mazor, Reut Falach, Anita Sapoznikov, Chanoch Kronman and Tamar Sabo Treatments for Pulmonary Ricin Intoxication: Current Aspects and Future Prospects Reprinted from: Toxins 2017 , 9 , 311, doi: 10.3390/toxins9100311 . . . . . . . . . . . . . . . . . . . 180 Yoav Gal, Anita Sapoznikov, Reut Falach, Sharon Ehrlich, Moshe Aftalion, Chanoch Kronman and Tamar Sabo Total Body Irradiation Mitigates Inflammation and Extends the Therapeutic Time Window for Anti-Ricin Antibody Treatment against Pulmonary Ricinosis in Mice Reprinted from: Toxins 2017 , 9 , 278, doi: 10.3390/toxins9090278 . . . . . . . . . . . . . . . . . . . 208 Sarah J. C. Whitfield, Gareth D. Griffiths, Dominic C. Jenner, Robert J. Gwyther, Fiona M. Stahl, Lucy J. Cork, Jane L. Holley, A. Christopher Green and Graeme C. Clark Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action Reprinted from: Toxins 2017 , 9 , 329, doi: 10.3390/toxins9100329 . . . . . . . . . . . . . . . . . . . 224 Amanda Y. Poon, David J. Vance, Yinghui Rong, Dylan Ehrbar and Nicholas J. Mantis A Supercluster of Neutralizing Epitopes at the Interface of Ricin’s Enzymatic (RTA) and Binding (RTB) Subunits Reprinted from: Toxins 2017 , 9 , 378, doi: 10.3390/toxins9120378 . . . . . . . . . . . . . . . . . . . 243 Gregory Hall, Shinichiro Kurosawa and Deborah J. Stearns-Kurosawa Shiga Toxin Therapeutics: Beyond Neutralization Reprinted from: Toxins 2017 , 9 , 291, doi: 10.3390/toxins9090291 . . . . . . . . . . . . . . . . . . . 259 Aleksander Rust, Lynda J. Partridge, Bazbek Davletov and Guillaume M. Hautbergue The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations Reprinted from: Toxins 2017 , 9 , 344, doi: 10.3390/toxins9110344 . . . . . . . . . . . . . . . . . . . 277 Letizia Polito, Daniele Mercatelli, Massimo Bortolotti, Stefania Maiello, Alice Djemil, Maria Giulia Battelli and Andrea Bolognesi Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells Reprinted from: Toxins 2017 , 9 , 182, doi: 10.3390/toxins9060182 . . . . . . . . . . . . . . . . . . . 292 Ka-Yee Au, Wei-Wei Shi, Shuai Qian, Zhong Zuo and Pang-Chui Shaw Improvement of the Pharmacological Properties of Maize RIP by Cysteine-Specific PEGylation Reprinted from: Toxins 2016 , 8 , 298, doi: 10.3390/toxins8100298 . . . . . . . . . . . . . . . . . . . 310 vi Preface to ”Ribosome Inactivating Toxins” Ribosome inactivating proteins (RIPs) form a vast family of hundreds of toxins from plants, fungi, algae and bacteria. RIP activities have also been detected in animal tissues. They exert an N-glycosydase catalytic activity that is targeted to a single adenine of a ribosomal RNA, thereby blocking protein synthesis and leading intoxicated cells to apoptosis. In many cases they perform additional depurinating activities that act against other nucleic acids, such as viral RNA and DNA, or genomic DNA. Although their role remains only partially understood, their functions may be related to plant defense against predators and viruses, plant senescence or bacterial pathogenesis. Most RIPs are no threat to human or animal health. However, several bacterial RIPs are major virulence factors involved in severe epidemic diseases such as dysentery or the hemolytic uremic syndrome that may occur in patients suffering from Shiga toxin-producing entero hemorrhagic Escherichia coli infection. A few RIPs synthesized in plant seeds have been involved in accidental or criminal poisonings, political intimidation or bio-suicides. Tremendous progress has been made in their detection, identification and characterization. However, the pathophysiologies of these intoxications seem much more complicated than being solely linked to cell death and are still far from being fully understood. There are no commercially available products to specifically prevent or block RIP action, although research progress has been made in the development of antibodies, small molecule inhibitors and vaccines. Finally, RIPs have been engineered into immunotoxins by conjugating them to antibodies or other targeting moieties. Numerous clinical trials have shown great promise, also with regard to the difficulties in developing such therapies to destroy cancer cells. This Special Issue of Toxins presents the most recent data on all aspects of RIPs: new RIPs, structure, function, mechanism of action, pathophysiology, anti-RIP drug development and RIP engineering into anticancer treatments. Julien Barbier, Daniel Gillet Special Issue Editors v ii toxins Editorial Ribosome Inactivating Proteins: From Plant Defense to Treatments against Human Misuse or Diseases Julien Barbier and Daniel Gillet * Service d’Ing é nierie Mol é culaire des Prot é ines (SIMOPRO), CEA, Universit é Paris-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette, France; julien.barbier@cea.fr * Correspondence: Daniel.gillet@cea.fr; Tel.: +33-1-69-08-76-46 Received: 11 April 2018; Accepted: 13 April 2018; Published: 18 April 2018 Ribosome inactivating proteins (RIPs) form a vast family of hundreds of toxins from plants, fungi, algae, and bacteria. RIP activities have also been detected in animal tissues. They exert an N-glycosydase catalytic activity that is targeted to a single adenine of a ribosomal RNA, thereby blocking protein synthesis and leading intoxicated cells to apoptosis. In many cases, they have additional depurinating activities that act against other nucleic acids, such as viral RNA and DNA, or genomic DNA. Although their role remains only partially understood, their functions may be related to plant defense against predators and viruses, plant senescence, or bacterial pathogenesis. In this Special Issue, a review by Fabbrini and colleagues [ 1 ] addresses our current knowledge about the function and mechanisms of action of plant type I and type II RIPs. In particular, they emphasize the diversity found in their mechanisms of action, although they share sequence and structural identities in their catalytic chain. In another review, De Zaeytijd and Van Damme focus on the heterogeneity of cereal RIPs from an evolutionary perspective, their differences from non-cereal RIPs, and their variety of roles in addition to defense against pathogens and insects [2]. Most RIPs are no threat to human or animal health. However, several bacterial RIPs are major virulence factors involved in severe epidemic diseases such as dysentery or the hemolytic uremic syndrome that may occur in patients suffering from Shiga toxin-producing entero-hemorrhagic Escherichia coli infection. A few RIPs synthesized in plant seeds such as ricin toxin, abrin, or sarcin have been or may be involved in accidental or criminal poisonings, political intimidation, or bio-suicides. In this Special Issue, four contributions address the most recent advances in understanding the three major steps of the intoxication process of cells by Shiga toxins, ricin, and/or sarcin: receptor-binding and triggering of endocytosis, the components of the intracellular trafficking machinery involved in intoxication and binding, and depurination of the ribosome. Johannes describes how the pentavalent binding of Shiga toxins to Gb3 gangliosides in lipid rafts induces membrane structural changes and stress leading to the internalization of the toxin-receptor complexes [ 3 ]. Becker and colleagues set up an elegant screening method in yeast, enabling them to not only to confirm the importance of the GARP complex and other protein partners in ricin A chain intracellular trafficking, but also to identify seven new proteins involved along the pathway [ 4 ]. This method can now be applied to identify trafficking components used by other RIPs. Li and Tumer analyze the differences in ribosome binding and catalytic activities of the non-activated and activated Shiga and ricin holotoxins, showing opposite behaviors for these two toxins [ 5 ]. Finally, Shi et al. describe the structure of the complex of ricin A chain with the C-terminal peptide of the ribosome stalk protein P2 [ 6 ]. They discuss the differences in this interaction with that of other RIPs with the same ribosomal target. The pathophysiology of intoxication by the most dangerous RIPs, such as Shiga toxins and ricin toxin, seem much more complicated than a sole link to circulation in the bloodstream and cell death, and is still far from being understood. Diana Karpman and her group review their recent work that brings important progress in the understanding of the mechanisms underlying the hemolytic uremic syndrome provoked by Shiga toxins [ 7 ]. They show that Shiga toxins are internalized by red blood Toxins 2018 , 10 , 160; doi:10.3390/toxins10040160 www.mdpi.com/journal/toxins 1 Toxins 2018 , 10 , 160 cells and then released in microvesicles. It is these toxin-containing microvesicles that participate in the prothrombotic lesions, hemolysis, and transfer of the toxin from the circulation into the kidney, that are characteristic of this deadly syndrome. Furthermore, a research article from the groups of Lee and Park describes for the first time the apoptotic processes induced by Shiga toxins in human retinal pigment epithelial cells, suggesting the mechanisms leading to blindness in severe cases of hemolytic and uremic syndrome [ 8 ]. Gal et al. extend their characterization of the crucial role of inflammation in ricin toxin pathogenesis by showing that total body irradiation of mice decreases inflammation markers and extends time to death [ 9 ]. Second after ricin toxin, the plant RIP abrin is considered an increasing risk of malevolent and suicidal use. Thus, there is a need for detection and decontamination tools. Tam et al. set up a two-monoclonal antibody-based ELISA that can detect as low as 1 ng/mL of abrin and that shows no false positive detection of other plant RIPs [ 10 ]. The same team showed that while various pH treatments of the toxin did not affect its activity, heating above 74 ◦ C completely inactivated its capacity to kill cells and mice. However, they showed that this treatment affects the lectin part of the toxin rather than its catalytic chain [ 11 ]. Interestingly, this article sets a correspondence between cytotoxicity testing and the mouse bioassay, which should help reduce the use of the mouse model for the evaluation of abrin and other RIPs. Due to sporadic but recurrent cases of biosuicide and biothreats with ricin toxin, there is an urgent need for a treatment of human intoxication. The group of Kronman gives us a thorough review of existing data on potential countermeasures and treatment strategies, although none are approved for medical use [ 12 ]. While antibodies represent the most realistic approach in the case of early post-intoxication intervention, the review stresses the importance of not only eliminating the toxin but also downregulating the explosive inflammatory response triggered by the toxin, as additionally described in a research article by the same group [9]. Interestingly, Whitfield and colleagues describe in detail an F(ab’) 2 polyclonal ovine antitoxin and its performance in a mouse model of ricin inhalation that is intended to be pharmaceutically qualified for human use [ 13 ]. Protection is mediated both by reducing the amount of circulating toxin and blocking its intracellular trafficking to the Golgi apparatus. Many studies in the past showed that a fraction only of the antibodies generated in the course of an immune response against ricin toxin was neutralizing. Here, the group of Nicholas Mantis identifies the presence of a supercluster of neutralizing epitopes at the interface between the A and B chains of the toxin by analyzing a series of V H H camelid antibody fragments from a phage library generated against ricin toxin [ 14 ]. Interestingly, these antibodies do not interfere with the binding of the toxin to the galactose and N-acetyl-galactosamine residues of cell surface glycosylation. This is a step forward in understanding the basis for antibody-mediated protection against this toxin. Hall et al. review the attempts to develop antibodies or other antitoxin strategies to treat the hemolytic and uremic syndrome caused by Shiga toxins, none of which have reached approval [ 15 ]. They suggest that the rarity of this disease is a major limit to achieving the necessary clinical trials. Then they advocate the development of drugs targeting the unfolded protein response and the ribotoxic stress response triggered by Shiga toxins as these pathways are involved in many other conditions, which may decrease the barriers to commercial development. The final aspect of research on RIPs covered by this Special Issue concerns their use in the engineering of immunotoxins to target cancer or cells infected by Human immunodeficiency virus (HIV) by conjugation of antibodies or other targeting moieties. Two reviews by Fabbrini et al. and Rust et al. discuss the difficulties that have been encountered in the development of several generations of immunotoxins, none of which have been approved after clinical trials [ 1 , 16 ]. They also present the future trends of immunotoxin development. Two examples of the complexities of such development are given in the articles of Polito et al. and Au et al. The former analyzes the difference in the mechanism of killing of two closely related saporin-containing immunotoxins targeting different markers on B-cell lymphomas, CD20 and CD22 [ 17 ]. The latter addresses the effect of PEGylation on the 2 Toxins 2018 , 10 , 160 pharmacology, biological activity, and antibody induction of a TAT-maize RIP construction designed to target HIV-infected cells [18]. Overall, this Special Issue of Toxins presents the most recent data on all aspects of RIPs, including function, diversity, evolution, as well as mechanism, pathophysiology, medical countermeasures, and engineering into anticancer drugs. Acknowledgments: D.G. and J.B. are supported by CEA, the Joint Ministerial Program of R&D against CBRNE risks, ANR Anti-HUS grant ANR-14-CE16-0004, and The Swedish Research Council grant K2015-99X-22877-01-6. Conflicts of Interest: The authors declare no conflict of interest. References 1. Fabbrini, M.S.; Katayama, M.; Nakase, I.; Vago, R. Plant ribosome-inactivating proteins: Progesses, challenges and biotechnological applications (and a few digressions). Toxins (Basel) 2017 , 9 , 314. [CrossRef] [PubMed] 2. De Zaeytijd, J.; Van Damme, E.J. Extensive evolution of cereal ribosome-inactivating proteins translates into unique structural features, activation mechanisms, and physiological roles. Toxins (Basel) 2017 , 9 , 123. [CrossRef] [PubMed] 3. Johannes, L. Shiga toxin-a model for glycolipid-dependent and lectin-driven endocytosis. Toxins (Basel) 2017 , 9 , 340. [CrossRef] [PubMed] 4. Becker, B.; Schnoder, T.; Schmitt, M.J. Yeast reporter assay to identify cellular components of ricin toxin a chain trafficking. Toxins (Basel) 2016 , 8 , 366. [CrossRef] [PubMed] 5. Li, X.P.; Tumer, N.E. Differences in ribosome binding and sarcin/ricin loop depurination by shiga and ricin holotoxins. Toxins (Basel) 2017 , 9 , 133. [CrossRef] [PubMed] 6. Shi, W.W.; Tang, Y.S.; Sze, S.Y.; Zhu, Z.N.; Wong, K.B.; Shaw, P.C. Crystal structure of ribosome-inactivating protein ricin a chain in complex with the c-terminal peptide of the ribosomal stalk protein p2. Toxins (Basel) 2016 , 8 , 296. [CrossRef] [PubMed] 7. Villysson, A.; Tontanahal, A.; Karpman, D. Microvesicle involvement in shiga toxin-associated infection. Toxins (Basel) 2017 , 9 , 376. [CrossRef] [PubMed] 8. Park, J.Y.; Jeong, Y.J.; Park, S.K.; Yoon, S.J.; Choi, S.; Jeong, D.G.; Chung, S.W.; Lee, B.J.; Kim, J.H.; Tesh, V.L.; et al. Shiga toxins induce apoptosis and er stress in human retinal pigment epithelial cells. Toxins (Basel) 2017 , 9 , 319. [CrossRef] [PubMed] 9. Gal, Y.; Sapoznikov, A.; Falach, R.; Ehrlich, S.; Aftalion, M.; Kronman, C.; Sabo, T. Total body irradiation mitigates inflammation and extends the therapeutic time window for anti-ricin antibody treatment against pulmonary ricinosis in mice. Toxins (Basel) 2017 , 9 , 278. [CrossRef] [PubMed] 10. Tam, C.C.; Cheng, L.W.; He, X.; Merrill, P.; Hodge, D.; Stanker, L.H. A monoclonal-monoclonal antibody based capture elisa for abrin. Toxins (Basel) 2017 , 9 , 328. [CrossRef] [PubMed] 11. Tam, C.C.; Henderson, T.D.; Stanker, L.H.; He, X.; Cheng, L.W. Abrin toxicity and bioavailability after temperature and ph treatment. Toxins (Basel) 2017 , 9 , 320. [CrossRef] [PubMed] 12. Gal, Y.; Mazor, O.; Falach, R.; Sapoznikov, A.; Kronman, C.; Sabo, T. Treatments for pulmonary ricin intoxication: Current aspects and future prospects. Toxins (Basel) 2017 , 9 , 311. [CrossRef] [PubMed] 13. Whitfield, S.J.C.; Griffiths, G.D.; Jenner, D.C.; Gwyther, R.J.; Stahl, F.M.; Cork, L.J.; Holley, J.L.; Green, A.C.; Clark, G.C. Production, characterisation and testing of an ovine antitoxin against ricin; efficacy, potency and mechanisms of action. Toxins (Basel) 2017 , 9 , 329. [CrossRef] [PubMed] 14. Poon, A.Y.; Vance, D.J.; Rong, Y.; Ehrbar, D.; Mantis, N.J. A supercluster of neutralizing epitopes at the interface of ricin’s enzymatic (RTA) and binding (RTB) subunits. Toxins (Basel) 2017 , 9 , 378. [CrossRef] [PubMed] 15. Hall, G.; Kurosawa, S.; Stearns-Kurosawa, D.J. Shiga toxin therapeutics: Beyond neutralization. Toxins (Basel) 2017 , 9 , 291. [CrossRef] [PubMed] 16. Rust, A.; Partridge, L.J.; Davletov, B.; Hautbergue, G.M. The use of plant-derived ribosome inactivating proteins in immunotoxin development: Past, present and future generations. Toxins (Basel) 2017 , 9 , 344. [CrossRef] [PubMed] 3 Toxins 2018 , 10 , 160 17. Polito, L.; Mercatelli, D.; Bortolotti, M.; Maiello, S.; Djemil, A.; Battelli, M.G.; Bolognesi, A. Two saporin-containing immunotoxins specific for cd20 and cd22 show different behavior in killing lymphoma cells. Toxins (Basel) 2017 , 9 , 182. [CrossRef] [PubMed] 18. Au, K.Y.; Shi, W.W.; Qian, S.; Zuo, Z.; Shaw, P.C. Improvement of the pharmacological properties of maize rip by cysteine-specific pegylation. Toxins (Basel) 2016 , 8 , 298. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 4 toxins Review Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions) Maria Serena Fabbrini 1 , Miku Katayama 2,3 , Ikuhiko Nakase 2 and Riccardo Vago 4,5, * 1 MIUR, Italian Ministry of Instruction, University and Research, 20090 Monza, Italy; msfabbrini@gmail.com 2 NanoSquare Research Institution, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Osaka 599-8570, Japan; sxc04031@edu.osakafu-u.ac.jp (M.K.); i-nakase@21c.osakafu-u.ac.jp (I.N.) 3 Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Osaka 599-8531, Japan 4 Urological Research Institute, Division of Experimental Oncology, IRCCS San Raffaele Hospital, 20132 Milan, Italy 5 University Vita-Salute San Raffaele, 23132 Milan, Italy * Correspondence: vago.riccardo@hsr.it; Tel.: +39-02-2643-5664 Academic Editors: Julien Barbier and Daniel Gillet Received: 31 August 2017; Accepted: 3 October 2017; Published: 12 October 2017 Abstract: Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N -glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α -sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications. Keywords: plant ribosome inactivating proteins; ER-stress; saporin; targeted drug delivery; nanovectors 1. Prologue The first time I heard the term ribosome-inactivating protein “RIP” was in 1987 when we were attending “GENE87” at the Milan University and one of the invited speakers was Prof. Fiorenzo Stirpe from the University “Alma Mater” of Bologna [ 1 ]. The speech was fascinating to all of us coming to attend the symposium from a Plant Biology institute. I had just started my own experimental thesis and it was even more intriguing that Prof. Stirpe was coming from a Medical School and not from a Botanical Institute. The bright idea of using plant-derived toxins to eliminate transformed cells was pioneered at that time. The two seminal papers by Endo and Tsurugi on the mechanism of action of ricin and type I RIPs acting on eukaryotic ribosomes were published this very same year [ 2 , 3 ]. Curiously, some researchers from an Italian pharmaceutical company came to our lab to get some advice on how to achieve the cloning of a RIP cDNA from Saponaria officinalis tissues. The dry seeds they were trying to use for preparing the cDNA library stored plenty of saponins that during the mashing procedures were producing huge amounts of bubbles (L. Benatti, personal communication). Toxins 2017 , 9 , 314; doi:10.3390/toxins9100314 www.mdpi.com/journal/toxins 5 Toxins 2017 , 9 , 314 This is the main reason why the first saporin cDNA was then cloned starting from fresh leaves [ 4 ], allowing me just by chance to meet the person with whom we still are sharing our lives. To end these digressions, we must certainly acknowledge the great amount of experimental work done by the group of Mike Lord and Lynne Roberts in Warwick while studying ricin, the prototype type II RIP, one of the most potent poisons known at that time, which was strikingly used to assassinate in a “rocambolesque” way a dissident in London during the heavy years of the cold war. Plant ribosome-inactivating proteins may be viewed as very special tools from the Plant Kingdom that allowed us to shed light on certain peculiar intracellular pathways, such as the retrograde transport along the secretory route or more recent findings about some RIP signal peptide(s) acting as stress-sensors. Still intracellular pathways of delivery need to be elucidated in detail to allow in the future more efficient uses in targeted anticancer therapy. 2. Biochemical and Structural Considerations Several plant species belonging to 17 different families, among them Cucurbitaceae, Euphorbiaceae, and Poaceae, and families belonging to the superorder Caryophyllales, produce plant Ribosome-Inactivating Proteins (RIPs), although many others do not, including the plant type model Arabidopsis thaliana [ 5 ]. They are found in most plant species as gene families, reflecting their differential distribution in plant tissues (roots, leaves and seeds) and may share among major functions the protection against viral or fungal infections and possibly be relevant for the physiologic responses during plant senescence or following stress inducers [ 6 , 7 ]. RIPs belong to the N-glycosidase family of toxins (EC3.2.2.22) able to specifically and irreversibly inactivate the large ribosomal subunits depurinating a specific adenine base (A4324 in the rat 28S ribosomal rRNA) located in a universally conserved GAGA-tetraloop, also known as the α -sarcin/ricin loop, present in 23S/26S/28S rRNA. Plant RIPs can be divided into three main classes: type I like saporin from Saponaria officinalis are composed of a single polypeptide chain of approximately 30 KDa, type II as ricin from Ricinus communis [ 8 ] are heterodimers consisting of an A chain, functionally equivalent to the type I polypeptide linked via a disulphide bridge to a B subunit endowed with lectin-binding properties [ 9 ]. For a long time, all type 2 RIPs were considered to be highly potent toxins, but, so far, there are also known type II RIPs, which are not or only less toxic in vivo , and therefore they are denominated as non-toxic type II RIPs [ 10 , 11 ]. Finally, type III RIPs are polypeptides, which are synthesized as inactive precursors (ProRIPs) that will require proteolytic processing events to form an active RIP [12]. Residues that are highly conserved among RIPs (shown in Figure 1 with an asterisk), besides the main residues at the catalytic cleft (arrowed in Figure 1), are those belonging to the “N-glycosidase signature”, which include Tyr80, Tyr123, and the key active site residues Glu177, Arg180, and Trp211 in RTA (Figure 2) and a few others surrounding this active site. The protein sequence identities between ricin A chain (RTA) and type I RIPs (Figure 1) are generally low and found to be respectively: saporin 22%, Gelonin 30%, pokeweed antiviral protein (PAP) 29%, thricosanthin 35%, dianthin 19%, bouganin 29%, and momordin / momorcharin, 33%. Despite the differences in amino acid sequences, their overall three-dimensional fold is well conserved as estimated by the superimposition of the 3D structures of several type I RIPs with the one of RTA (Figure 3), which clearly demonstrates that RTA and type I RIPs all share a common “RIP fold” characterized by the presence of two major domains: an N-terminal domain, which is mainly beta-stranded, and a C-terminal domain that is predominantly alpha–helical. 6 Toxins 2017 , 9 , 314 ȱ Figure 1. Amino acid sequence alignment of different type I RIPs compared to ricin (RTA), abrin and cinnamomin catalytic A chains by T-Coffee. Color shades indicate levels of amino acid homology between the aligned sequences. Conserved amino acids are identified with an asterisk and residues crucial for the catalytic activity are arrowed: Tyr72, Tyr120, Glu176, Arg179 and Trp208 in the sequence of saporin. 7 Toxins 2017 , 9 , 314 ȱ Figure 2. Three-dimensional reconstruction of catalytic cleft of saporin obtained by Swiss PDB Viewer (v4.0.4, SIB—Swiss Institute of Bioinformatics, Lausanne, Switzerland). Conserved residues crucial for the RIP signature are colored: Tyr72 (yellow), Tyr120 (red), Glu176 (orange), Arg179 (green) and Trp208 (blue). Hydrogen bonds among key residues are shown in orange. Insertions and deletions, as compared to PAP, momordin from Momordica charantia L . and RTA were found to lay mainly in random coil regions. Glu177 and Arg180 in RTA (as Glu176 and Arg179 in saporin) are directly involved in the mechanism of catalysis. However, while a RTA Glu177 mutant was 20-fold less active than wild-type A chain in inhibiting translation in a reticulocyte lysate, the Arg179 saporin mutant was found 200-fold less active [ 13 ]. Double mutants at the catalytic site have been investigated for several type I RIPs, since for heterologous expression studies less active site mutants were needed to allow elucidating their biosynthesis. A loss of in vitro and in vivo saporin cytotoxicity can be achieved when Glu176 and Arg179 are both mutated to lysine and glutamine residues, respectively. This double saporin mutant (termed KQ) is, indeed, devoid of all the detrimental effects associated with RIP expression in several hosts [ 14 – 17 ]. Interestingly, mutation of Trp208 in saporin did not impair its in vitro enzymatic activity nor cytotoxicity [ 18 ], but this same residue has been demonstrated to be crucial for PAP structural integrity [ 19 ]. A negative electrostatic potential, arising from both the negatively charged phosphodiester backbone and conserved solvent-exposed acidic patches on the ribosomal proteins, covers much of the ribosomal surface [ 20 ]. The net positive charge of saporin and its high content in basic residues (around 10% lysine residues) are likely to be critical for the recognition of the ribosomal surface. In RTA, a set of arginine residues around the active site are involved in electrostatic interactions with the phosphodiester backbone of the α -sarcin/ricin loop [ 21 , 22 ]. Both RTA- and saporin-catalyzed rRNA modification shows a net dependence on salt and ion concentrations, indicating that these toxins can exploit multiple electrostatic interactions with their target ribosomes [ 23 ]. Extra enzymatic activities have been putatively ascribed to RIPs, but apart the Polynucleotide: adenosineglycosidase (PAG) activity documented as a DNA multiple depurination, DNAse-like and RNAse-like activities seem to be due to cross-contaminations of the protein preparation [ 15 , 24 – 27 ]. Similar observations were also recently made [ 28 ], when comparing saporin-6 to the leaf isoform L1/L3 (which behaves differently from all other isoforms studied to date), they showed that saporin-6 enzymatic activity released two adenines from ribosomes, the major fraction being the one deriving from the N-glycosidase activity while L1/L3 was able to “multidepurinate” ribosomes. Characterization of the kinetic parameters indicated that poly(A) 8 Toxins 2017 , 9 , 314 RNA depurination proceeds with a Km of 639 ± 32 μ M and a kcat of 61 ± 1 min − 1 at pH 7.8 and 25 ◦ C. The catalytic efficiency of L1 on this substrate appears therefore to be considerably lower if compared to the action of a typical RIP, such as ricin A chain, on intact rat ribosomes which has been reported to occur with a Km of 2.6 μ M and a kcat of 1777 min − 1 [ 29 ]. The biological significance of the activity against rRNA at sites different from SRL, and on substrates other than the ribosomes (DNA, viral RNA, poly(A) mRNA) remains to be firmly established. When rRNA was extracted from mammalian cells exposed either to seed saporin or ricin, rRNA was found to be depurinated at a single site presumably corresponding to the one targeted by ricin [ 30 ]. Analysis of the in vivo activity of L1/L3 saporin will be required to assess whether multiple depurination plays any major role in the intoxication process and to clarify the mechanism of 80S ribosome inactivation by L1. The strong spatial similarities between type I RIPs, as shown in Figure 3, might suggest that different specificities/enzymatic activities could only reside in a restricted polypeptide area while assessment of whether these regions may contribute to altered activity would require either site-directed mutagenesis or protein domain swapping experiments. Figure 3. Three-dimensional structure of different type I RIPs and ricin A chain (RTA). Superimposition of secondary structure elements of Saporin ( red , PDB code 1QI7), Gelonin ( pink , 3KU0), PAP ( magenta , 1GIK), Trichosanthin ( cyan , 1QD2), Dianthin ( yellow , 1RL0), Bouganin ( grey , 3CTK), Momordin ( orange , 1 MOM), Momorcharin ( blue , 1AHA), RTA ( green , 1J1 M), modified from [31]. Native ricin A chain carries two N-glycosylation (Asn-X-Ser/Thr) sites and is very poor in lysines (only two residues), a feature that has been linked to the cytosolic entry route of ricin A chains (see Section 5) [ 32 ]. Mature saporin has no oligosaccharide side chains similar to most type I RIPs that are hypothesized to be internalized by animal cells mainly passively by fluid-phase pinocytosis, with some relevant exceptions (see Section 5). Recently, a variant PAP from asiatic Phytolacca acinosa , PAP-S1aci, was resolved at 1.7 Angstroms (sharing 95% identity with PAP-S, one seed isoform of Phytolacca americana ) and was found to have a proline substitution in the active site (Pro174) together with a rare type of N-glycosylation consisting of N-acetyl-D-glucosamine residues linked to the Asn10- Asn44- Asn255 sites, corresponding to putative rRNA-binding regions, mapped through computer modelling studies based on their structural data [ 33 ]. The authors hypothesize that these GlcNAc modifications may have evolved to exploit Mannose/GlcNAc-receptor-mediated endocytosis to enhance cytotoxic activity against seed predators. Their computer simulation studies suggest that 9 Toxins 2017 , 9 , 314 PAP-S1aci depurination activity would be adversely affected by larger, more typical oligosaccharide chains. The presence of short mannosyl residues in PAP-S1aci may thus confer an advantage to these seeds without compromising their RIP catalytic activity. The absence of carbohydrate side chains on PAP-I, a cell-wall protein may reflect its specialized antiviral role for “local suicide” of the virus- infected cells (see below). 3. Biotechnological Application in Agriculture To explore the potential antimicrobial activity, different RIPs, including pokeweed antiviral protein (PAP), trichosanthin (TC) from Trichosanthes kirilowii Maxim and the antiviral protein from Phytolacca insularis Nakai have been expressed in transgenic plants successfully leading to plant resistance against various viral and/or fungal proteins [ 34 – 37 ]. Transformed tobacco plants with the nontoxic C-terminal deletion mutant PAPW237* of Phytolacca americana and the active site mutant PAPE176V showed both that the ribosomes from these transgenic tobacco plants were not found depurinated. Interestingly, extracts from transgenic plants expressing PAPW237* protected tobacco plants from Potato Virus X infection, while the plant extracts from catalytic site mutant PAPE176V did not. PAP proteins have been studied since the nineties by the group headed by Tumer focusing on their potential applications to agriculture [ 38 ]. They also found that transgenic Arabidopsis (Line 512) plant expressing PAPW237* displayed an enhanced resistance to a strain of Tobacco Etch virus (TEV) and detected several genes upregulated, including auxin-responsive genes (more than 4-fold vs. basal), as well as genes involved in immunity and plant defense when performing a transcript profiling analysis using the Affymetrix Arabidopsis microarray. Ribosomal RNA is not the only substrate for PAP enzymatic activity, and capped mRNAs and uncapped RNAs are subject to inactivation by PAP. Additionally, double-stranded (ds) supercoiled DNA could be cleaved by this toxin [ 38 ]. The catalytic site required for rRNA depurination is the same required for DNA cleavage, as the PAPE176V mutant also could not cleave dsDNA, which, when treated with PAPwt, contained apurinic/apyrimidinic (AP) sites due to the removal of adenines. This same PAG activity has also been reported for different other type I RIPs including Gelonin, momordin I, PAP-S and saporin [ 26 ], but not for the ricin A chain. However, its role in the intoxication process by RIPs has yet to be elucida