Nucleotide sequence of cDNA coding for saporin-6, a type-1 ribosome-inactivating protein from Saponaria officinalis

We have isolated and sequenced partial cDNA clones that encode SO-6, a ribosome-inactivating protein from Saponaria officinalis. A cDNA library was constructed from the leaves of this plant and screened with synthetic oligonucleotide probes representing various portions of the protein. The deduced amino acid sequence shows the signal peptide and a coding region virtually accounting for the entire amino acid sequence of SO-6. The sequence reveals regions of similarity to other ribosome-inactivating proteins, especially in a region of the molecule where critical amino acid residues might participate in the active site.     

The amino acid sequence of a ribosome-inactivating protein from Saponaria officinalis seeds

The complete primary structure of saporin SO-6, a ribosome-inactivating protein extracted from Saponaria officinalis seeds, has been determined. The sequence was reconstructed following purification and analysis of peptides obtained after digestion of the protein with different proteolytic agents. The protein is composed of 253 amino acids, corresponding to a molecular weight of 28,621 Da. Comparison of the primary structure of SO-6 with the sequence deduced from cDNA, shows amino acid substitutions in 11 positions, suggesting a tissue-related genetic variability. When the sequence of saporin is compared to those of two related proteins, ricin A chain and trichosanthin, a low degree of similarity (12%) is found; nevertheless some considerations about structure-function relationships and evolution of RIPs are possible.     

Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.     

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.     

N-terminal sequence of some ribosome-inactivating proteins

The N-terminal portion of some type 1 ribosome-inactivating proteins (RIPs) isolated from the seeds of Gelonium multiflorum, Momordica charantia, Bryonia dioica, Saponaria officinalis and from the leaves of Saponaria officinalis are reported in the present paper. Their relationship with other RIPs is discussed.     

肥皂草素类核糖核蛋白体失活蛋白的纯化及其性质研究

 利用强阳离子交换柱从Saponaria officinalis L种子中分离出一种肥皂草素(Saporin)成分。它在无细胞体系中显示了较强的抑制蛋白合成的活性,与抗体连接后能特异性杀伤靶细胞。     

Ribosome-inactivating proteins from plant cells in culture.

1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg.     

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.     

美洲商陆抗病毒蛋白的研究

美洲商陆抗病毒蛋白(pokeweed antiviral proteins.PAP)是一种具有多种生物功能活性的蛋白质,其在广谱抗病毒方面有重要的应用价值。综述美洲商陆抗病毒蛋白的分子结构与功能,抗病毒的分子机理及其在植物基因工程中的应用。     

Expression and characterisation in E. coli of mutant forms of saporin

In the present communication, we report on the expression and characterisation in Escherichia coli of mutant derivatives of saporin, a type 1 ribosome-inactivating protein from Saponaria officinalis L. The effects of substitution of Glu 176 with Lys and those of deletion of 19 amino acids at the C-terminal were evaluated both in vivo, testing the influence of expressed proteins on bacterial growth and in vitro measuring their N-glycosidase and supercoiled DNA relaxation activities. Results indicate that both modifications of the wild-type protein abolish its toxicity to bacterial cells and impair its enzymatic activity on polynucleotide substrates, either RNA or DNA.     

Cytotoxicity of pokeweed antiviral protein

Pokeweed antiviral protein, a plant protein which inactivates eukaryotic ribosomes, was found to be cytotoxic to both HeLa and Vero cells. Cellular protein synthesis was inhibited by exposure of the cells to microM concentrations of the antiviral protein for 24 h periods or longer. The extent of the inhibition of cellular protein synthesis was dependent upon the time of exposure to pokeweed antiviral protein and was partially reversed by washing the cells at various times prior to the measurement of protein synthesis. The antiviral protein was also observed to bind nonspecifically to cells at both 4 degrees and 34 degrees C. The data indicate that the pokeweed antiviral protein is capable of slowly entering mammalian cells which results in the inhibition cellular protein synthesis.     

Ribosome inactivating protein saporin induces apoptosis through mitochondrial cascade, independent of translation inhibition

Ribosome inactivating proteins (RIPs) are toxic translation inhibitors that kill eukaryotic cells by arresting protein synthesis at the translocation step. Saporin-6, expressed in the seeds of Saponaria officinalis plant, is a type I RIP comprising of a single polypeptide chain. Saporin is a specific RNA N-glycosidase and it removes a specific adenine residue from a conserved loop of the large rRNA of eukaryotic cells. Saporin-6 is one of the most potent of several isoforms of saporin, obtained from different tissues of the Saponaria plant. In addition to potently inhibiting translation, saporin has been also shown to induce cell death by apoptosis in different cellular models. To elucidate the mechanism of apoptosis induction by saporin, we have investigated the apoptotic pathway triggered by saporin. We have also analyzed whether the inhibition of protein synthesis by the toxin is the trigger for induction of apoptosis. We demonstrate that saporin-6 induces caspase-dependent apoptosis in U937 cells via the mitochondrial or intrinsic pathway. Unlike many other toxins the catalytic N-glycosidase activity of saporin is not required for apoptosis induction, and the apoptosis onset occurs before any significant inhibition of protein synthesis ensues.     

Culture senescence and abscisic acid induce saporin production in cultured roots of Saponaria officinalis

The presence and variation of activity of the type 1 ribosome-inactivating protein saporin has been evaluated in cultured roots of the soapwort Saponaria officinalis . Results from western analysis and in vitro protein synthesis inhibition indicate that saporin production is increased in senescing cultures, reaching a maximum value during the late stationary phase. Accordingly, cultures treated with the senescence-related hormone abscisic acid show a significant increase in saporin activity, independently from the culture growth phase. Stress conditions, such as the presence of hydrogen peroxide in the culture medium, had no effect on the modulation of enzymatic activity. The putative regulation of saporin production by abscisic acid and its possible role in accomplishing the ageing programme is discussed.     

Chemical modifications of pokeweed antiviral protein: effects upon ribosome inactivation, antiviral activity and cytotoxicity

Pokeweed antiviral protein (PAP) is a protein known to inactivate eukaryotic ribosomes by an unknown enzymatic action and inhibit the production of mammalian viruses in tissue culture. This protein was subjected to a variety of chemical modifications to determine their effects upon ribosomal inactivation, antiviral action, and cytotoxicity. It was found that modifications of a number of different amino acid residues had similar effects upon all 3 activities. Also the inactivation of PAP with diethylpyrocarbonate was not due to its reaction with a histidine residue but to a modification of an unidentified amino acid residue.     

AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis

AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated. Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided. The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data. The congruency of the experiments was analyzed using the Bionumerics software.     

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.     

YP3：食用菌榆黄蘑中新的植物病毒抑制物蛋白

食用菌中含有多种抗病毒蛋白,可用于植物保护.采用硫酸铵分级沉淀、DEAE-纤维素离子交换层析和SephacrylTMS-200凝胶层析方法,从食用菌榆黄蘑新鲜子实体中提取到一单亚基蛋白,命名为YP3.利用SDS-聚丙烯酰胺不连续凝胶电泳初步估计其分子量大约为27.6 kDa.氨基酸组成分析表明该蛋白非常类似其它的抗植物病毒蛋白,并且几乎不含糖.其N-末端序列为NRDVAACARFIDDFCDTLTP,在GenBank中没有找到同源序列.浓度为0.24 mg/L时蛋白YF3对烟草花叶病毒(TMV 20 mg/L)侵染心叶烟的抑制率为50%.同时还发现YP3对供试的细菌和真菌没有抑制活性,对胃癌细胞株MGC-803、肝癌细胞株SMMC-7721、肺癌细胞株SPC-A1的细胞增殖具有一定的抑制作用,其IC50大约为20 mg/L.     

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.     

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome

The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.     

Reproduction and Pollination in Central European Populations of Silene and Saponaria Species

Flower morphology, flowering phenology, flower visitors, reproductive systems, and stigmatic receptivity of six species of Silene and Saponaria (Silene alba, S. dioica, S. vulgaris, S. nutans, S. noctiflora. Saponaria officinalis) were studied from April to October 1993 and from April to June 1994 in natural populations around Giessen in Hessen/Central Germany and, additionally, in individuals grown from seeds in the Botanical Garden of the University of Giessen. With the exception of Saponaria officinalis and S. noctiflora, all species were regularly visited and pollinated by crepuscular and nocturnal moths and hawkmoths, but only one species, S. alba, was exclusively pollinated by these night-active insects. The other species showed mixed pollination syndromes in which nocturnal and diurnal insects both promoted pollen transfer. Geitonogamy or even autogamy occurred in the gynodioecious and hermaphrodite species S. vulgaris, S. nutans, S. noctiflora, and Saponaria officinalis. Silene noctiflora, the only annual species, is pseudocleistogamous; the majority of its flowers did not open, and fruit set occurred after selfing in bud.