Korean mistletoe lectin (KML-IIU) and its subchains induce nitric oxide (NO) production in murine macrophage cells

Synthesis of nitric oxide (NO) is one of the important effector functions of innate immune cells. Although several reports have indicated mistletoe lectins induce immune cells to produce cytokines, studies regarding the activities of the lectins in the production of NO have been very limited. Here, we report on the induction of NO synthesis in a murine macrophage cell line, RAW264.7, by Korean mistletoe lectin (KML-IIU). When the macrophage cells were treated with KML-IIU in the presence of a suboptimal concentration of IFN-γ, NO production was induced in a concentration-dependent manner. Significantly higher levels of NO were induced by subchains of the KML-IIU (A and B), which have lower toxicities, as compared to the hololectin. Furthermore, expression of the inducible nitric oxide synthase (iNOS) gene was elevated in accordance with the level of NO production. When the synthase was inhibited by iNOS inhibitors (L-NIL and L-NAME), NO production was specifically reduced in a concentration-dependent manner. Our studies demonstrate that the KML-IIU and its subchains induce NO production in murine macrophage cells via activation of the iNOS gene expression, suggesting that the KML-IIU subchains may be used as an immunomodulator to enhance the effector functions of innate immune cells.     

Isolation and Characterization of Isolectins from Talisia esculenta Seeds

Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm × 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (M_r 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Mistletoe lectin (Viscum album coloratum) modulates proliferation and cytokine expressions in murine splenocytes

It is well documented that an extract of European mistletoe has a variety of biological effects, such as the stimulation of cytokine production from immune cells, and additional immunoadjuvant activities. While the European mistletoe has been studied intensively, we know less about Korean mistletoe as a therapeutic plant, especially as a possible immunomodulating drug. This study will investigated the effects of Korean mistletoe lectin (Viscum album L. var. coloratum agglutinin, VCA) on murine splenocytes to investigate whether VCA acts as an immunomodulator, which could lead to improved immune responses in these cells. The results showed that VCA inhibited cell proliferation at higher concentrations (at 1-8 ng/ml) and enhanced cell proliferation at lower concentrations (at 4-32 pg/ml). Further studies were carried out to determine if the proproliferative or anti-proliferative activity exhibited by VCA was correlated with cytokine secretion. Consequently, interferon (IFN)-gamma secretion was decreased in concanavalin A (ConA)-stimulated murine splenocytes by VCA (4-64 ng/ml), but there was no change in IL-4 levels. This suggests that VCA has the ability to modulate murine splenocyte proliferation and can possibly act on the balance of Th1/Th2 cellular immune responses.     

Alpha-galactoside-binding isolectins from wild jack fruit seed (Artocarpus hirsuta): purification and properties

Five isolectins with marked specificity for alpha-linked galactose were purified from the wild jack (Artocarpus hirsuta) seeds by affinity chromatography on cross-linked guar gum. They were composed of a glycosylated subunit A (Mr = 16 kDa) and a nonglycosylated subunit B (Mr = 11 kDa) in noncovalent association. The isolectins which eluted as a single peak of Mr 45 kDa on gel filtration in Biogel P-100 and in a TSK G-3000 SW high pressure column, were resolved into five peaks on electrophoresis at pH 4.5. Sodium dodecyl sulphate polyacrylamide gel electrophoreogram of the major isolectin band suggested that the isolectins may be the five possible tetrameric combinations of A and B subunits. The combined isolectins bound only two molecules of 4-methyl umbelliferyl alpha-D-galactoside with a binding constant of 4.75 x 10(4) M-1. The pH optimum of sugar binding was 7.0. The isolectins specifically bound to human IgG and IgA but not to IgM.     

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.     

Mitogenic properties of structurally related Lathyrus lectins

The mitogenic properties of ten phylogenetically related Lathyrus lectins have been studied. Despite a close structural resemblance and similar carbohydrate specificities, the lectins exhibited significant differences in their ability to induce cell proliferation of human lymphocytes. The differences in optimal dose were in the range 10-30 times. L. ochrus (whole lectin) also had an ability to induce cellular growth 3-10 times better than that of the isolated isolectins, L. ochrus I and II, illustrating the complexity behind the structure-function relation of lectin mitogens.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Purification and characterization of complex carbohydrate specific isolectins from wild legume seeds: Acacia constricta is (vinorama) highly homologous to Phaseolus vulgaris lectins

Vinorama isolectins (VL2-VL4) were purified from seeds of Acacia constricta (vinorama) using affinity chromatography on a fetuin-fractogel column followed by cationic-exchange chromatography. Each isolectin fraction presented a characteristic isoelectric point range from 5.5 to 8.4. Under native conditions, VL containing fractions migrated as tetramers of 133 kDa, while in SDS-PAGE, in presence of 2-mercaptoethanol, a single subunit band with M(r) of 34 kDa was observed. VL was found to be a glycoprotein with a 7.5% neutral sugar content. Antibodies to Phaseolus vulgaris lectins PHA and other wild legume lectins as Olneya tesota (palo fierro) PF2 and PF3, and Parkinsonia aculeate (palo verde) PV reacted with VL, but not with anti Glycine max agglutinin SBA or anti Lotus tetragonolobus agglutinin LTA. Furthermore, direct analysis of VL peptides showed sequences homologous to those reported in different lectins of the Phaseolus genus. VL2-VL4 did not have ABO serological or simple sugar specificity, but were inhibited by complex carbohydrates from fetuin and thyroglobulin. Asialofetuin carbohydrates strongly interacted with VL4 and VL3. Vinorama isolectins could be classified as "complex lectins".     

Isolation and partial characterization of 3 nontoxic d‐galactose–specific isolectins from seeds of Momordica balsamina

Three isolectins denoted hereforth MBaL‐30, MBaL‐60, and MBaL‐80 were isolated from seeds extract of Momordica balsamina by 30%, 60%, and 80% ammonium sulfate saturations, respectively. The native molecular weights of these lectins, as judged by gel filtration, were 108, 56, and 160 kDa, respectively. On SDS‐PAGE, under reduced condition, 27 kDa band was obtained for all isolectins. The lectins hemagglutinating activities were variably inhibited by d ‐galactose (minimum inhibitory concentrations = 12.5mM, 50mM, and 0.391mM, respectively). MBaL‐30 and ‐60 could agglutinate all human blood types with slight preference for the A and O blood groups, whereas MBaL‐80 did not agglutinate B and AB blood types. The 3 isolectins were purified from crude seeds extract, collectively, in a single step on the affinity matrix Lactamyl‐Seralose 4B; this purified lectin fraction, which contains all isolectins, is termed MBaL. The N‐terminal of MBaL till the 25th amino acid was NLSLSELDFSADTYKSFIKNLRKQL, which shares 88% sequence identity with Momordica charantia lectin type‐2 ribosomal inactivating protein from Momordica charantia and 50% with momordin II from Momordica balsamina . MBaL retained 100% activity at up to 50°C for 30 minutes. MBaL‐30 and MBaL‐60 exhibited maximum activities in the pH range between 4 and 8, while MBaL‐80 was showing maximum activity in the pH range between 3 and 5. Treatment of MBaL‐30 and MBaL‐60 with EDTA completely abolished their hemagglutinating activities. Addition of Zn and Fe ions to the ethylenediaminetetraacetic acid–treated MBaL‐30 and MBaL‐60 lectins did not only regained the loss of activity but also resulted in 200% to 300% increase in activity, respectively. MBaL‐30 and ‐60 agglutinated gram positive Listeria monocytogenes and Staphylococcus aureus, whereas MBaL‐30 could merely agglutinate Escherichia coli . None of these lectins could arrest bacterial growth. Addition of MBaL to cancer cell lines (Gastric cancer cell line (AGS) and Gastric cencer cell line (MKN45), Glioblastoma (ECV‐304), and Human urinary bladder cancer cell line (U87‐MG)) at varying concentrations did not cause statistically significant changes on cell growth and viability.     

Transpiration and stomatal conductance of mistletoe (<Emphasis Type="Italic">Loranthus europaeus</Emphasis>) and its host plant,downy oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis>)

Sap flow rate was measured in the crown of a solitary specimen of downy oak (Quercus pubescens) infested by mistletoe (Loranthus europaeus). Five oak branches and two mistletoe plants were selected for analysis. The seasonal sum of transpired water expressed per leaf area unit was five times higher in the mistletoe than in the oak. In addition, the diurnal curves of sap flow were different between the plants. In the morning, the sap flow measured in the mistletoe lagged one hour behind the sap flow measured in an oak branch unencumbered by mistletoe. In contrast, no time lag was observed in the evening. The proportion of water transpired at night relative to the total transpiration was 7% in both species. The stomatal conductances derived from the inverted Penman-Monteith equation and their dependence on global radiation and the vapour pressure deficit (D) revealed that D exerts a different behaviour in stomatal control of transpiration in the mistletoe. We also determined that the concentration of calcium in the leaf mass could serve as a proxy for transpiration rate, however the relationship was not proportional.     

Stage- and tissue-specific expression of a β-1,4-galactosyltransferase in the embryonic epidermis

Changes in oligosaccharide structures of glycoconjugates have been observed, and are postulated to have key roles in embryonic development and differentiation. N-Acetylglucosamine (GlcNAc) beta-1,4-galactosyltransferase (beta4GalT) AKI showed different expression patterns in time and space, and different enzymatic activity from the other known family members. The epidermis of mouse embryo included a high level of AKI activities, which transferred galactose (Gal) to endogenous glycoprotein (molecular weight 130 kDa) (GP130). The maximum activity was for 13.5-d postcoitum embryos. Specific antibody against AKI inhibited 81% of GlcNAc betaGalT activities, which indicates that AKI represents the major part of the embryonic epidermis enzymes. AKI shows 2.2 times higher galactosyltransferase activity toward Gal-acceptor glucose with alpha-lactalbumin (alpha-LA) than toward GlcNAc without alpha-LA. AKI is also expressed in mouse melanoma and leukemia cell lines and in human basal cell carcinoma specimens. The GP130 Gal acceptor once galactosylated by AKI may be directly involved in epidermal differentiation and oncogenesis.     

Synergistic anticancer effects of lectin and doxorubicin in breast cancer cells

We studied the effects, either combined or alone, of lectin from Korean mistletoe (Viscum album var. coloratum agglutinin, VCA) and doxorubicin (DOX) in MCF-7 (estrogen receptor-positive) and MDA-MB231 (estrogen receptor-negative) human breast cancer cells. When VCA and DOX were combined, a strong synergistic effect was shown in cell growth inhibition, compared to VCA or DOX treatment alone. In quantitative apoptosis studies analyzed by flow cytometry, a combination of two agents showed an increase in apoptosis in both cells, compared to agents alone. Also, pro-apoptotic proteins including Bax, Bik, and Puma were increased in both cells, and the survival factor Bcl-2 was inhibited in MCF-7 cells when drugs were combined. Furthermore, VCA combined with DOX mediated S phase arrest, accompanied with a decrease of cell number at G0/G1 phase. This suggests that VCA and DOX combination may possibly lead to a novel strategy for the treatment of breast cancer.     

A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a ~(51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (ⅰ) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner; (ⅱ) ML increased γδ T cell cytotoxicity; (ⅲ) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (ⅳ) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.     

Lectin variability in Phaseolus coccineus

Lectin variability within Phaseolus coccineus is revealed by non-denatured electrophoretic patterns and immunological labelling of total seed protein extracts, showing that the different cultivars and wild varieties studied can be classified into three main categories according to the number of isolectins (three, two or one) present in each extract. Attempts in the purification of these isolectins were performed on three different affinity systems in which ligands were thyroglobulin (known to purify the P. vulgaris isolectins), pig red cell membrane ghosts (stroma) or antibodies against the P. vulgaris cv. Contender E₂L₂ isolectin. The P. coccineus isolectins exhibit varied affinities towards thyroglobulin and stroma, the cathodic and anodic (pH 4.5) isolectins being respectively retained by the two systems, whereas the antibody affinity system is the only one able to purify the totality of the isolectins present in an extract.     

Isolation and characterization of mistletoe extracts (Viscum album L.). II. Effect of agglutinating and cytotoxic fractions on mouse ascites tumor cells

Lectin from mistletoe (Viscum album L.) was studied for its relations with the toxins from Viscum album, ascites tumor cells of mouse, and human immunoglobulins. Using affinity chromatography on glutaraldehyde-crosslinked IgG (human) from viscum crude extract, a fraction was isolated which exhibited full agglutination capacity and high toxicity. The supernatant showed no agglutination capacity but a strong toxic effect on mouse ascites tumor cells. This toxic effect could not be influenced by further additions of insolubilized IgG. Chromatography on DEAE cellulose also gave agglutinating fractions with toxic effects and a non-agglutinating toxic portion. Column chromatography on Sephadex G 75 allowed separation of toxic from agglutinating components. The molecular weight of the toxin remaining after lectin removal was above 10,000. Lectin was found to bind more readily to mouse ascites tumor cells than to erythrocytes.     

Purification and characterization of four isoforms of Himalayan mistletoe ribosome-inactivating protein from Viscum album having unique sugar affinity

Ribosome-inactivating proteins having antitumor and immunomodulatory properties constitute the active principle of widely used mistletoe therapy in Europe. This is the first report of the four isoforms of Himalayan mistletoe ribosome-inactivating proteins (HmRips) from Viscum album parasitized on wild apple inhabiting NW Himalayas. HmRips were purified by affinity chromatography and four isoforms were separated by ion-exchange chromatography. HmRip 1, 2, 3, and 4 have isoelectric points of 6.6, 6.1, 5.2, and 4.7, respectively. Disulfide linked toxin and lectin subunits of HmRip 1 and 2 isoforms have molecular weights of 28 and 34kDa while those of HmRip 3 and 4 have 28 and 32kDa. The isoforms lacked blood group specificity and showed positive activity with seven mammalian erythrocyte types but did not show any activity with avian erythrocyte type. Lectin activity of HmRips remained unchanged for a wide range of temperatures (0-65 degrees C) and pH (3-9). Unlike other type II Rips, the HmRip 1, 2, and 4 showed unique affinity towards l-rhamnose, meso-inositol, and l-arabinose while HmRip 3 has specificity to gal/galNAc. Sugar binding studies with 22 sugars also suggested that the C-4 hydroxyl of galactose might be the critical site involved in sugar binding of HmRips. Type II Rips are known to be galactoside specific and do not have affinity for l-rhamnose and meso-inositol. However, HmRip 1, 2, and 4 having equal affinity for galactose and l-rhamnose do not strictly fit into any of the four structural classes of the lectins and represent a new class of type II Rips and plant lectins.     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.