Ribosome-inactivating proteins: from toxins to useful proteins.

Ribosome-inactivating proteins (RIPs) either single-chain (type 1) or two-chain (type 2) are frequent in plants, often in multiple forms. They are RNA N-glycosidases, have antiviral, antifungal and insecticidal activity. Their expression in plants is increased under stressful conditions. They are investigated for practical applications in medicine and in agriculture. In medicine, RIPs have been linked to, or fused with, appropriate antibodies or other carriers to form "immunotoxins" or other conjugates specifically toxic to the cells target of the carrier, with the aim of eliminating malignant or other undesired cells. In agriculture, it has been observed that an enhanced expression of RIPs confers to plants an increased resistance to viruses, fungi, insects, and also to drought and salinity.

Type 1 ribosome-inactivating proteins - entomotoxic, oxidative and genotoxic action on Anticarsia gemmatalis (Hübner) and Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

Ribosome-inactivating proteins (RIPs) from plants inhibit protein synthesis by inactivating ribosomes. Some two-chain (type 2) RIPs are highly toxic and may play a role in plant defense. The lower toxicity of single-chain (type 1) RIPs reflects the lack of a protein domain able to bind to, and translocate the toxin across cell membranes. We studied the effect of single-chain RIPs, lychnin, momordin, gelonin, PAP-S and saporin S-6, in larvae of Anticarsia gemmatalis and Spodoptera frugiperda. After ingesting a total dose of 20 or 40 microg of the toxins, weight gain, survival rate, lesions in DNA and oxidative status (catalase and superoxide dismutase activities and lipidic peroxidation) of RIP-treated insects were assayed. Momordin was the less toxic in the biossays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. RIP-induced mortality reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. RIP-treated insects showed a 2-3-fold increase in DNA lesions as assessed by the comet assay, but there were no correlations between stress markers and DNA damage. We conclude that single-chain RIPs are entomotoxic to lepidopteran insects causing extensive DNA lesions.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.

In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells.

Lanceolin and stenodactylin, new type 2 ribosome-inactivating proteins (RIPs) from Adenia plants were recently isolated and their high cytotoxicity was described. Present experiments were performed to investigate the effect of these toxins on neural cells in culture and their in vivo retrograde transport and neurotoxicity in the central nervous system. The concentrations of lanceolin and stenodactylin inhibiting by 50% protein synthesis were in the 10(-11) and 10(-12) (cerebellar granule neurons), 10(-12) and 10(-13) (astrocytes), and 10(-13) (microglia) molar range, respectively. Both RIPs resulted toxic for glial cells in culture by MTT test, killing 50% of microglia, the most sensitive cell type, at concentrations around 10(-14)M. Stenodactylin was highly neurotoxic in vivo, when injected intracerebrally, and was retrogradely transported through axons projecting to the injected region. Stereotaxic injection of 1.3 ng toxin into the left dorsal hippocampus resulted in loss of cholinergic neurons in the ipsilateral medial septal nucleus, where cell bodies of neurons providing cholinergic input to the hippocampus are located. The retrograde transport of RIPs along neurons allows to perform experiments of target-selective lesioning, and can be exploited also to perform specific experiments of immunolesioning of selected neuronal populations.

Cloning and expression of the B chain of volkensin, type 2 ribosome inactivating protein from Adenia volkensii harms: co-folding with the A chain for heterodimer reconstitution.

Type 2 ribosome inactivating proteins (RIPs) include some potent plant toxins, among which ricin from Ricinus communis and abrin from Abrus precatorius seeds, have been known for more than a century. Two other type 2 RIPs belong to this class of proteins, both isolated from plants of the same family (Passifloraceae), modeccin and volkensin, from Adenia digitata and Adenia volkensii roots, respectively. Volkensin is probably the most potent plant toxin known, with an LD50 for rats of 50-60 ng/kg. Here we report the cloning, expression and renaturation of recombinant volkensin B chain. Furthermore, starting from separately expressed A and B chains, a co-association procedure was set-up, leading to in vitro heterodimeric volkensin reconstitution. The recombinant heterodimer was characterized by N-terminal sequence analysis and its hemagglutinating activity assessed. In parallel, we have explored the carbohydrate-binding properties of native volkensin with the aim to correlate toxin-specific properties (i.e., axonal transport along neurons) to lectin's sugar-binding preferences.

Sequence determination of lychnin, a type 1 ribosome-inactivating protein from Lychnis chalcedonica seeds.

The complete amino acid sequence of lychnin, a type 1 ribosome-inactivating protein (RIP) isolated from Lychnis chalcedonica seeds, has been determined by automated Edman degradation and ESI-QTOF mass spectrometry. Lychnin consists of 234 amino acid residues with a molecular mass of 26 131.14 Da. All amino acid residues involved in the formation of the RIP active site (Tyr69, Tyr119, Glu170, Arg173 and Trp203) are fully conserved. Furthermore, a fast MALDI-TOF experiment showed that two out of three cysteinyl residues (Cys32 and Cys115) form a disulfide bridge, while Cys214 is in the thiol form, which makes it suitable for linking carrier molecules to generate immunotoxins and other conjugates.

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay.

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata.

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 IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 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.

Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae).

The caudices of 10 Adenia species contain galactose-binding lectins that were purified by affinity chromatography. All lectins but three agglutinate human erythrocytes. Six lectins consist of two unequal chains, which can be separated by reduction, and inhibit protein synthesis both by a rabbit reticulocyte lysate and by HeLa and Raji cells. The lectins from A. goetzii, A. lanceolata and A. stenodactyla had the highest cytotoxicity, inhibiting cell protein synthesis with IC50s (concentration inhibiting by 50%) below 0.1 ng/ml, and deadenylate DNA, thus being type 2 ribosome-inactivating proteins.

Lesions caused by ricin applied to rabbit eyes.

PURPOSE: Ricin, a highly potent toxin from castor beans, is a potential biological weapon that could be dispersed in the air as dust or aerosol. In these forms, ricin, besides being inhaled, could reach unprotected eyes. The present research was performed to ascertain the lesions that the toxin causes when applied to rabbit eyes. METHODS: Ricin was applied to rabbit eyes in solution, in quantities ranging from 1 to 100 mug. Animals were observed until death, when eyes and internal organs were removed and fixed. Sections were stained and examined microscopically. RESULTS: Ricin caused inflammation of the eyes and adnexa, visible both macroscopically and histologically. The damage was greatly reduced by rinsing the eyes with 10% lactose, provided the rinsing was done almost immediately after application of the toxin. Rinsing with phosphate-buffered saline (PBS) had no effect. With the highest dosage, congestion of internal organs was also apparent. CONCLUSIONS: Application of ricin to eyes causes local damage, mainly of the inflammatory type. The ineffectiveness of rapid rinsing with PBS and the partial efficacy of rapid rinsing with lactose indicate that the toxin quickly binds to and is taken up by cells. The lesions of internal organs show that ricin applied to the eyes can be absorbed, pass into the circulation, and, at least at some dosages, damage internal organs.

The 1.4 anstroms structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Shiga toxin 1 and ricin inhibit the repair of H2O2-induced DNA single strand breaks in cultured mammalian cells.

A growing body of evidence suggests that ribosome-inactivating proteins (RIPs) remove adenine moieties not only from rRNA, but also from DNA--an effect leading to DNA damage in cultured cells. We herein report that two distinct RIPs of bacterial (shiga toxin 1, Stx1) and plant (ricin) origin, inhibit the repair of the DNA lesions generated by hydrogen peroxide in cultured human cells. This effect is unrelated either to inhibition of protein synthesis or to depletion of cellular antioxidant defenses and is likely to derive from direct interactions with cellular DNA repair machinery. Therefore, the genotoxicity of these toxins on mammalian cells seems to be a complex phenomenon resulting from the balance between direct (DNA damaging activity), indirect (DNA repair inhibition) effects and the eventual presence of other DNA damaging species. In particular, with regard to Stx1, it could be hypothesized that Stx-producing bacteria increase the risk of transformation of surrounding, inflamed tissues in the course of human infections.

Ribosome-inactivating proteins.

The main results of the research performed in the last 30 years on ribosome-inactivating proteins (RIPs) are reviewed, with emphasis on the new, controversial and uncertain aspects. The nature, distribution, mechanism of action and properties of these proteins are briefly reported, together with their possible applications. A pattern appears of a still largely unexplored subject, whose role in nature is probably important, and not limited to the biology of plants, since RIPs have been found also in other organisms.

Description, distribution, activity and phylogenetic relationship of ribosome-inactivating proteins in plants, fungi and bacteria.

Ribosome-Inactivating Proteins (RIPs) are enzymes that trigger the catalytic inactivation of ribosomes and other substrates. They are present in a large number of plants and have been found also in fungi, algae and bacteria. RIPs are currently classified as type 1, those formed by a single polypeptide chain with the enzymatic activity, and type 2, those formed by 2 types of chains, i.e. A chains equivalent to a type 1 RIPs and B chains with lectin activity. Type 2 RIPs usually contain the formulae A-B, (A-B)2 and less frequent (A-B)4 and polymeric forms of type 2 RIPs lectins. RIPs are broadly distributed in plants, and are present also in fungi, bacteria, at least in one alga; recently RIP-type activity has been described in mammalian tissues. The highest number of RIPs has been found in Caryophyllaceae, Sambucaceae, Cucurbitaceae, Euphorbiaceae, Phytolaccaceae and Poaceae. However there are no systematic screening studies to allow generalisations about occurrence. The most known activity of RIPs is the translational inhibitory activity, which seems a consequence of a N-glycosidase on the 28 S rRNA of the eukaryotic ribosome that triggers the split of the A(4324) (or an equivalent base in other ribosomes), which is key for translation. This activity seems to be part of a general adenine polynucleotide glycosylase able to act on several substrates other than ribosomes, such as tRNA, mRNA, viral RNA and DNA. Other enzymatic activities found in RIPs are lipase, chitinase and superoxide dismutase. RIPs are phylogenetically related. In general RIPs from close families share good amino acid homologies. Type 1 RIPs and the A chains of type 2 RIPs from Magnoliopsida (dicotyledons) are closely related. RIPs from Liliopsida (monocotyledons) are at the same time closely related and distant from Magnoliopsida. Concerning the biological roles played by RIPs there are several hypotheses, but the current belief is that they could play significant roles in the antipathogenic (viruses and fungi), stress and senescence responses. In addition, roles as antifeedant and storage proteins have been also proposed. Future research will approach the potential biological roles played by RIPs and their use as toxic effectors in the construction of immunotoxins and conjugates for target therapy.

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

Volkensin from Adenia volkensii Harms (kilyambiti plant), a type 2 ribosome-inactivating protein.

Volkensin, a type 2 ribosome-inactivating protein from the roots of Adenia volkensii Harms (kilyambiti plant) was characterized both at the protein and nucleotide level by direct amino acid sequencing and cloning of the gene encoding the protein. Gene sequence analysis revealed that volkensin is encoded by a 1569-bp ORF (523 amino acid residues) without introns, with an internal linker sequence of 45 bp. Differences in residues present at several sequence positions (reproduced after repeated protein sequence analyses), with respect to the gene sequence, suggest several isoforms for the volkensin A-chain. Based on the crystallographic coordinates of ricin, which shares a high sequence identity with volkensin, a molecular model of volkensin was obtained. The 3D model suggests that the amino acid residues of the active site of the ricin A-chain are conserved at identical spatial positions, including Ser203, a novel amino acid residue found to be conserved in all known ribosome-inactivating proteins. The sugar binding site 1 of the ricin B-chain is also conserved in the volkensin B-chain, whilst in binding site 2, His246 replaces Tyr248. Native volkensin contains two free cysteinyl residues out of 14 derived from the gene sequence, thus suggesting a further disulphide bridge in the B chain, in addition to the inter- and intrachain disulphide bond pattern common to other type 2 ribosome-inactivating proteins.

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

Twenty years of hunting for proteins with Carlo Alfonso Rossi.

The research accomplished in more thanr twenty years of collaboration with Carlo Alfonso Rossi is reviewed. Several lectins and toxic and non-toxic ribosome-inactivating proteins were identified, purified and characterized. The general properties of the proteins are described.

Crystallization and preliminary X-ray diffraction analysis of two ribosome-inactivating proteins: lychnin and dianthin 30.

Lychnin from the seeds of Lychnis chalcedonica and dianthin 30 from the leaves of Dianthus caryophyllus belong to the type 1 ribosome-inactivating proteins (RIPs). They have been crystallized by the vapour-diffusion method and the crystals diffracted to 1.7 and 1.3 A, respectively, using a synchrotron source. Lychnin and dianthin 30 crystals both belong to space group P2(1) with one protein chain in the asymmetric unit. The structure of dianthin 30 has been solved by molecular replacement using the coordinates of saporin-S6 as a model. The structure determination of lychnin, the sequence of which is not yet available, is in progress using the coordinates of other RIPs as models for molecular replacement.

Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts.

It has been known that ribosome-inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non-ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin-L2, saporin-S6, gelonin and momordin depurinate also poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase (auto modified enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of fibroblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system.