Specific dose-dependent damage of Lieberkühn crypts promoted by large doses of type 2 ribosome-inactivating protein nigrin b intravenous injection to mice.

Nigrin b is a non-toxic type 2 ribosome-inactivating protein as active as ricin at ribosomal level but 10(5) and 5 x 10(3) times less toxic for animal cell cultures and mice, respectively, than ricin. The purpose of the present study was to analyze the effects of intravenous injection of large amounts of nigrin b to the mouse. Injection through the tail vein of 16 mg/kg body weight killed all mice studied before 2 days. Analysis of several major tissues by light microscopy did not reveal gross nigrin b-promoted changes, except in the intestines which appeared highly damaged. As a consequence of the injury, the villi and crypt structures of the small intestine disappeared, leading to profuse bleeding and death. In contrast, intravenous injection of 5 mg/kg body weight was not lethal to mice but did trigger reversible toxic effects. In both cases, lethal and sub-lethal doses, the target of nigrin b appeared to be the highly proliferating stem cells of the intestinal crypts, which had undergone apoptotic changes. In contrast to nigrin b, the injection of 3 mug/kg of ricin kills all mice in 5 days but does not trigger apoptosis in the crypts. Therefore, the effect seen with sub-lethal nigrin b concentrations seems to be specific. Nigrin b killed COLO 320 human colon adenocarcinoma cells with an IC(50) of 3.1 x 10(-8) M and the effect was parallel to the extent of DNA fragmentation of these cells. Accordingly, despite the low general toxicity exerted by nigrin b as compared with ricin, intravenous injection of large amounts of nigrin b is able to kill mouse intestinal stem cells without threatening the lives of the animals, thereby opening a door for its use for the targeting of intestinal stem cells.

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

Among 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 x 10(5)/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.

Non-toxic type 2 ribosome-inactivating proteins (RIPs) from Sambucus: occurrence, cellular and molecular activities and potential uses.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.

Evidence for distinct cellular internalization pathways of ricin and nigrin b.

Nigrin b and ricin are type 2 (two chain) ribosome-inactivating proteins that exhibited nearly the same strong inhibitory activity on cell-free protein synthesis. Incubation of HeLa cells for 6 hr with ricin at 37 degrees C promoted protein synthesis inhibition with an IC50 of 0.2 ng/ml. Incubation of the cells for 6 hr at 18 degrees C abolished completely the inhibition. Incubation of HeLa cells with nigrin b for 6 hr at 37 degrees C was nearly 10(5) times less inhibitory than ricin. In contrast to the effects observed with ricin, incubation of HeLa cells with nigrin b at 18 degrees C slightly increased the inhibitory action on protein synthesis as compared with incubation at 37 degrees C. These results strongly support the hypothesis that the internalization of ricin and nigrin b could involve different receptors and therefore they could follow different intracellular pathways.

Effects of chronic administration of either ethanol or pentanol on rat duodenum morphology.

The morphology of the rat duodenum after chronic treatment with 15% (v/v) ethanol and 4% (v/v) pentanol was studied. Male Wistar rats of experimental groups were given ethanol and pentanol for 15 weeks with food and fluid freely available. Ethanol-15% and 4% pentanol-fed rats showed a significantly reduced fluid and food intake as compared with control rats. The study of the mucosa indicated that the number of chronic inflammatory infiltrating (mononuclear cells) and goblet cells was higher in the groups of the ethanol- and pentanol-fed rats than in the control group. There was an increase in the thickness of the brush border in pentanol-fed rats. Intervillus adhesion was concurrently observed in the pentanol-fed rats but not in the control or ethanol-fed rats. After ethanol feeding many of the villi developed blebs at the apex of the villus or laterally on its upper half. These blebs generally remained intact. In contrast, after pentanol feeding no bleb formation was appreciated. The intake of ethanol and other short chain alcohols present in alcoholic beverages leads to mainfold disturbances on the rat duodenum. These findings suggest that the chronic ingestion of pentanol seems to promote cellular changes but less important than those observed after chronic ethanol ingestion.

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.

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 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.

Differences in cytotoxicity of native and engineered RIPs can be used to assess their ability to reach the cytoplasm.

Ricin is a heterodimeric cytotoxin composed of RTB, a galactose binding lectin, and RTA, an enzymatic N-glycosidase. The toxin is endocytosed, and after intracellular routing, RTA is translocated to the cytoplasm where it inactivates ribosomes resulting in a loss of host cell protein synthesis and cell death. We show for the first time that the cytotoxicity against cultured T cells by several RTA mutants is directly proportional to the enzyme activity of RTA, suggesting this is a reliable system to measure translocation effects. Large discrepancies between cytotoxicity and enzyme action for a given pair of toxins are therefore attributable to differences in cell binding, uptake, or membrane translocation. Fluid phase uptake and cytotoxicity of isolated RTA are essentially identical to that of the single chain toxin PAP. This important finding suggests that RTA, and the A chain of class 2 RIPs in general, has not evolved special translocation signals to complement the increased target cell binding facilitated by RTB. Experiments with the lectin RCA and with ebulin suggest those toxins have diminished cytotoxicity probably mediated by comparative deficiencies in B chain binding. Addition of a KDEL sequence to RTA increases fluid phase uptake, consistent with the notion that transport to the ER is important for cytotoxicity. Fusion of MBP or GST to the amino terminus of RTA has little effect on enzyme action or cytotoxicity. This result is not altered by protease inhibitors, suggesting the fusion proteins are probably not cleaved prior to translocation of the toxic A chain and implying that the toxins can carry large passenger proteins into the cytoplasm, an observation with interesting potential for analytical and therapeutic chemistry.

Ribosome-inactivating proteins (RIPs) of wild Oregon cucumber (Marah oreganus).

Two type 1 RIPs, designated as MOR-I and MOR-II, have been isolated from Marah oreganus (manroot) seed extract. They are similar but not identical to trichosanthin, a type 1 RIP in the same family. MOR-I and MOR-II are monomeric proteins with molecular weights of 27989.0 and 27632.8 respectively and have pI values greater than 8.8. MOR-I and MOR-II inhibit cell-free protein synthesis with IC50s of 0.063 and 0.071 nM, respectively, and are relatively stable with respect to temperature and pH variations. They share a conserved N-terminal amino acid sequence (D-SF-LS) and cross-react with goat anti-trichosanthin polyclonal serum.

Constitutive and inducible type 1 ribosome-inactivating proteins (RIPs) in elderberry (Sambucus nigra L.).

Two novel highly basic type 1 (single chain) ribosome-inactivating proteins (RIPs) with N-glycosidase activity have been found in elderberries (the fruits of Sambucus nigra L.). Mass spectrometry of these RIPs, which we named nigritins f1 and f2, gave Mr values of 24095 and 23 565, respectively. Both proteins strongly inhibited protein synthesis in rabbit reticulocyte lysates but were inactive against plant ribosomes. Both nigritins have a similar topological activity on pBlueScript SK+ DNA as that displayed by dianthin 30. Nigritin f1 is a constitutive RIP since it is present in both green and mature intact elderberries at nearly the same proportion with respect to total fruit protein. By contrast, nigritin f2 is inducible and only appeared in mature intact elderberries. Elderberries also contain two isoforms of a basic nigrin equivalent to the recently found basic nigrin b in elder bark (De Benito et al., FEBS Letters 413 (1997) 85-91). Our results indicate that probably not all plant RIPs exert the same biological function and that this may be determined by the physiological state of the tissue.

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.

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 homodimeric 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.

Analysis of human ocular mucus: effects of neuraminidase and chitinase enzymes.

PURPOSE: Our goal was to establish the characteristic migration pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of high molecular weight mucins from human ocular mucus and the effects of treatment with exo- and endoglycosidases. METHODS: Chromatography by gel filtration with Sepharose CL-4B was performed on samples collected from normal subjects. Human ocular mucins from the high molecular weight fraction were digested with exoglycosidases (neuraminidase, N-acetyl-beta-D-glucosaminidase, beta-D-glucosidase) and endoglycosidases (chitinase, lysozyme); and the resulting products were analyzed by electrophoresis. Carbohydrate identification was performed using lectin probes. RESULTS: The migration of the ocular mucins on SDS-PAGE stopped after treatment with neuraminidase, which removes the terminal negatively charged sialic acid residues from mucin. Chitinase (beta(1-4)N-acetylglucosaminidase) treatment increased the electrophoretic migration of mucins. Staining with wheat germ agglutinin and Maackia amurensis agglutinin lectins showed that these mucins contain beta(1-4)NAcGlc and SAa(2-3)Gal linkages. CONCLUSIONS: These studies demonstrate that the mobility of human ocular mucins on SDS-PAGE is determined by their intrinsic total negative charge and is not dependent on SDS treatment. It is interesting to note that human ocular mucus contains chitinous material resistant to lacrimal lysozyme, which is accessible to chitinase, an enzyme now found to degrade human ocular mucins. These chitinous linkages could be in part responsible for the mucus resistance.

A novel type-1 ribosome-inactivating protein isolated from the supernatant of transformed suspension cultures of Trichosanthes kirilowii.

A type-1 ribosome-inactivating protein (RIP) designated TK-35 has been purified from the supernatant of suspension cultures of Agrobacterium rhizogenes-transformed stem sections of Trichosanthes kirilowii. The protein was purified from the supernatant by PerSeptive SH/M cation exchange and Sephadex G-75 S gel permeation chromatography. The protein occurs as a monomer, with a molecular weight of 35,117, and is glycosylated. A protein translation inhibition assay indicates that TK-35 has an IC50 value of 2.45 nM and is able to release the rRNA N-glycosidase diagnostic fragment from rabbit reticulocyte lysate. TK-35 is quite thermally stable. Analysis of its N-terminal sequence and two lys-C-protease-digested polypeptides (internal) amino acid sequence indicates that this protein is not homologous to trichosanthin and other type-1 RIPs in Cucurbitaceae family.

Isolation and partial characterization of a novel and uncommon two-chain 64-kDa ribosome-inactivating protein from the bark of elder (Sambucus nigra L.).

A novel, strongly basic, two-chain ribosome-inactivating protein (RIP) with an apparent Mr of 64000 by SDS-PAGE and 63469 by mass spectrometry analysis, that we have named basic nigrin b, has been found in the bark of elder (Sambucus nigra L.). The new protein does not agglutinate red blood cells, even at high concentrations and displays an unusually and extremely high activity towards animal ribosomes (IC50 of 18 pg/ml for translation by rabbit reticulocyte lysates). However, it is inactive against plant and HeLa cells protein synthesis. Our functional and structural data are consistent with a heterodimeric structure for basic nigrin b of the type A-B*, B* being a truncated lectin lacking functional binding domains equivalent to the B (lectin) chain of the type 2 RIP SNA I and nigrin b present also in elder bark.

Characterization of a new non-toxic two-chain ribosome-inactivating protein and a structurally-related lectin from rhizomes of dwarf elder (Sambucus ebulus L.).

A new N-glycosidase ribosome-inactivating protein (RIP) belonging to the novel family of the nontoxic type 2 RIPs from Sambucaceae has been isolated from rhizomes of dwarf elder (Sambucus ebulus L.) and named ebulin r. Dwarf elder rhizomes also contain a novel monomeric N-Ac-galactosamine-binding lectin that we named SEAII. Ebulin r and SEAII have two isoforms each one, which were readily resolved by ion exchange. Both isoforms of ebulin (ebulins r1 and r2) strongly inhibited protein synthesis in mammalian but not in plant ribosomes by promoting depurination of sensitive ribosomes. Ebulin r and SEAII have apparent molecular masses of 56 and 33.5 kDa, respectively. Ebulins r1 and r2 are composed of two dissimilar subunits (types A-B) of apparent molecular masses of 26 and 30 kDa by disulphide bridges. The rhizome SEAII and the lectins SNA II and SNA III from elder (Sambucus nigra L.) share good amino acid sequence homology. This rhizome ebulin-A chain is more sequence-related to RIP members of cucurbitaceae than to any other plant family. The rhizome ebulin B chain shares a large homology in amino acid sequence with ebulin 1-B chain and SEAII. Anti-ebulin 1 polyclonal antibodies raised in rabbits reacted better with ebulin r1 than with ebulin r2, thus suggesting that both RIP isoforms could have some differences.

Elderberry (Sambucus nigra) bark contains two structurally different Neu5Ac(alpha2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins.

A second NeuAc(alpha2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sambucus nigra) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(alpha2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate-binding specificity and ribosome-inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.

Isolation, cDNA cloning, biological properties, and carbohydrate binding specificity of sieboldin-b, a type II ribosome-inactivating protein from the bark of Japanese elderberry (Sambucus sieboldiana).

A type II ribosome-inactivating protein (RIP) was isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) and named sieboldin-b. Sieboldin-b is a heterodimeric protein consisting of 27- and 33-kDa subunits and showed strong ribosome-inactivating activity in vitro but did not show in vivo toxicity. The amino acid sequence of sieboldin-b deduced from the structure of the cDNA showed that both subunits of sieboldin-b are encoded on a single precursor polypeptide. Sieboldin-b has a structure homologous with the Neu5Ac(alpha 2-6)Gal/GalNAc-specific bark lectin from S. sieboldiana (SSA) and also typical type II RIPs such as ricin and abrin. Detailed analyses of carbohydrate binding properties of sieboldin-b revealed that sieboldin-b binds to Gal/GalNAc, similar to ricin/abrin, in spite of its highly homologous structure with SSA. The biological properties of these toxins/lectins are compared, and the possible explanation for such diversity is discussed.

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

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 degrees 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.

Primary structure of omega-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibition of protein synthesis in eukaryotic and prokaryotic cell-free systems.

A new sulfur-rich basic polypeptide, so called omega-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified omega-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of omega-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteolytic cleavage. Omega-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by electrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues, and has strong sequence identity (66%) with the sorghum SI alpha 1 alpha-amylase inhibitor. Omega-hordothionin, like gamma-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to gamma-hordothionin, omega-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. Gamma-hordothionin also inhibited the alpha-amylase activity from human saliva, while omega-hordothionin and the other different genetic variants of thionins, alpha-hordothionin and beta-hordothionin, failed to show any inhibitory effect.

Recent advances in the uses and applications of ribosome-inactivating proteins from plants.

Plant ribosome-inactivating proteins (RIPs) are inhibitors present in all parts of plants that irreversibly inactivate eukaryotic ribosomes, thus impairing protein synthesis. RIPs are enzymes with N-glycosidase activity on the large rRNA. Their powerful inhibitory activity has been made use of advantageously to construct conjugates with suitable carriers targeted to altered specific cells. RIPs may be used to inhibit replication of both animal and plant viruses. The introduction of genes coding for RIPs into the genome of plants leads to an increase in resistance towards fungal pathogens and viruses. RIPs are important tools for the treatment of cancer and AIDS and for the protection of crop production.