Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata Luigi Barbieri a , Letizia Polito a , Andrea Bolognesi a, ⁎ , Marialibera Ciani a , Emanuele Pelosi a , Valentina Farini a , Ajay K. Jha b , Neelam Sharma b , Jorge M. Vivanco b , Angela Chambery c , Augusto Parente c , Fiorenzo Stirpe a a Dipartimento di Patologia sperimentale, Università di Bologna, I-40126 Bologna, Italy b Department of Horticulture, Colorado State University, Fort Collins, CO 80523-1173, USA c Dipartimento di Scienze della Vita, Seconda Università di Napoli, I-81100 Caserta, Italy Received 6 October 2005; received in revised form 23 December 2005; accepted 4 January 2006 Available online 26 January 2006 Abstract The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC 50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml -1 ) and by HeLa, HT29 and JM cells with IC 50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml -1 , all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities. © 2006 Elsevier B.V. All rights reserved. Keywords: Cucurbita moschata; DNA glycosylase; Plant defence; Pumpkin; Ribosome-inactivating protein; Tomato 1. Introduction Ribosome-inactivating proteins (RIPs) is the denomination given to plant proteins that were found to damage ribosomes in an irreversible manner, with consequent arrest of protein syn- thesis (recent reviews in [1–4]). Ribosome-inactivating proteins are classically divided into type 1, single-chain proteins, and type 2, in which an enzymatically active A chain is linked to a B chain with lectin properties. A type 3 has been proposed, in- cluding some peculiar RIPs [5–7], in which the A chain is attached to peptidic segments with unknown function. The lec- tinic B chain of type 2 RIPs binds to carbohydrate structures present on the cell surface, allowing and sometimes facilitating the internalization of the enzymatic A chain into cells. Some type 2 RIPs are potent toxins (ricin and related toxins, review in [8]), whereas some others have a low toxicity. Type 1 RIPs, being devoid of the B chain, cannot bind to cells in which they enter with difficulty and consequently their toxicity is much lower than that of ricin and related toxins. Ribosome-inactivating proteins possess numerous proper- ties: besides the toxicity of some type 2 RIPs, they have antiviral activity against plant and animal viruses, (review in [9]), are allergenic [10,11] and have transforming activity [12]. Ribosome-inactivating proteins were identified in several plant tissues (seed, leaf, sarcocarp, bark) and latices, and ribo- some-inactivating activity was observed in many, but not all plants examined [13]. However, in the majority of the screening Biochimica et Biophysica Acta 1760 (2006) 783 – 792 http://www.elsevier.com/locate/bba ⁎ Corresponding author. Tel.: +39 051 20 94700; fax: +39 051 20 94746. E-mail address: andrea.bolognesi@unibo.it (A. Bolognesi). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.01.002