Tomás Girbés

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.

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.

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l.

Ebulin l is a type‐II ribosome‐inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type‐II RIP, ebulin is a disulfide‐linked heterodimer composed of a toxic A chain and a galactoside‐specific lectin B chain. A normal level of ribosome‐inactivating N‐glycosidase activity, characteristic of the A chain of type‐II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type‐II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type‐II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A‐chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside‐binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2γ subdomain of the lectin B chain primarily causes the reduced affinity. Proteins 2001;43:319–326.

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.

Use of Ribosome-Inactivating Proteins from Sambucus for the Construction of Immunotoxins and Conjugates for Cancer Therapy

The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively.

Targeting cancer cells with transferrin conjugates containing the non-toxic type 2 ribosome-inactivating proteins nigrin b or ebulin l

Nigrin b and ebulin l are type 2 ribosome-inactivating proteins (RIPs) with 10(4) times less cellular and in vivo toxicity than ricin that are currently being considered for the construction of anti-cancer conjugates. Here we provide evidence that both RIPs can be used for the construction of conjugates directed to a target such as the transferrin receptor (TfR), which is over-expressed in cancer cells. Nigrin b- and ebulin l-transferrin conjugates were constructed with no substantial reduction in the translational inhibitory molecular activity of either RIPs. Conjugation with transferrin decreased the IC(50) of the proteins from 3 x 10(-7)M (nigrin b) and 1.5 x 10(-8)M (ebulin l) to 3.5 x 10(-10)M in HeLa cells. Thus, both conjugates could be considered as useful tools for targeting TfR-over-expressing cancer cells.

Interaction of volkensin with HeLa cells: binding, uptake, intracellular localization, degradation and exocytosis

AbstractAmong two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.

Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins. Proposal of an extended classification of plant ribosome-inactivating proteins (rRNA N-glycosidases).

The four chain agglutinins from Abrus precatorius, Viscum album and Ricinus communis promote depurination of the 28 S rRNA from rabbit reticulocyte ribosomes characteristic of the common ribosome‐inactivating proteins (RIPs). These agglutinins inhibited mammalian protein synthesis at nanomolar concentrations but they do not affect plant protein synthesis under the same conditions. Therefore, they should also be considered as true RIPs but of a new class, the four‐chain RIPs. An extended classification of RIPs is presented based on the former one from Stirpe et al. [Bio/technology 10 (1992) 405‐412].

Ebulitins: a new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1.

A new family of single chain (type 1) ribosome‐inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non‐toxic two chain (type 2) RIP named ebulin 1 in its leaves. Ebulitins are basic proteins of M r 32,000, 29,000 and 29,000 for ebulitins α, β and γ, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.

Ribosomal translocation promoted by guanylylimido diphosphate and guanylyl-methylene diphosphonate.

It has been known for a number of years that ribosomal translocation requires the participation of elongation factor G (EF-G) and the hydrolysis of GTP ([1 ], review). However, the significance of this hydrolytic reaction, a fundamental question in protein biosynthesis, has not yet been satisfactorily explained. It has been traditionally assumed that the energy liberated in the reaction assists the rearrangements of tRNA, mRNA and ribosomal subunits that result in the movement of mRNA and, probably, ofpeptidyltRNA [1 ]. Very recently, however, an alternative view has been proposed [2,3] : the binding of EF-G plus GTP to the ribosome promotes these rearrangements, and the hydrolysis of GTP merely allows completion of the translocation process by inducing the release of EF-G from the ribosome. This second alternative is supported by the finding that the nonhydrolyzable analog of GTP guanylyl-methylene diphosphonate (Gpp(CH2)p) can, under some conditions, replace GTP in translocation [2,3]. However, since earlier research showed Gpp (CH2)p to be incapable of promoting translocation [4 -6 ] , we have reexamined its effect on this process using ribosomes complexed with N-acetyl-Phe-tRNA and poly (U), and endogenous E. coli polysomes. Moreover, to study further the role of GTP hydrolysis in translocation we have investigated the effect of a different nonhydrolyzable analog [7] : guanylylimido diphosphate