Abstract
Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in plants, fungi, and bacteria. RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively cleaving a specific adenine residue from a highly conserved, surface-exposed, stem-loop (S/R loop) structure in the 28S rRNA of ribosomes. Some RIPs also exhibit a number of other enzymatic activities such as RNase, DNase, phospholipase, and superoxide dismutase (SOD). RIPs are considered to be plant defense-related proteins as they are able to inhibit the multiplication and growth of several pathogenic virus, fungi, and bacteria either alone or in conjugation with other defense-related proteins. The mechanism of inhibitory activity of RIPs against fungal pathogens seems to be by directly inhibiting fungal growth rather than depurinating host plant ribosomes and causing cell death as previously envisaged. This chapter describes the protocol used to isolate and purify RIPs from plant tissues.
The first two authors contributed equally to this work.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nielsen, K. and Boston, R. S. (2001) Ribosome-inactivating proteins: a plant perspective. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 785–816.
Endo, Y. and Tsurugi, K. (1987) RNA N-glycosidase activity of ricin A-chain: mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol.Chem. 263, 8735–8739.
Mundy, J., Leah, R., Boston, R., Endo, Y., and Stirpe, F. (1994) Genes encoding ribosome-inactivating proteins. Plant Mol. Biol. Rep. 12, S60–S62.
Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: the site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J. Biol. Chem. 262, 5908–5912.
Barbieri, L., Gorini, P., Valbonesi, P., Castiglioni, P., and Stirpe, F. (1994) Unexpected activity of saporins. Nature 372, 624.
Park, S. W., Vepachedu, R., Owens, R. A., and Vivanco J. M. (2004) The Nglycosidase activity of the ribosome-inactivating protein ME1 targets singlestranded regions of nucleic acids independent of sequence or structural motifs. J. Biol. Chem. 279, 34,165–34,174.
Park, S. W., Vepachedu, R., Sharma, N., and Vivanco, J. M. (2004) Ribosomeinactivating proteins in plant biology. Planta 219, 1093–1096.
Lam, S. K. and Ng, T. B. (2001) Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hyprizigus marmoreus. Biochem. Biophys. Res. Comm. 285, 1071–1075.
Lam, S. K. and Ng, T. B. (2001) First simultaneous isolation of a ribosome-inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence for synergism of their antifungal effects. Arch. Biochem. Biophys. 393, 271–280.
Park, S. W., Lawrence, C. B., Linden, J. C., and Vivanco, J. M. (2002) Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots. Plant Physiol. 130, 164–178.
Jach, G., Gornhardt, B., Munday, J., et al. (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J. 8, 97–109.
Maddaloni, M., Forlani, F., Balmas, V., et al. (1997) Tolerance to the fungal pathogen Rhizoctonia solani AG4 of transgenic tobacco expressing the maize ribosomeinactivating protein b-32. Transgenic Res. 6, 393–402.
Tumer, N.E., Hudak, K., Di, R., Coetser, C., Wang, R., and Zoubenko, O. (1999) Pokeweed antiviral protein and its applications. Curr. Top. Microbiol. Immunol. 240, 139–158.
Zoubenko, O., Uckun, F., Hur, Y., Chet, I., and Tumer, N. E. (1997) Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants. Nature Biotechnol. 15, 992–996.
Hudak, K. A., Dinman, J. D., and Tumer, N. E. (1999) Pokeweed antiviral protein accesses ribosomes by binding toL3. J. Biol. Chem. 274, 3859–3864.
Hudak, K. A., Wang, P., and Tumer, N. E. (2000) A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template. RNA 6, 369–380.
Wang, P., Zoubenko, O., and Tumer, N. E. (1998) Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II. Plant Mol. Biol. 38, 957–964.
Vivanco, J. M., Savary, B. J., and Flores, H. E. (1999) Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the Andean crop Mibilis expansa. Plant Physiol. 119, 1447–1456.
Ng, T. B. and Parkash, A. (2002) Hispin, a novel ribosome-inactivating protein with antifungal activity from hairy melon seeds. Protein Expres. Purification 26, 211–217.
Park, S. W., Stevens, N. M., and Vivanco, J. M. (2002) Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity. Planta 216, 227–234.
Ramakrishnan, S. and Houston, L. L. (1984) Inhibition of human acute lymphoblastic leukemia cells by immunotoxins: potentiation by chloroquine. Science 223, 58–61.
Jansen, B., Uckun, F. M., Jaszcz, W. B., and Kersey, J. H. (1992) Establishment of a human t(4;11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin. Cancer Res. 52, 406–412.
Bruland, O. S., Fodstad, O., Stenwig, A. E., and Pihl, A. (1988) Expression and characteristics of a novel human osteosarcoma-associated cell surface antigen. Cancer Res. 48, 5302–5309.
Battelli, M. G., Polito, L., Bolognesi, A., Lafleur, L., Fradet, Y., and Stirpe, F. (1996) Toxicity of ribosome-inactivating protein-containing immunotoxins to a human bladder carcinoma cell line. Int. J. Cancer 65, 485–490.
Bolognesi, A., Tazzari, P. L., Olivieri, F., Polito, L., Falini, B., and Stirpe, F. (1996) Induction of apoptosis by ribosome-inactivating proteins and related immunotoxins. Int. J. Cancer 68, 349–355.
Surolia, N. and Misquith, S. (1996) Cell surface receptor directed targeting of toxin to human malaria parasite, Plasmodium falciparum. FEBS Lett. 396, 57–61.
Acknowledgments
Work reported in this communication was supported by a CAREER award from the National Science Foundation (MCB-0093014) to JMV, and by the Colorado State University Agricultural Experiment Station (JMV).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Park, SW., Prithiviraj, B., Vepachedu, R., Vivanco, J.M. (2006). Isolation and Purification of Ribosome-Inactivating Proteins. In: Loyola-Vargas, V.M., Vázquez-Flota, F. (eds) Plant Cell Culture Protocols. Methods in Molecular Biology™, vol 318. Humana Press. https://doi.org/10.1385/1-59259-959-1:335
Download citation
DOI: https://doi.org/10.1385/1-59259-959-1:335
Publisher Name: Humana Press
Print ISBN: 978-1-58829-547-7
Online ISBN: 978-1-59259-959-2
eBook Packages: Springer Protocols