M. Angeles Rojo

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.

Isolation and partial characterization of nigrin b, a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (Mr 58 000) contains two subunits, A (Mr 26 000) and B (Mr 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.

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].

Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein nigrin b

The cytotoxicity of the type 2 ribosome-inactivating proteins (RIPs) ricin and nigrin b was determined in a variety of cancer cells. Nigrin b, considered to be a novel non-toxic type 2 RIP as compared with ricin, was approximately 10(4)-10(5) times less toxic than ricin in all cancer cells studied, with the exception of melanoma cells. Cancer cells displayed considerable heterogeneity in their sensitivity to ricin, melanoma cells being the least sensitive. Rabbit polyclonal anti-nigrin b antibodies did not cross-react with ricin as analyzed by enzyme-linked immunosorbent assays. The low non-specific toxicity of nigrin b as compared with that of ricin and the lack of immunological cross-reaction between anti-nigrin b antibodies and ricin supports the use of nigrin b in the construction of cytotoxic conjugates as an alternative to ricin when anti-ricin antibodies are produced during cancer therapy.

Cusativin, a new cytidine-specific ribonuclease accumulated in seeds of Cucumis sativus L.

Dry seeds of Cucumis sativus L. were found to contain a heat-sensitive endoribonuclease of a novel type which we have named cusativin. It was purified to apparent electrophoretic homogeneity by chromatography through S-Sepharose Fast Flow, Sephadex G-75, CM-Sepharose, Superdex 75-FPLC (fast protein liquid chromatography) and Mono S-FPLC. It is a single unglycosylated polypeptide chain with an apparent molecular mass (Mr) of 22900. Polyclonal anti-cusativin antibodies raised in rabbits only reacted with melonin, the translation inhibitor from Cucumis melo L. Functional, Western blot and enzyme-linked immunosorbent assay (ELISA) analyses indicated that cusativin is present in the coat and cotyledons of dry seeds, but not in embryonic axes. Cusativin is accumulated in maturing seeds. By contrast, after seed germination there is degradation of the cusativin present in cotyledons but not that present in the seed coat. The preference of cusativin for polynucleotide cleavage was poly(C)≫poly(A) acids, poly(U) and poly(G) being unaffected by cusativin. Under the denaturing conditions used for RNA sequencing, cusativin acted only on poly(C). Cusativin proved to be useful for RNA sequencing, in particular, complementing the data obtained with RNase CL3. Cusativin represents a new class of plant RNase and, as far as we are aware, is the first plant enzyme that shows cleavage specificity for cytidine under the denaturing conditions of RNA sequencing.

cDNA molecular cloning and seasonal acumulation of an ebulin l-related dimeric lectin of dwarf elder (Sambucus ebulus L.) leaves

SELld is a dimeric D-galactose and mucin-binding lectin (apparent Mr 68000) which coexists with the non-toxic type 2 ribosome-inactivating protein (RIP) ebulin l in dwarf elder (Sambucus ebulus L.) leaves. To ascertain a potential structural correlation with ebulin l molecular cloning of a cDNA coding for SELld was performed. SELld shared a 76% of identity with the ebulin l-B chain. Notably, it was found that SELld has Tyr present in the high affinity 2gamma sugar-binding domain of ricin which is absent in ebulin l-B chain and which seems responsible of the low cell and in vivo toxicities of ebulin l. The concentration of ebulin l in leaves decreased along the developmental stage of dwarf elder and almost disappeared in senescence while the content in SELld changed in the opposite way. Our results suggest that SELld and ebulin l play different biological roles in dwarf elder leaves.

Musarmins: three single-chain ribosome-inactivating protein isoforms from bulbs of Muscari armeniacum L. and Miller.

Three new ribosome-inactivating protein (RIP; EC 3.2.2.22) isoforms that we have named musarmins (MUs) 1, 2 and 3 have been isolated from the bulbs of Muscari armeniacum L. and Miller by ion-exchange chromatography and gel filtration. Analysis by electrophoresis revealed that they are single-chain proteins and mass spectrometry analysis afforded Mr values of 28,708, 30,003 and 27,626 for MUs 1, 2 and 3, respectively. Musarmins strongly inhibited protein synthesis carried out by mammalian ribosomes, with IC50 values in the 0.14-0.24nM range but not that carried out by plant cell-free systems or HeLa cells. MUs promote the single depurination of rabbit reticulocyte 28S rRNA. cDNA cloning of genes coding for musarmins revealed that they contain open reading frames of 298, 294 and 295 aminoacids for MU1, MU2 and MU3, respectively. Mature MU1, MU2 and MU3 contain 277, 273 and 273 aminoacids, respectively suggesting post-translational C-terminal processing. An untranslated mRNA coding for an ORF very similar to that of MU3 was detected in leaves. Each of the four MU genes contains an intron. In contrast to other RIPs, MUs are present only in bulbs and are not induced in leaves either by senescence, or by treatment of leaves with H2O2 or salicylic acid, or by growth in darkness. Therefore, these proteins could play a non-vital role in plants; for instance, as anti-pathogens and protective agents only in some stages of the plant life cycle (237).

Enzymic activity of melonin, a translational inhibitor present in dry seeds of Cucumis melo L.

Abstract Melonin, a protein from the dry seeds of Cucumis melo L. that inhibits protein synthesis in cell-free systems, is a ribonuclease (RNase). Melonin degrades poly(C) but not poly(U) at 55°C and in the presence of 7 M urea, melonin degrades both poly(C) and poly(U). Reflecting this, melonin degrades bonds containing cytidine and, to a lesser extent, uridine in non-synthetic RNA substrates. The NH 2 -terminal amino acid of purified melonin was blocked, but the amino acid sequences of some tryptic peptides indicate that melonin has sequence similarity to cusativin, an RNase isolated from Cucumis sativus L., and to the RNases from Petunia inflata and Nicotiana alata that determine gametophytic self-incompatibility.

Isolation and Characterization of a new Dgalactose- Binding Lectin from Sambucus Racemosa L.

A new acidic lectin from red elder (Sambucus racemosa L.) bark has been isolated by affinity chromatography and gel filtration. Noteworthy, and in contrast to other Sambucus species, red elder bark lacks acidic non-toxic type 2 ribosome-inactivating proteins but has basic ribosome-inactivating protein activities. The new lectin (SRLbm) shows specificity for N-Ac-Galactosamine/D-Galactose and has an apparent Mr of 30,000. The N-terminal amino acid sequence displays a close homology with other lectins and B chains of non-toxic type 2 ribosome-inactivating proteins nigrins and ebulins present in other Sambucus species. SRLbm triggers red blood cell agglutination in the range 4-12 micro g/ml.

Fusidic acid-dependent wheat germ ribosomal complexes require unphosphorylated elongation factor 2

Abstract The effects of the antibiotic fusidic acid on translation and the phosphorylation pattern in a wheat Triticum aestivum germ cell-free system were investigated. The antibiotic reduces phosphorylation of elongation factor 2 (EF-2) at concentrations at which it efficiently inhibits translation. The interaction of EF-2 with the ribosome occurs in its non-phosphorylated state, as proved by the formation of the fusidic acid-dependent ribosomal complexes.