High sensitivity of cultured human trophoblasts to ribosome-inactivating proteins.

Many plant proteins possessing abortifacient activities were identified as ribosome-inactivating proteins (RIPs). The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. Protein synthesis of human trophoblasts and BeWo cells was lowered by RIPs more than that of other cells. The trophoblastic receptors for estradiol were not affected by treatment of the cells with momordin. The binding and uptake of saporin 6 and momordin by BeWo and HeLa cells were not correlated to cell toxicity.     

Effect of ribosome-inactivating proteins on ribosomes from Tetrahymena pyriformis and Acanthamoeba castellanii

The effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by Tetrahymena pyriformis and Acanthamoeba castellanii ribosomes has been studied. The reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivating proteins type 1 (dianthin 32, saporin 6 and bryodin) and by two toxins (abrin and volkensin).     

Dianthins, ribosome-damaging proteins with anti-viral properties from Dianthus caryophyllus L. (carnation).

1. Dianthin 30 and dianthin 32, two proteins isolated from the leaves of Diathus caryophyllus (carnation), were purified to homogeneity by chromatography on CM-cellulose. 2. The mol.wt. of dianthin 30 is 29 500 and that of dianthin 32 is 31 700. Both dianthins are glycoproteins containing mannose. 3. Dianthins inhibit protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 9.15 ng/ml (dianthin 30) and 3.6 ng/ml (dianthin 32). They act by damaging ribosomes in a less-than-equimolar ratio. Protein synthesis by intact cells is partially inhibited by dianthins at a concentration of 100 microgram/ml. 4. Dianthins mixed with tobacco-mosaic virus strongly decrease the number of local lesions on leaves of Nicotiana glutinosa.     

Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Purification and characterisation of gelonin from seeds of Gelonium multiflorum

Gelonin, a ribosome-inactivating protein has been isolated from the seeds of Gelonium multifluorum of Euphorbiaceae family by two methods and the results are compared. In method-I conventional aqueous extraction, cation-exchange and gel-filtration chromatography has been used. In method-II S-Sepharose fast flow gel has been used to purify the proteins from the seed extract, followed by ammonium sulfate fractionation, cation-exchange and gel-filtration chromatography. Extensive physico-chemical and immunological characterizations show that molecular weight of gelonin as determined by gel-filtration chromatography and SDS-PAGE is approximately 30 kDa. The non-proteinous material which binds to CMC-gel in association with gelonin in method-I is substantially removed when gelonin is purified by method-II. Cation exchange, G-100 chromatography, RP-HPLC and SDS-PAGE show that method-II yields 50% more purified gelonin when compared to the yield by method-I. The immunoreactivity of gelonin obtained by methods I and II vary from 22-26% and 50-66% respectively and the ribosome-inactivating property vary from 46-56% and 70-87% respectively.     

Anti-CD30 immunotoxins with native and recombinant dianthin 30

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein fromDianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30⁺ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus). L428 and L540 (both from Hodgkin's lymphoma). IC₅₀ values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30^– cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC₅₀ higher than nM).     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Cofactor requirement of ribosome-inactivating proteins from plants

A large number of type 1 ribosome-inactivating proteins (RIPs)from plants (families of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae,Phytolaccaceae, and Poaceae) were examined for their requirementfor ATP and supernatant factors for full activity. A markedrequirement was observed with agrostin among Caryophyllaceae,gelonin among Euphorbiaceae, and with both barley RIP and tritin-Samong Poaceae. The distribution of cofactor requirement in Phytolaccaceaediscriminates leaf forms (cofactor-independent) from seed androot forms (cofactor-dependent). The results are discussed onthe basis of the present knowledge on the tissue localizationof RIPs and on the sensitivity of ribosomes to conspecific RIPs. Key words: Cofactors, ribosome-inactivating proteins, RNA-N-glycosidase, up-regulation     

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.     

Cytoxicity of erythrocyte ghosts loaded with ribosome-inactivating proteins following fusion with CHO cells

The ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, pokeweed antiviral protein, and one from Saponaria officinalis were enclosed in human erythrocyte ghosts. The proteins once trapped in ghosts and fused with CHO cells inhibited colony formation at concentrations of approximately 1 ng/ml (3 X 10(-11) M), whereas the free proteins only had an effect at concentrations of greater than 1 microgram/ml.     

Nucleotide sequence of cDNA coding for dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus.

Rabbit antibodies raised against dianthin 30, a ribosome inactivating protein from carnation (Dianthus caryophyllus) leaves, were used to identify a full length dianthin precursor cDNA clone from a lambda gt11 expression library. N-terminal amino acid sequencing of purified dianthin 30 and dianthin 32 confirmed that the clone encoded dianthin 30. The cDNA was 1153 basepairs in length and encoded a precursor protein of 293 amino acid residues. The first 23 N-terminal amino acids of the precursor represented the signal sequence. The protein contained a carboxy-terminal region which, by analogy with barley lectin, may contain a vacuolar targeting signal.     

单链核糖体失活蛋白的核糖核酸酶活性

以芹菜4.5SRNA为底物, 在pH5.0的条件下, 5种纯核糖体失活蛋白:天花粉蛋白、苦瓜子蛋白、肥皂草蛋白、丝瓜素毒蛋白和多花白树毒蛋白均显示出核糖核酸酶活性, 放射自显影图显示出它们对RNA分子中的各种碱基具有不同的敏感性.     

Role of positive charge of lysine residue on ribosome-inactivating property of gelonin

The report that gelonin cross-linked with monoclonal antibodies with the use of 2-iminothiolane (2-IT) exhibited higher cytotoxicity than the conjugates prepared with the use of N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) alone, has prompted us to investigate the effect of epsilon-NH2 group modification with 2-IT on the ribosome-inactivating property (RIP) of gelonin. The purified gelonin was modified with 2-IT at a different molar ratio and their effects on immunoreactivity and ribosome-inactivating property were compared with those of N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (long chain-SPDP) and SPDP modified gelonin derivatives. Modification of single amino group with 2-IT results in about 25-50% inhibition of immunoreactivity and 60-70% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampers both immunoreactivity and protein synthesis inhibition property of gelonin. Both the long chain-SPDP with SPDP modifications showed more pronounced effects on immunoreactivity and RIP activity as compared to the similar ratio of 2-IT modification(s). It may, therefore, be concluded that the positive charge plays an important role in the immunological as well as the protein synthesis inhibitory effect of gelonin.     

In vitro production of dianthin from crown gall lines of carnation (Dianthus caryophyllus L.)

Tumorous crown gall lines of carnation (Dianthus caryophyllus L.) were obtained by transforming leaf explants by the co-cultivation procedure; transformed lines were checked for their ability to produce in vitro the type 1 ribosome-inactivating proteins dianthins. Crude extracts from cultured callus were able to inhibit protein synthesis in a cell-free system based on rabbit reticulocyte lysate. A protein with an apparent molecular mass comparable to that of dianthin 30 from leaves was identified from its chromatographic behaviour and by Western analysis. Dianthin from callus showed a specific activity comparable to that reported for the leaf isoform. The presence and accumulation in the tumorous line of a 37 kDa protein with no ribosome-inactivating activity and strong antigenic reactivity to dianthins is discussed. Received: 18 July 1997 / Revision received: 10 January 1998 / Accepted: 5 February 1998     

Uptake and intracellular fate of gelonin,a ribosome-inactivating protein,in rat liver

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property

Gelonin, purified from the seeds of Gelonium multiflorum, using cation-exchange and gel-filtration chromatography was characterised for its purity, homogeneity and molecular weight by reverse-phase HPLC (RP-HPLC) and SDS-PAGE analysis. The HPLC purified gelonin was used for entrapment studies in the liposomes. Liposomes were prepared by reverse phase evaporation (REV) technique using three different types of lipid composition in the same molar ratio. The method resulted in 75–80% entrapment efficiency of gelonin in the liposomes. Entrapped and unentrapped gelonin was characterized for physico-chemical, immunochemical and biological properties. The immunoreactivity of entrapped gelonin was fully preserved but the ribosome-inactivating property was slightly inhibited. The method involved mild conditions, highly reproducible and the liposomes produced appeared to be stable for several months. It has important implications in the development of cell type specific cytotoxic agents where a chemical cross-linking is involved which significantly inhibits both immunoreactivity and ribosome-inactivating ability of the toxin.     

Antibody-complement-mediated cytotoxicity is enhanced by ribosome-inactivating proteins

The antibody-mediated cytotoxicity of complement can be increased by the ribosome-inactivating proteins gelonin and PAP-S. Treatment of human lymphoid cells that express CALLA with an anti-CALLA monoclonal antibody, J5, and then with rabbit complement, leaves about 6% of the cells alive. The same treatment in the presence of a sublethal concentration of gelonin or PAP-S leaves only about 0.02 and 0.3% of the cells alive respectively. This synergistic effect has potential implications for the in vitro elimination of malignant cells or of immunocompetent cells from bone marrow before its transplantation.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.