Phaseolus Vulgaris Agglutinin Research Articles

Lectin-induced oxidative stress in human platelets

Previously we have shown that wheat germ agglutinin (WGA) and, with minor potency, Phaseolus vulgaris agglutinin (PHA), but not lens culinarian agglutinin (LCA), induce platelet aggregation, through the PLCƴ2 activation by the concerted action of src/syk and PI3K/BTK pathways. In this study, we have investigated platelet oxidative stress induced by lectins. Several parameters indicative of oxidative stress, such as reactive oxygen species (ROS), superoxide anion, lipid peroxidation and the efficiency of the aerobic metabolism, have been measured. It was found that ROS, superoxide anion formation and lipid peroxidation are significantly increased upon platelet treatment with WGA and PHA while LCA is ineffective. WGA is always more effective than PHA in all experimental conditions tested. In addition, the involvement of NADPH oxidase 1, syk and PI3K in oxidative stress induced by WGA and PHA has been shown. Concerning the lectins effect on aerobic metabolism, WGA and PHA, but not LCA, act as uncoupling agents, determining an increase of oxygen consumption and a decrease of ATP synthesis, with a consequent decrease of P/O value. These results are confirmed by the impairment of platelets proton gradient formation, evaluated by membrane potential, in platelets treated with WGA and PHA. In conclusion lectins, especially WGA, induce oxidative stress in platelets and decrease energy availability through modifications of membrane structure leading to the inefficiency of the aerobic machinery that steers platelets toward death as suggested by the decreased metabolic activity of platelets and the increased lactic dehydrogenase release.

The Ca2+/calmodulin kinase/AMP-activated protein kinase pathway regulates the lectin phaseolus vulgaris agglutinin induced NO production in human platelets

The Ca2+/calmodulin kinase/AMP-activated protein kinase pathway regulates the lectin phaseolus vulgaris agglutinin induced NO production in human platelets

The molecular mechanisms involved in lectin-induced human platelet aggregation.

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.

Alteration of salivary glycopatterns in oral lichen planus

Background: Oral lichen planus (OLP) is one of the most common oral mucosal lesions affecting 0.5–2% of the adult population. It is difficult to distinguish between OLP and other oral mucosal diseases. Structural changes in the glycans of saliva proteins might be reliable indicators of OLP. However, little is known about the alteration of salivary glycopatterns during OLP.Objective: We aimed to investigate the alterations of salivary protein glycosylation related to OLP.Material and methods: Twenty-eight patients with OLP and 30 age- and sex-matched healthy volunteers (HVs) were enrolled in the test group to probe the difference of salivary glycopatterns using lectin microarrays. The lectin blotting were further utilized to validate the expression of certain glycans.Results: The glycoproteins recognized by three lectins [Aleuria aurantia lectin (AAL); Phytolacca americana (PWM); Phaseolus vulgaris agglutinin (E + L), (PHA-E + L)] were mainly increasing in the saliva of OLP. Meanwhile, these glycoproteins also exhibited significant age-associated alterations.Conclusions: This study provided a new basic insight into salivary glycopatterns in OLP and helped to develop new potential biomarkers for diagnosis of OLP.

Lectin affinity electrophoresis.

An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

Toxic and deterrent effects of phytohemagglutinin on the grain aphid Sitobion avenae

The toxicity and deterrent effects of phytoheamagglutinin (Phaseolus vulgaris agglutinin, PHA) to the grain aphid (Sitobion avenae F.) was investigated in the laboratory by use of liquid diets or gels containing various PHA concentrations. The addition of PHA to the liquid diet increased the aphid pre-reproductive period, mortality, and generation time and decreased aphid fecundity and intrinsic rate of natural increase. Aphids fed on sucrose-agarose gels with higher concentrations of PHA exhibited no phloem-feeding behavior. The number of gel penetrations was reduced and duration of the pathway phase was increased as PHA concentration increased. These results indicate that PHA is detrimental to S. avenae and that the effect of PHA is at least partially explained by suppression of feeding behaviour. PHA could be useful for managing S. avenae.

Distribution of Lectin‐Bindings in the Testis of the Lesser Mouse Deer, Tragulus javanicus

The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d-galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N-acetyl-d-galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N-acetyl-D-glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), D-mannose and d-glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), L-fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.

Electrokinetic Potential in B-Thalassemia Erythrocyte Membranes: Effect of Static Magnetic Field (2T)

ABSTRACTThe effects of the static magnetic field (SMF) on zeta potential of erythrocytes from patients with heterozygous ‘thalassemia’ and healthy subjects were studied by means of cell micro-electrophoresis. Electrophoretic mobility (EPM) of ‘thalassemic’ erythrocytes from patients in pregnancy decreased significantly after SMF exposure (0.1 - 2 T) compared to the same membranes without SMF pre-exposure. Significantly decreased extent of lipid peroxidation was observed in ‘thalassemic’ erythrocytes from patients in pregnancy increasing the dose of SMF pre-treatment. SMF exposure increased the aggregate's formation in erythrocyte suspension from healthy subjects as well as in ‘thalassemic’ erythrocytes at the expositions (0.5–2 T). Lectin—membrane interaction was tested on the EPM of erythrocytes in norm and pathology. Thus lectin binding may provide information about changes in glycoprotein complexes of the membrane. We studied the effect of Phaseolus vulgaris agglutinin (Phytohemagglutinin, PHA-M) on the surface charge density and 90° light scattering (LS) of erythrocytes in physiological pH (PBS, pH 7.6). PHA-M induced an increase in EPM of normal erythrocytes at doses of 60 pg PHA/ml and 150 pg PHA/ml. There was an enhancement in zeta potential of thalassemic erythrocytes upon pre-treatment with the 200 pgPHA/ml.SMF (0.1–2T) altered electrokinetic properties of ‘thalasemic’ erythrocytes with increased agglutinability, which could be due to reduction of zeta potential as well as decrease in lipid peroxidation products of erythrocyte membranes.

Biochemical and Genomic Analysis of Neoculin Compared to Monocot Mannose-Binding Lectins

Neoculin occurring in an edible tropical fruit is a heterodimeric protein which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Both the primary and the overall tertiary structures of neoculin resemble those of monocot mannose-binding lectins. This study investigated differences in biochemical properties between neoculin and the lectins. Structural comparison between the mannose-binding sites of lectins and the corresponding regions of neoculin showed that there is at least one amino acid substitution at each site in neoculin, suggesting a reason for the lack of its mannose-binding ability. This was consistent with hemagglutination assay data demonstrating that neoculin had no detectable agglutinin activity. DNA microarray analysis indicated that neoculin had no significant influence on gene expression in Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions. These data strongly suggest that neoculin has no lectin-like properties, encouraging its practical use in the food industry.

Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.

Age Effect on Human Aortic Valvular Glycoproteins

The aortic valve has been the subject of many hemodynamic studies but, to our knowledge, posttranslational modification of human valve proteins has not yet been studied. Thus, the aim of this study was to determine whether any age-related changes in the protein composition of normal human aortic valves and their glycosylation pattern could be observed. Aortic valves harvested from male cadaveric donors free of cardiovascular diseases were divided into four age groups: I, mean age 21 years; II, 30 years; III, 41 years; IV, 51 years. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Identification of monosaccharide moieties or oligosaccharide units was performed with the use of eight lectins of narrow specificity: Galantus nivalis agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin, Datura stramonium agglutinin, Aleuria aurantia agglutinin, Arachis hypogeae agglutinin, Phaseolus vulgaris agglutinin, and Lycopersicon esculentum agglutinin. Isolated proteins showed no age-related changes in SDS-PAGE protein profile, contrary to their glycosylation. Protein sialylation, number of tri/tetraantennary complex glycans, proteins having terminal galactose and polylactosaminyl units increased with age, whereas protein fucosylation showed the opposite relationship. Moreover, groups III and IV possessed a larger number of proteins bearing high-mannose and/or hybrid-type glycans, and the quantity of these structures seemed to change, in particular proteins, with the age of donors. Our results clearly demonstrate that glycosylation profile in human aortic proteins is associated with the age of the donor.

Direct targeting of cancer cells: A multiparameter approach

Lectins have been widely used in cell surface studies and in the development of potential anticancer drugs. Many past studies that have examined lectin toxicity have only evaluated the effects on cancer cells, not their non-cancer counterparts. In addition, few past studies have evaluated the relationship between lectin-cell binding and lectin toxicity on both cell types. Here we examine these parameters in one study: lectin-cell binding and lectin toxicity with both cancer cells and their normal counterparts. We found that the human colon cancer cell line CCL-220/Colo320DM bound to agarose beads derivatized with Phaseolus vulgaris agglutinin (PHA-L) and wheat germ agglutinin (WGA), while the non-cancer human colon cell line CRL-1459/CCD-18Co did not. When these lectins were tested for their effects on cell viability in culture, both cell lines were affected by the lectins but at 6, 48 and 72 h incubation times, PHA-L was most toxic to the cancer cell line in a concentration dependent manner. At 48 h incubation, WGA was more toxic to the cancer cell line. The results suggest that it may be possible to develop lectin protocols that selectively target cancer cells for death. In any case, examination of both malignant cells and their non-malignant counterparts, analysis of their binding characteristics to immobilized lectins, and examination of the toxicity of free lectins in culture, provides a multiparameter model for obtaining more comprehensive information than from more limited approaches.

Effect of orally administered plant lectins on intestinal liquor accumulation and amylase activity in rats.

Short-term effects of orally administered plant lectins, with special reference to the Phaseolus vulgaris agglutinin (phytohaemagglutinin, PHA), were studied in growing rats. The orally administered PHA elicited a dose-dependent accumulation of liquor with elevated pH in the proximal small intestine. Although the concentration of alpha-amylase activity did not change, total alpha-amylase activity slightly, but significantly increased in the gut. When a panel of plant lectins with different carbohydrate binding specificities was tested at the dose of 100 mg/kg body weight, most of them stimulated the secretion of liquor, but the total alpha-amylase activity was increased only by PHA, ConA or WGA.

Lectinocytochemical detection of apoptotic murine leukemia L1210 cells.

Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about its changes during apoptosis. We investigated whether specific changes in the expression of plasma membrane glycoproteins take place during apoptosis and whether these changes could be used for a quantitative estimation of apoptosis. Lectin cytochemical study of normal and apoptotic murine leukemia cells of the L1210 line was done. Horseradish peroxidase-labeled Laburnum anagyroides bark agglutinin, Phaseolus vulgaris agglutinin, Pisum sativum lectin (PSL), Ricinus communis agglutinin (RCA-120; 120 kDa), Solanum tuberosum agglutinin, Triticum vulgaris lectin (wheat germ agglutinin), Viscum album agglutinin, Canavalia ensiformis lectin (concanavalin A), and Helix pomatia lectin were used to compare specific glycoprotein expressions in normal and apoptotic murine leukemia cells of the L1210 line sensitive (L1210) and resistant (L1210R) to apoptosis induction by cisplatin. The data demonstrated significantly increased binding of alpha-D-mannose-specific PSL lectin (P<0.01) and beta-D-galactose-specific RCA-120 lectin (P<0.001) by the apoptotic cells of the L1210 and L1210R lines in comparison with the intact cells. That binding was shown to be specific because it was blocked by the corresponding inhibitory sugars. Lectins specific to alpha-D-mannose (PSL) and beta-D-galactose (RCA-120) can be used to distinguish between native and apoptotic murine leukemia L1210 cells and to quantitatively estimate apoptosis in a population of these cells.

Effect of plant lectins on the host-finding behaviour of Radopholus similis

AbstractThe motility and the chemotactic response towards plant roots of Radopholus similis, after treatment with novel types of lectins, were examined in vitro by analysing movement tracks on agar plates. Six plant lectins belonging to five different lectin families and a banana thaumatin-like protein (BanTLP) were included in the experiment. A 1% concentration of Phaseolus vulgaris agglutinin (PHA) had an adverse effect on the motility of R. similis females: 63% showed no or very little movement on agar plates compared to an average of 33% for other lectins and 3% for the control treatment. A 0.05% concentration of PHA still reduced the motility of R. similis females by 75%. Concanavalin A and wheat germ agglutinin did not alter the chemotactic response towards plant roots, despite binding of both lectins to R. similis. In contrast, Galanthus nivalis agglutinin (GNA) reduced orientated movement of R. similis towards plant roots. Subsequently, secretions of R. similis were stained with Coomassie Brilliant Blue R. Nematodes treated with GNA produced secretions less abundantly compared to the control treatment and BanTLP. The other lectins in the experiment had variable effects on secretion.

Effect of germanium, poly-trans-[2-carboxyethyl] germasesquioxane on natural killer (NK) activity in dogs.

In an attempt to study the immunological effects on normal and adenocarcinoma (AC) dogs, the natural killer (NK) activity was determined. Augmentation of NK cell activities in responsive normal and AC dogs that indicatedanergy in the phaseolus vulgaris agglutinin (PHA) skin-test was manifested when the animals were accordingly treated with germanium (GN).

Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines.

BackgroundThe aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins.ResultsHuman non-malignant epithelial cell of ureter HCV29, v-raf transfected HCV29 line (BC3726) and transitional cell cancers of urine bladder Hu456 and T24 were grown in cell culture. Equal amounts of protein from each cell extracts were separated by SDS-PAGE electrophoresis and were blotted on an Immobilon P membrane. Cadherins were immunodetected using anti-pan cadherin mAb and lectin blotting assays were performed, in parallel. N-oligosaccharides were analysed by specific reaction with Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Datura stramonium agglutinin (DSA), Aleuria aurantia agglutinin (AAA), Phaseolus vulgaris agglutinin (PHA-L) and wheat germ agglutinin (WGA). The cadherin from HCV29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans, some of which were α2,6-sialylated. The cadherin from BC3726 cell line exhibited exclusively high mannose type glycans. Cadherins from Hu456 and T24 cell lines expressed high mannose type glycans as well as β1,6-branched oligosaccharides with poly-N-acetyllactosamine structures and α2,3-linked sialic acid residues. Additionally, the presence of fucose and α2,6-sialic acid residues on the cadherin from T24 cell line was detected.ConclusionsThese results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis.

Spontaneous eosinophilic granulated round cell tumors in rats.

Morphologic and histochemical characteristics were noted for three spontaneous tumors with eosinophilic cytoplasmic granules that occurred in aged Fischer 344 rats. Macroscopic lesions were widely distributed in the body, mainly involving the intra-abdominal adipose tissue, pancreas, and mesenterium. These lesions were generally hard swellings with nodular and sclerosing areas. Bloody ascites was a concomitant finding. Histologically, the tumor cells were round, from 9 to 30 microm in diameter with one or two round to oval nuclei, and characterized by eosinophilic granules (0.5-2.0 microm) that stained definitely to weakly positive with the periodic acid-Schiff reaction and demonstrated no metachromasia with toluidine blue stain. Furthermore, the granules were characterized by a positive reaction with lectin histochemistry for concanavalin A (Con A), wheat germ agglutinin (WGA), phaseolus vulgaris agglutinin (PHA-E4), lens culinaris agglutinin (LCA), and recinus communis agglutinin (RCA-I) in all tumors and for ulex europaeus agglutinin (UEA-I), peanut agglutinin (PNA), and soybean agglutinin (SBA) in one tumor. Positive reactions for anti-rat mast cell protease II and CD8 were not demonstrated immunohistochemically. Abundant glycogen was noted in the large tumor cells from one rat. With electron microscopy, the cytoplasmic granules were identified as electron-dense homogenous bodies bounded by a single unit membrane. These characteristics are similar to those of granulated metrial gland cells, but further study is needed to clarify the cell of origin for these tumors.

Low-dose intragastric administration of Phaseolus vulgaris agglutinin (PHA) does not induce immunoglobulin E (IgE) production in Sprague-Dawley rats.

Native Phaseolus vulgaris agglutinin (PHA) poses a potential health threat, when ingested with improperly cooked red kidney beans. Since PHA triggers human basophilic granulocytes in culture to rapidly release considerable amounts of interleukin-(IL-)4 and IL-13, key cytokines for inducing immunoglobulin E (IgE) production, the question was addressed whether this lectin can evoke in vivo IgE production. IgE-low-responder (Sprague-Dawley) rats received PHA (6 mg/rat/day) intragastrically by gavage over a period of 10 days. Up to day 35, there was no IgE induction regardless of whether the animals were boostered subcutaneously with PHA or not, indicating that PHA cannot be regarded as a general IgE inducer in rats.

Differences of alpha3beta1 integrin glycans from different human bladder cell lines.

Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.