J. Miguel Ferreras

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.

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra : comparison with ricin

Abstract Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome-inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 °C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.

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.

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.

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.

Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally related new homodimeric lectin in fruits of Sambucus ebulus L.

Abstract. Mature leaves of dwarf elder (Sambucus ebulus L.) contain the non-toxic type 2 ribosome-inactivating protein ebulin 1 (Girbés et al., 1993b, J. Biol. Chem. 268: 18195–18199). We have now found that the green fruits of dwarf elder contain both free and polymerized forms of ebulin (ebulin f) and a new homo-dimeric D-galactose-binding lectin (SELfd). Polymerized material containing ebulin and lectin is composed of aggregates of variable relative molecular mass, some of them being close to 250 000. These aggregate forms are maintained in part by reducible disulphide bridges and reconstitute from reductant-free dialyzed material previously reduced with 2-mercaptoethanol. Direct incubation of free ebulin f with the free SELfd did not lead to polymerization, thus indicating that polymerization triggers some kind of substantial and perhaps catalyzed change in the structure of these proteins. Ebulin-containing polymerized material reacts with anti-ebulin f antibodies. Our results indicate that ebulin f is a fruit-form of ebulin 1. In contrast to green fruits, mature fruits lack both polymerized material and ebulin f, thus indicating some kind of reserve role for them in green fruits. Polymerization of ebulin and the dimeric lectin may represent a novel means of storing the non-toxic type 2 ribosome-inactivating proteins and lectins found in highly metabolic tissues, such as green fruits.

Cytotoxicity of an ebulin l-anti-human CD105 immunotoxin on mouse fibroblasts (L929) and rat myoblasts (L6E9) cells expressing human CD105.

Tumour growth is characterised by the formation of a fine vessel network or neovasculature which nourishes tumour cells. Two kinds of novel anti-angiogenic therapies are based on the prevention of vessels growth and on the destruction of those vessels already formed. We report here on the design and construction of a novel immunotoxin formed with the non-toxic type II ribosome-inactivating protein ebulin l and the mouse anti-human CD105 monoclonal antibody 44G4. The 44G4-ebulin immunotoxin was formed by covalent linking of both proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and was purified by chromatography on Superdex 200 HiLoad. The analysis of the anti-ribosomal effects in a cell-free translation system indicated that conjugation does not affect the activity of ebulin l. The immunotoxin displays cytotoxicity with nanomolar IC50 values on human CD105+ cells like the mouse fibroblasts L929 cells transfected with the short form of human CD105 and the rat myoblasts L6E9 transfected with the long form of human CD105. In contrast, cells lacking human CD105 were 2-2.5 logs less sensitive to the immunotoxin. Free ebulin displays IC50 values in the range 10(-6) M. Since CD105 is being considered as a potential target for the anti-vascular therapy of tumours, the present immunotoxin could be a promising tool for the anticancer therapy, especially due to the very low in vivo toxicity of ebulin l as compared ricin and other toxins used for immunotoxins.

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.