Characterization and cytotoxic mechanisms of toxic and non-toxic type 2 ribosome-inactivating proteins

Abstract

Ribosome inactivating proteins (RIPs) are a family of plant proteins that irreversibly arrest protein synthesis through depurination of rRNA. The anticancer, antiviral and neurotoxic properties allow RIPs to have useful applications in agriculture and in many biomedical fields, including clinical drug development. Galactose–binding lectins contained in caudices of four Adenia species were purified by affinity chromatography on CL Sepharose 6B. All lectins agglutinate human erythrocytes. The lectin from A. kirkii, called kirkiin, had the highest toxicity, inhibiting protein synthesis both by cell-free system and by whole cells, and deadenylates mammalian and yeast ribosomes. Kirkiin is highly toxic to cells, in which it induces apoptosis with EC50 (concentration reducing viability by 50%) of 10-14M after 72h. Analysis of the genomic clones encoding kirkiin reveals a high degree of sequence similarity to other type 2 RIPs. Molecular modeling confirmed that kirkiin have a similar structure to ricin. Thus, kirkiin shows all the properties of a toxic type 2 RIP and is amongst the most potent plant toxins. The pathogenesis of cell intoxication induced by the non-toxic type 2 RIPs from Sambucus, ebulin l and nigrin b, was investigated in a neuroblastoma-derived cell line (NB100), which showed to be very sensitive to RIPs. Despite the high translational inhibition activity in cell-free systems, both RIPs showed a low cytotoxicity in NB100 cells with respect to both type 1 and type 2 RIPs. The results revealed that the mechanism of cell death induced by Sambucus RIPs (10-8M) is partly due to caspase-dependent apoptosis and suggest the existence of a further cell death pathway that would be prevalent at the early 24h of intoxication and that is different from necrosis, necroptosis and autophagy. A block of cell cycle can be excluded. After 24h, the apoptotic pathway becomes the main route of cell death.