Fiorenzo Stirpe

Ribosome inactivating proteins from plants: present status and future prospects

Plant ribosome–inactivating proteins (RIPs) N–glycosidases which cleave the N–glycosidic bond of adenine in a specfic ribosomal RNA sequence. Most commonly RIPs are single–chain proteins (type 1 RIPs), but some (type 2 RIPs) possess a galactose–specific lectin domain that binds to cell surfaces. The latter RIPs are potent toxins, the best known of which is ricin. RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotixins or conjugates specifically toxic to a given type of cell.

Ribosome‐inactivating proteins up to date

Ribosome‐inactivating proteins (RIPs) from plants inactivate eukaryotic ribosomes, as far as studied by rendering their 60 S subunit unable to bind elongation factor 2. These proteins seem widely distributed and possibly ubiquitous in plants. They are either type 1, those consisting of a single polypeptide chain, or type 2 (ricin and related toxins), those consisting of two chains, one of which is a galactose‐binding lectin. The literature on RIPs from 1982 has been reviewed with respect to (i) the chemical and biological properties of RIPs, (ii) their use for the preparation of immunotoxins and (iii) new perspectives.

Ribosome-inactivating proteins: progress and problems

Abstract.Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies (‘immunotoxins’) or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.

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.

Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells

Ribosome‐inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline‐halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical ≤ 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA‐free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.—Brigotti, M., Alfieri, R., Sestili, P., Bonelli, M., Petronini, P. G., Guidarelli, A., Barbieri, L., Stirpe, F., Sperti, S. Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells. FASEB J. 16, 365–372 (2002)

Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae).

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endos diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Induction of apoptosis by ribosome‐inactivating proteins and related immunotoxins

Immunotoxins have been prepared with 3 ribosome‐inactivating proteins (RIPs), namely, momordin, pokeweed anti‐viral protein from seeds (PAP‐S) and saporin, linked to the Ber‐H2 monoclonal antibody directed against the CD30 antigen of human lymphocytes. Either the RIPs or the immunotoxins induced apoptosis in the CD30+ L540 cell line, as shown by the morphological aspects of the cells, by the DNA fragmentation visible at the electrophoresis, and by the formation of DNA breaks evidenced by 2 cytofluorometric techniques (propidium‐iodide staining and fluoresceine‐isothiocyanate conjugate dUTP incorporation). The AC50 (concentration causing apoptosis in 50% of the cells) is in the range 10‐8 to 10‐7 M in the case of RIPs, and 10‐11 to 10‐10 M in the case of the immunotoxins.

In vitro anti-tumour activity of anti-CD80 and anti-CD86 immunotoxins containing type 1 ribosome-inactivating proteins

Immunotoxins specific for the CD80 and CD86 antigens were prepared by linking three type 1 ribosome‐inactivating proteins (RIPs), namely bouganin, gelonin and saporin‐S6, to the monoclonal antibodies M24 (anti‐CD80) and 1G10 (anti‐CD86). These immunotoxins showed a specific cytotoxicity for the CD80/CD86‐expressing cell lines Raji and L428. The immunotoxins inhibited protein synthesis by target cells with IC50s (concentration causing 50% inhibition) ranging from 0·25 to 192 pmol/l as RIPs. The anti‐CD80 immunotoxins appeared 1–2 log more toxic for target cells than the anti‐CD86 ones. Immunotoxins containing saporin and bouganin induced apoptosis of target cells. The toxicity for bone marrow haemopoietic progenitors of these conjugates was also evaluated. Bouganin and related immunotoxins at concentrations up to 100 nmol/l did not significantly affect the recovery of committed progenitors or of more primitive cells. The saporin‐containing immunotoxins at concentrations ≥ 1 nmol/l showed some toxicity on colony‐forming unit cells (CFU‐C). The expression of the CD80 and CD86 molecules is prevalently restricted to antigen‐presenting cells and is also strong on Hodgkin and Reed–Sternberg cells in Hodgkins disease. Present results suggest that immunotoxins targeting type 1 ribosome‐inactivating proteins to these antigens could be considered and further studied for the therapy of Hodgkins disease or other CD80/CD86‐expressing tumours.

Ber-H2 (anti-CD30)-saporin immunotoxin : a new tool for the treatment of Hodgkin's disease and CD30+ lymphoma : in vitro evaluation

An immunotoxin containing an anti‐CD30 monoclonal antibody (Ber‐H2) and saporin, a ribosome‐inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 × 10–12 M to 5.10∼14 M, assaporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkins disease and CD304+ lymphomas.

Activities associated with the presence of ribosome‐inactivating proteins increase in senescent and stressed leaves

The ribosome‐inactivating proteins (RIPs) from Hura crepitans and Phytolacca americana release adenine from herring sperm DNA. Leaf extracts from these plants show the same enzymatic activities as the RIPs. The translation inhibitory activity and the activity on DNA are both increased in the leaves of both plants during senescence or when subjected to heat or osmotic stress. It is proposed that a physiological role of RIPs could be to intervene in the death of plant cells.