De novo synthesis and lectin binding studies of unsaturated carba-pyranoses.

Starting from branched para-benzoquinones a practical and highly flexible route is described for the preparation of unsaturated carbapyranoses. The potential of the synthesized galactose analogues to act as competitive inhibitors in lectin-carbohydrate interactions is investigated by means of Surface Plasmon Resonance (SPR) Spectroscopy.

Mistletoe lectin binds to multidrug resistance-associated protein MRP5.

BACKGROUND: Mistletoe lectins (MLs) are the active components of aqueous mistletoe extracts widely used in complementary cancer therapy, however, it is not clear if they bind to carbohydrate residues only or whether they interact with proteins as well. Protein-protein interactions do not seem unlikely as MLs act at very low molar concentrations usually observed with peptide-peptide interactions only and not seen with lectin-sugar interactions. MATERIALS AND METHODS: In order to detect protein-protein interactions a random peptide library was screened for the ability to bind to MLs. RESULTS: MLs bound to peptides showing homologies to multidrug resistance-associated protein 5 (MRP5). However, the MLs only slightly modified the MRP5 efflux pump, while periodate treatment to inhibit cell membrane binding via glycan completely abolished the ML-I binding sites in MRP5 overexpressing cells. CONCLUSION: The protein sequence is not important for ML-I binding, indicating that the biological activity of MLs can most likely be attributed to the sugar chains.

European and Far East Mistletoes: Potential in Adjuvant Cancer Therapy / 针灸推拿医学（英文版）

Semiparasitic plants, mistletoes, distributed in Europe and East-Asia mainly, China, Korea and Japan have long been recognised as therapeutic herbs. Since the 1920s, extracts from European mistletoe (Viscum album L) have been popular in Europe as an unconventional cancer treatment. These extracts have been used in adjuvant cancer therapy because their immunostimulatory, cytostatic/cytotoxic and DNA stabilising activities. The main biological activities are addressed to sugar binding proteins, the Mistletoe Lectins, and to micro proteins named viscotoxins. The mistletoe lectins are members of the family of toxic lectins and recognize sugars and glycoconjugates containing galactose and/or N-acetyl-galactosamine groups. In vitro and in vivo assays confLrm that the mistletoe lectins, isolated from the plant and compared with recombinant forms, play an important role as biologically active principles in mistletoe extracts. Limited experimental evidence indicates that other components like glycosides, polysaccharides, amines etc, may also have anticancer activity. Under discussion in Europe is the principal question of using holo plant extracts or single component lectin preparations: wild type iectins or recombinant ones in cancer therapy. Finally problems of preparation, characterization and standardization of commercial mistletoe preparations and evaluation of biological activities are discussed.

Solubility studies of oleanolic acid and betulinic acid in aqueous solutions and plant extracts of Viscum album L.

Mistletoe (Viscum album L.) contains the triterpene acids oleanolic acid (OA) and betulinic acid (BA), which were found to have anti-tumour properties. In this study, the solubilities of OA and BA were studied in water (up to 0.02 microg/mL in each case) and in alkaline solutions of 10 mM trisodium phosphate (pH 11.5; OA: 77.2 microg/mL; BA: 40.1 microg/mL). Furthermore, triterpene acids were quantified in aqueous mistletoe extracts (pH 7.3; drug to extract ratio 1:25). OA (1.1 microg/mL) and BA (0.9 microg/mL) were extracted with a yield of less than 5%. Preparing plant extracts with basic pH values resulted in a triterpene acid content of 9.3 microg/mL OA and 5.2 microg/mL BA (pH 12.1), reaching neither the solubility limits nor a complete extraction of the plant material. The triterpene acid content of neutral plant extracts above the solubility limit could be due to interactions with biocolloids. Interaction studies were performed by gel permeation chromatography. Different mechanisms of the dissolution at pH 7.3 and pH 10.2 are discussed.

The proteasome inhibitor bortezomib augments anti-proliferative effects of mistletoe lectin-I and the PPAR-gamma agonist rosiglitazone in human melanoma cells.

BACKGROUND: The NFkappaB signalling pathway plays an important role in chemoresistance and decreased apoptosis. One indirect way to inhibit the NFkappaB pathway is to slow down the proteasomal degradation of its inhibitor IkappaB, thus preventing NFkappaB from translocation into the nucleus. Hence, the effect of the proteasome inhibitor bortezomib (Velcade) on the cell proliferation of the MV3, FemX-1 and G361 human melanoma cell lines and its action in combination with the PPAR-gamma agonist rosiglitazone or the mistletoe lectin ML-I, both having anti-proliferative effects on melanoma cells in single agent use, was investigated. MATERIALS AND METHODS: Proliferation of melanoma cells under the different treatment regimes over a broad concentration range (0.0001-100 microg/ml) was assessed by means of the XTT cell proliferation assay. RESULTS: At a concentration of 0.1 microg/ml bortezomib significantly reduced the proliferation rate of all melanoma cells to 1-13% of the control, which was mediated through increased apoptosis and inhibition of NFkappaB expression. Furthermore, the combination of bortezomib and rosiglitazone was the most potent and increased the effectiveness against melanoma cell growth by 63-71% (compared to single use of rosiglitazone) and by 27-39% (compared to single use of bortezomib), respectively. CONCLUSION: This combination strategy might be a promising approach for future melanoma therapy.

Glycoconjugate profiling of primary melanoma and its sentinel node and distant metastases: implications for diagnosis and pathophysiology of metastases.

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

Antifungal effects and mechanism of action of viscotoxin A3.

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine-rich peptides of approximately 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal-derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion-channel-like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3-induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.

Influence of mistletoe lectins and cytokines induced by them on cell proliferation of human melanoma cells in vitro.

Although aqueous mistletoe extracts are widely used in complementary cancer therapy, the precise mode of action of their main therapeutic agents, the three mistletoe lectins (MLs), is poorly understood as they act both as cytotoxic agents and as immunomodulators due to their cytokine release by mononuclear cells. Thus, this study aims to investigate both the direct and the indirect effects of MLs on the growth of human melanoma cells in vitro. Proliferation of six human melanoma cell lines under ML treatment and additionally under the influence of cytokines induced by them (TNF-alpha, IL-1, IL-6) was assessed by means of the tetrazolium derived reduction (XTT) assay. Furthermore, ML binding patterns were analysed and correlated with the biological effects. All three MLs inhibited melanoma cell proliferation in a dose-dependent manner starting at very low ML concentrations (0.001-100 ng/ml) with ML-I being the most cytotoxic lectin (significant inhibition of ultra-sensitive cell line MV3 at 1 x 10(-13) ng ML-I/ml). Even if applied in a broad concentration range (0.0001-100 ng/ml) cytokines had no influence on cell proliferation at all. For ML-I, no association between binding intensity and cytotoxicity was observed, while for ML-II and -III an association between binding and toxicity was established. In conclusion, this study emphasises the direct anti-proliferative effect of the mistletoe lectins on melanoma cells with ML-I being superior to MLs-II and -III. The observation of an ultra-sensitivity of one cell line towards ML-I toxicity may serve as an explanation for the therapeutic success in anecdotal case reports and needs further investigations.

Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity.

Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.

A new gene encoding the ribosome-inactivating protein from mistletoe extracts.

Extracts from mistletoe (Viscum album L.) contain three main toxic proteins--the lectins MLI (also known as viscumin), MLII and MLIII. A catalytic subunit of the mistletoe plant toxic lectin MLIII has been cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII A-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Ribosome-inactivating activity of the recombinant MLIII A-subunit was determined in a cell-free system exhibiting inhibition of endogenous protein synthesis. The comparative analysis of nucleotide and deduced amino acid sequences of the cloned MLIII A and the native MLI A-subunits was performed, revealing the main differences in the primary structure of these proteins. Antigenicity analysis of the MLIII A-subunit has revealed a new epitope D179-E184 that is not present in viscumin. The role of toxic lectins with respect to the immunological properties of mistletoe extracts is discussed.

Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. album.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi.

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA(3) and VtB, at a final concentration of 10 micro M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 micro M of VtA(3) and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 micro M, which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA(3) and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA(3) to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA(3) produces rapid changes in fungal membrane permeability. It also induces H(2)O(2) production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.

A mistletoe lectin (ML-1)-containing diet reduces the viability of a murine non-Hodgkin lymphoma tumor.

In this study we show that the characteristics of non-Hodgkin lymphoma (NHL) tumors in female Naval Medical Research Institute (NMRI, USA) mice fed mistletoe lectin (ML)-containing diets were different from those in mice fed control diet. The non-Hodgkin lymphoma tumor was originally established from a spontaneous tumor which developed in the inguinal region of a male mouse. Mice (five animals per group) were fed a lactalbumin (LA)-based control diet or a diet which provided up to 10 mg lectin per day. At the highest daily intake (10 mg lectin) the degree of mitotic activity in tumors was reduced by 75% and the nuclear area had diminished by 21%. The overall level of lymphocyte infiltration (CD3 positive cells) in tumors from mistletoe lectin fed mice was increased by a factor of two. Other morphological studies showed a high incidence of apoptotic bodies in non-Hodgkin lymphoma tumors obtained from mice fed mistletoe lectin diets. The feeding of such diets thus produced several identifiable changes in the morphology of non-Hodgkin lymphoma tumors. These were consistent with the observed reduction in tumor mass. In 4/15 mice fed a mistletoe lectin diet for 11 days there was no longer evidence of viable tumor. The results show that this lectin exerts powerful anti-tumor effects when provided by the oral route.

Interaction of viscotoxins A3 and B with membrane model systems: implications to their mechanism of action.

Viscotoxins are small proteins that are thought to interact with biomembranes, displaying different toxic activities against a varied number of cell types, being viscotoxin A(3) (VtA(3)) the most cytotoxic whereas viscotoxin B (VtB) is the less potent. By using infrared and fluorescence spectroscopies, we have studied the interaction of VtA(3) and VtB, both wild and reduced ones, with model membranes containing negatively charged phospholipids. Both VtA(3) and VtB present a high conformational stability, and a similar conformation both in solution and when bound to membranes. In solution, the infrared spectra of the reduced proteins show an increase in bandwidth compared to the nonreduced ones indicating a greater flexibility. VtA(3) and VtB bind with high affinity to membranes containing negatively charged phospholipids and are motional restricted, their binding being dependent on phospholipid composition. Whereas nonreduced proteins maintain their structure when bound to membranes, reduced ones aggregate. Furthermore, leakage experiments show that wild proteins were capable of disrupting membranes whereas reduced proteins were not. The effect of VtA(3) and VtB on membranes having different phospholipid composition is diverse, affecting the cooperativity and fluidity of the membranes. Viscotoxins interact with membranes in a complex way, most likely organizing themselves at the surface inducing the appearance of defects that lead to the destabilization and disruption of the membrane bilayer.

Lectin histochemistry of the lymphoid organs of the chicken.

Cellular interactions within the immune system are in part mediated via the carbohydrate-rich coat of the cell membrane, the glycocalyx, of which the terminal carbohydrate residues are of particular functional importance. Thus, these carbohydrate residues from thymus, bursa of Fabricius, spleen and bone marrow of 2- and 30-day-old chickens were investigated by lectin histochemistry. In the thymus, mannose as well as N-acetyl-glucosamine (glcNAc)-specific lectins labelled macrophages, epithelial reticulum cells and lymphocytes within the cortex. In the bursa of Fabricius, the brush border of the lining epithelium, the macrophages and the endothelium were labelled by mannose-specific lectins. The follicle-associated epithelium was labelled by a broad spectrum of lectins. Epithelial cells that separated the cortex from the medulla and large mononuclear cells in the cortex were only being labelled by N-acetyl-galactosamine (galNAc)-specific and glcNAc-specific lectins, respectively. In the spleen, lymphocytes of the peri-ellipsoid lymphocyte sheaths and macrophages of the red pulp were labelled by lectins of nearly all sugar specificities. In general, glycotopes of these organs were more intensively labelled in the 2-day-old chicken than in the 30-day-old chicken, indicating changes in glycotope expression during post-hatching development. Thus, cells of the avian immune system are as rich and diverse in their lectin binding sites as their mammalian counterparts, indicating that similar carbohydrate lectin interactions between cells and matrices take place in birds as well.

Crystal structure at 3 A of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose.

The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 A resolution to an R-factor of 19.9% using 21 899 reflections, with Rfree 24.0%. MLI forms a homodimer (A-B)2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and non-polar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 A apart on one side of the dimer, and two C-terminal sugar-binding sites are 87 A apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.

The cytotoxic effect of mistletoe lectins I, II and III on sensitive and multidrug resistant human colon cancer cell lines in vitro.

Multidrug resistance glycoprotein1 (MDR-1) eliminates amphiphilic chemotherapeutic agents out of tumour cells leading to therapeutic failures. The aim of this study was to investigate the cytotoxic effect of mistletoe lectins (MLs) I, II and III on the sensitive human colon cancer cell line HT 29(mdr-), its multidrug resistant variant HT 29(mdr+), the variant HT 29(SF1m) transfected with the MDR-1 gene and its sensitive control cell line HT 29(deltaSF). Both cell proliferation and ML binding pattern were analysed. Marked quantitative differences concerning the cytotoxic effect of the three MLs on the different cell lines were observed. All MLs showed the greatest cytotoxicity towards the HT 29(mdr+) cells, in which multidrug resistance (MDR) was induced by increasing concentrations of a MDR inducing agent. In contrast, MDR-1 and mock-transfected cells showed almost the same sensitivity towards the three MLs as the control cells (HT 29(mdr-)). FACS analysis showed that the HT 29(mdr+) cells were the cells with the highest density of ML binding sites. Thus, higher sensitivity of HT 29(mdr+) cells are not caused by the overexpression of MDR-1, but are caused by the general changes of the cellular glycosylation during the acquisition of the MDR phenotype.