MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis.

Increasing evidence shows that Traditional Chinese Medicine (TCM) has an obvious appeal for cancer treatment, but there is still a lack of scientific investigation of its underlying molecular mechanisms. Bitter melon or bitter gourd (Momordica charantia) is an edible fruit that is commonly consumed, and it is used to cure different diseases in various ancient folk medical practices. We report that a bioactive protein, MAP30, isolated from bitter melon seeds exhibited potent anticancer and anti-chemoresistant effects on ovarian cancer cells. Functional studies revealed that MAP30 inhibited cancer cell migration, cell invasion, and cell proliferation in various ovarian cancer cells but not normal immortalized ovarian epithelial cells. When administered with cisplatin, MAP30 produced a synergistic effect on cisplatin-induced cell cytotoxicity in ovarian cancer cells. When low doses of cisplatin and MAP30 were co-injected intraperitoneally, a remarkable reduction of tumor dissemination and tumor growth was observed in an ovarian cancer ascites mouse model. Notably, blood tests confirmed that MAP30 did not cause any adverse effects on liver and kidney functions in the treated mice. MAP30 activated AMP-activated protein kinase (AMPK) signaling via CaMKKß and induced cell cycle arrest in the S-phase. MAP30 modulated cell metabolism of ovarian cancer cells via suppression of GLUT-1/-3-mediated glucose uptake, adipogenesis, and lipid droplet formation in tumor development and progression. MAP30 also induced an increase in intracellular Ca2+ ion concentration, which triggered ROS-mediated cancer cell death via apoptosis and ferroptosis. Collectively, these findings suggest that natural MAP30 is a non-toxic supplement that may enhance chemotherapeutic outcomes and benefit ovarian cancer patients with peritoneal metastases.

Antitumor mechanism of MAP30 in bladder cancer T24 cells, and its potential toxic effects in mice.

To investigate the antitumor mechanism of MAP30 in human bladder cell line (T24) and its potential toxic effects in mice.  In this study, the biological behavior of MAP30's influence on bladder cell was investigated to reveal the antitumor mechanism and role of MAP30 in bladder cancer. MAP30 gene sequence optimized by gene synthesis codon was inserted into the prokaryotic expression vector pET-28a to produce a large amount of target protein in Escherichia coli. The protein product was obtained after purification. Membrane hydration method was used to prepare MAP30 liposome in order to enhance its membrane permeability. The effects of MAP30 on the viability, apoptosis and migration of T24 cell were assessed using 3­(4,5­dimethyl­thiazol­2­yl)­2,5­diphenyl­2H­tetrazolium bromide (MTT), flow cytometric and TUNEL assays, respectively. Mice were transfected with bladder cancer cells for 48 h. The expressions of apoptotic and non-apoptotic proteins were determined using Western blotting. Changes in tumor volume and occurrence of metastasis were assessed using luciferase assay. After 7 days, liver and kidney were excised for histological examination. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH), and activities of catalase and glutathione peroxidase (GPx) were determined in serum or homogenate using enzyme-linked immunosorbent assay (ELISA). The yield of MAP30 after purification was significantly increased. The results of MTT assay showed that MAP30 significantly and concentration-dependently inhibited the proliferation and migration of T24 cells (p < 0.05). The prepared liposomes had uniform hydrated particle size of 132.6 nm, with encapsulation efficiency of 78 %. The inhibitory effect of MAP30 liposome on T24 cells was significantly higher than that of MAP30, and MAP30 significantly increased the number of apoptotic cells (p < 0.05). Western blotting showed that MAP30 significantly promoted the expression of caspase 3 (p < 0.05), but did not significantly affect the expressions of bcl-2 and bax (p > 0.05). It also significantly down-regulated the expressions of NF-kB, JNK and MMP2 (p < 0.05). Tumor formation was significantly inhibited, and tumor volume reduced in bladder cancer-bearing mice after treatment with MAP30 (p < 0.05). Histological examination showed that MAP30 induced mild histological changes in the liver and kidney of mice, and significantly increased the level of MDA at day 1 (p < 0.05). It also significantly and time-dependently increased ROS, but reduced GSH levels and activities of catalase and GPx (p < 0.05). However, MAP30 had no significant effect on DNA (p > 0.05). The apoptotic effect of MAP30 in T24 cells is mediated via activation of caspase-3 signaling pathway. The protein produces mild histological changes in the liver and kidney of mice, but has no significant effect on DNA.

Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin.

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria.

Ebulin-RP, a novel member of the Ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains.

BACKGROUND: Sambucus ebulus is a rich source of ribosome-inactivating proteins (RIPs) and RIP-related lectins generated from multiple genes. These proteins differ in their structure, enzymatic activity and sugar binding specificity. METHODS: We have purified and characterized ebulin-RP from S. ebulus leaves and determined the amino acid sequence by cDNA cloning. Cytotoxicity was studied in a variety of cancer cells and a comparative study of the ability of ebulin-RP to bind sugars using "in vitro" and "in silico" approaches was performed. RESULTS: Ebulin-RP is a novel heterodimeric type 2 RIP present in S. ebulus leaves together with the type 2 RIP ebulin l, which displayed rRNA N-glycosidase activity but unlike ebulin l, lacked functional sugar binding domains. As a consequence of changes in its B-chain, ebulin-RP displayed lower cytotoxicity than ebulin l towards cancer cells and induced apoptosis as the predominant pattern of cell death. CONCLUSIONS: Ebulin-RP is a novel member of the ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains. Type 2 RIP genes from Sambucus have evolved to render proteins with different sugar affinities that may be related to different biological activities and could result in an advantage for the plant. GENERAL SIGNIFICANCE: The ebulin family of RIPs and lectins can serve as a good model for studying the evolutionary process which may have occurred in RIPs. The lack of cytotoxicity of ebulin-RP makes it a good candidate as a toxic moiety in the construction of immunotoxins and conjugates directed against specific targets.

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death.

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. © 2016 Wiley Periodicals, Inc.

Lectin Digestibility and Stability of Elderberry Antioxidants to Heat Treatment In Vitro.

Elderberry contains healthy low molecular weight nutraceuticals and lectins which are sequence-related to the elderberry allergen Sam n1. Some of these lectins are type II ribosome-inactivating proteins. The sensitivity of native lectins present in elderberry fruits and bark to the proteolysis triggered by in vitro simulated gastric and duodenal fluids has been investigated. It was found that these lectins are refractory to proteolysis. Nonetheless, incubation for 5-10 min in a boiling water bath completely sensitized them to the hydrolytic enzymes in vitro. Under these conditions neither total Folin-Ciocalteau's reagent reactive compounds, total anthocyanins and the mixture of cyanidin-3-glucoside plus cyanidin-3-sambubioside, nor antioxidant and free-radical scavenging activities were affected by more than 10% for incubations of up to 20 min. Therefore, short-time heat treatment reduces potential allergy-related risks deriving from elderberry consumption without seriously affecting its properties as an antioxidant and free-radical scavenging food.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

Escherichia coli-based expression system for the heterologous expression and purification of the elicitin ß-cinnamomin from Phytophthora cinnamomi.

Elicitins are sterol carrier proteins from the Oomycete genera Phytophthora and Phytium and elicit a hypersensitive response in many economically important plants, in some cases causing a systemic acquired resistance. Their recombinant expression in bacteria is complicated by the presence of three disulfide bonds in the elicitin structure. In consequence, elicitins have so far only been produced in soluble form by isolation from native Phytophthora or Phytium strains or by recombinant expression in the yeast Pichia pastoris. Here, for the first time, we report the soluble expression of the elicitin ß-cinnamomin from Phytophthora cinnamomi in Escherichia coli by secretion of the protein into the periplasm. ß-Cinnamomin yields have been significantly improved after careful selection of the optimum secretion signal sequence. In total, 17.6 mg ß-cinnamomin per liter cell culture have been obtained in shake flasks with the secretion signal sequence of the maltose-binding protein MalE from E. coli. Furthermore, by making use of a C-terminal His-tag, ß-cinnamomin purification has been significantly simplified with only one step of immobilized metal ion affinity chromatography yielding protein of high purity (>90%). The established protocol has further been successfully applied to the soluble expression of another elicitin.

Kirkiin: A New Toxic Type 2 Ribosome-Inactivating Protein from the Caudex of Adenia kirkii.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin.

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 A resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F(o)-F(c) electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin-ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.

Alpha-O-linked glycopeptide mimetics: synthesis, conformation analysis, and interactions with viscumin, a galactoside-binding model lectin.

Efficient cycloaddition of a silylidene-protected galactal with a suitable heterodiene yielded the basis for a facile diastereoselective route to a glycopeptide-mimetic scaffold. Its carbohydrate part was further extended by beta1-3-linked galactosylation. The pyranose rings retain their (4)C(1) chair conformation, as shown by molecular modeling and NMR spectroscopy, and the typical exo-anomeric geometry was observed for the disaccharide. The expected bioactivity was ascertained by saturation-transfer-difference NMR spectroscopy by using the galactoside-specific plant toxin viscumin as a model lectin. The experimental part was complemented by molecular docking. The described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational properties and bioactivity establish the prepared alpha-O-linked glycopeptide mimetics as promising candidates for further exploitation of this scaffold to give O-glycans for lectin blocking and vaccination.

Inhibition on Hepatitis B virus in vitro of recombinant MAP30 from bitter melon.

The gene encoding MAP30 protein was cloned from bitter melon and recombinant MAP30 was expressed and purified. The human hepatoma G2.2.15 cells were exposed to different concentrations of MAP30. MTT assay was used to evaluate the cytotoxicity of the drugs and real-time PCR and Southern hybridization were applied to quantify extracellular HBV DNA and replicative intermediates intracellular and cccDNA in nucleus. HBsAg and HBeAg were assessed by enzyme-linked immunosorbent assay (ELISA). The results showed that exposure of HepG2.2.15 cells to MAP30 resulted in inhibition of HBV DNA replication and HBsAg secretion. After exposed to three different concentrations of MAP30 for 2, 4, 6, and 8 days respectively, the inhibition rates of extracellular HBV DNA, HBsAg, and HBeAg of each concentration decreased significantly (P < 0.05). After 9 days of treatment, the inhibition rates of extracellular HBV DNA of the different concentrations differed greatly (P < 0.001). The MAP30 could inhibit the production of HBV (P < 0.01) dose-dependently. The expression of HBsAg was significantly decreased by MAP30 dose-dependently (P < 0.001) and time-dependently (P < 0.001). Lower dose of MAP30 (8.0 microg/ml) could inhibit the expression of HBsAg and HBeAg.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds.

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Effects of recombinant MAP30 on cell proliferation and apoptosis of human colorectal carcinoma LoVo cells.

MAP30, an attractive protein isolated from bitter melon, has been previously found to have the anti-tumor and anti-HIV activities. In this study, MAP30 was cloned and expressed and the effects of the recombinant protein on cell proliferation and apoptosis of human colorectal carcinoma LoVo cells were investigated. The results showed that the proliferation of LoVo cells were significantly suppressed by MAP30 in time- and dose-dependent manners at the concentration ranging from 0.67 to 4.67 muM. The apoptotic nuclei of LoVo cells induced by MAP30 were obviously observed, and the genomic degradation was detected by single-cell gel electrophoresis (comet assay). Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. The proportion of the periodic tumor cells was altered by MAP30. Sub-G1 curves were displayed by a flow cytometry analysis. Results of northern and western blots showed that the transcription and expression of Bax, a member of pro-apoptotic proteins, were gradually up-regulated as treated time increased. On the contrary, the transcription and expression of Bcl-2, an anti-apoptotic member, were down-regulated. These data provided powerful evidences for the first time that recombinant MAP30 can induce the apoptosis of the human colorectal carcinoma LoVo cells.

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

BACKGROUND: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. METHODS: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. RESULTS: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. CONCLUSION: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.

Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

Ebulin from dwarf elder (Sambucus ebulus L.): a mini-review.

Sambucus ebulus L. (dwarf elder) is a medicinal plant, the usefulness of which also as food is restricted due to its toxicity. In the last few years, both the chemistry and pharmacology of Sambucus ebulus L. have been investigated. Among the structural and functional proteins present in the plant, sugar-binding proteins (lectins) with or without anti-ribosomal activity and single chain ribosome-inactivating proteins (RIPs) have been isolated. RIPs are enzymes (E.C. 3.2.2.22) that display N-glycosidase activity on the 28S rRNA subunit, leading to the inhibition of protein synthesis by arresting the step of polypeptide chain elongation. The biological role of all these proteins is as yet unknown. The evidence suggests that they could be involved in the defense of the plant against predators and viruses or/and a nitrogen store, with an impact on the nutritional characteristics and food safety. In this mini-review we describe all the isoforms of ebulin that have to date been isolated from dwarf elder, as well as their functional characteristics and potential uses, whilst highlighting concern regarding ebulin toxicity.