Phaseolus vulgaris lectins: A systematic review of characteristics and health implications.

Legume lectins are carbohydrate-binding proteins of non-immune origin. Significant amounts of lectins have been found in Phaseolus vulgaris beans as far back as in the last century; however, many questions about their potential biological roles still remain obscure. Studies have shown that lectins are anti-nutritional factors that can cause intestinal disorders. Owing to their ability to act as toxic allergens and hemagglutinins, the Phaseolus vulgaris lectins are of grave concern for human health and safety. Nonetheless, their potential beneficial health effects, such as anti-cancer, anti-human immunodeficiency virus (anti-HIV), anti-microbial infection, preventing mucosal atrophy, reducing type 2 diabetes and obesity, promoting nutrients absorption and targeting drugs, are of immense interest. The significance of Phaseolus vulgaris lectins in biological researches and the potential biomedical applications have placed tremendous emphasis on the development of purification strategies to obtain the protein in pure and stable forms. These purification strategies entail considerations such as effects of proteolysis, heating, gamma radiation, and high-hydrostatic-pressure that can have crucial outcomes in either eliminating or improving bioactivities of the lectins. Thus, up-to-date research findings of Phaseolus vulgaris lectins on different aspects such as anti-nutritional and health impacts, purification strategies and novel processing trends, are systematically reviewed.

Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice.

Red kidney bean (Phaseolus vulgaris) is consumed worldwide as a vegetarian protein source. But, at the same time the allergenicity potential of red kidney bean is a matter of concern. This study is aimed towards purification, characterization, thermal stability, proteolytic digestion and allergenicity assessment of one of the clinically relevant allergens of red kidney bean. The purification of red kidney bean allergic protein was carried out with the help of column chromatography, IgE immunoblotting and reverse phase high-pressure liquid chromatography (RP-HPLC). The purified protein was characterized by peptide mass finger printing (PMF) and studied for its thermal stability, and proteolytic resistance using simulated gastric fluid (SGF) assay. The allergenicity potential of the purified protein was studied in BALB/c mice. The purified protein was identified as leucoagglutinating phytohemagglutinin (PHA-L) with molecular weight 29.5 kDa. The PHA-L showed resistance to heat as well as proteolytic enzyme. Higher levels of total IgE, specific IgE, and histamine were observed in PHA-L treated BALB/c mice when compared to control. Overall, PHA-L possesses characteristics of allergens and may play a potential role in the red kidney bean induced allergy.

Extraction and purification of a lectin from red kidney bean and preliminary immune function studies of the lectin and four Chinese herbal polysaccharides.

Reversed micelles were used to extract lectin from red kidney beans and factors affecting reverse micellar systems (pH value, ionic strength and extraction time) were studied. The optimal conditions were extraction at pH 4-6, back extraction at pH 9-11, ion strength at 0.15 M NaCl, extraction for 4-6 minutes and back extraction for 8 minutes. The reverse micellar system was compared with traditional extraction methods and demonstrated to be a time-saving method for the extraction of red kidney bean lectin. Mitogenic activity of the lectin was reasonably good compared with commercial phytohemagglutinin (extracted from Phaseolus vulgaris) Mitogenic properties of the lectin were enhanced when four Chinese herbal polysaccharides were applied concurrently, among which 50 µg/mL Astragalus mongholicus polysaccharides (APS) with 12.5 µg/mL red kidney bean lectin yielded the highest mitogenic activity and 100 mg/kg/bw APS with 12.5 mg/kg/bw red kidney bean lectin elevated mouse nonspecific immunity.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans).

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. "Anasazi beans" using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-14 and the temperature range of 0-80 degrees C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC(50) of 1.3 microM, and inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.6 microM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Long-term fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage.

The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.

Efecto inmunomodulador de la fitohemaglutinina de phaseolus vulgaris / Immunomodulator effect of fitohemaglutinin of phaseolus vulgaris

Se desarrolló un modelo experimental in vivo para evaluar la potencialidad de la fitohemaglutinina de 2 variedades de Phaseolus vulgaris de modular el sistema inmunológico. Ratas Wistar macho recibieron 10 y 20 mg/kg de peso de fitohemaglutinina obtenida de las variedades de frijol “Cueto” y “Judía roja Santo Domingo”. Ambos extractos fueron inocuos, incrementaron la masa corporal de los animales y no modificaron el hematócrito ni los conteos celulares de polimorfonucleares neutrófilos, eosinófilos y monocitos. La fitohemaglutinina BETERA obtenida del Phaseolus vulgaris de la variedad “Cueto” fue más efectiva para producir incrementos en el número total de leucocitos y linfocitos circulantes y estimular a los linfocitos B para la producción de anticuerpos(AU)

Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris.

The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris. To optimise yields of PHA-E, transformants of P. pastoris were selected for high-level production of the recombinant protein. A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported. PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography. The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa. Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein. The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E. The data presented here suggest that, using P. pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.

Elimination of artifacts on native western blots arising from endogenous lectin activity.

While studying the behavior of profilin from Phaseolus vulgaris seeds under native conditions, a high molecular weight species suggesting a complex of profilin and associated proteins was observed by Western immunoblotting. This putative complex was also observed when enzyme-linked secondary antibodies alone were used, and this apparently resulted from antibody association, through its glycosyl moieties, with the endogenous carbohydrate-binding activity from the seed extracts. This endogenous activity corresponded to that of purified phytohemagglutinin (PHA). In addition, the P. vulgaris lectin activity was very stable and was observed when the extracts were pretreated with varying concentrations of sodium dodecyl sulfate, Triton X-100, urea and beta-mercaptoethanol, or when membrane blots were boiled in water before incubation with antibody. The activity was abolished only if the membrane was boiled in 1% sodium dodecyl sulfate. This finding could also be useful to implement assays for carbohydrate-binding activity from cell or tissue extracts using different visualizable reagents bearing particular glycosyl moieties.

Isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (Phaseolus vulgaris) seeds.

A homodimeric lectin adsorbed on Affi-gel blue gel and CM-Sepharose and possessing a molecular weight of 67 kDa was isolated from red kidney beans. The hemagglutinating activity of this lectin was inhibited by glycoproteins but not by simple sugars. The lectin manifested inhibitory activity on human immunodeficiency virus-1 reverse transcriptase and alpha-glucosidase. The N-terminal sequence of the lectin exhibited some differences from previously reported lectins from Phaseolus vulgaris but showed some similarity to chitinases. It exerted a suppressive effect on growth of the fungal species Fusarium oxysporum, Coprinus comatus, and Rhizoctonia solani. The lectin had low ribonuclease and negligible translation-inhibitory activities.

Purification and characterization of a galactose-specific lectin from corn (Zea mays) coleoptile.

We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl-Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since beta-galactose residues are better recognized than those from the anomeric alpha-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.

Mechanisms of lectin (phytohemagglutinin)-induced growth in small intestinal epithelial cells.

BACKGROUND/AIMS: The lectin phytohemagglutinin is a mitogen for intestinal epithelial cells in vivo. The mechanisms of action are unknown and were therefore analyzed in vitro. METHODS: Human (Intestine-407) and rat (IEC-6; IEC-18) intestinal epithelial cell lines were stimulated with phytohemagglutinin. Proliferation was assayed by (3)H-thymidine incorporation, activation of mitogen-activated protein kinase (MAPK) by Western blotting, and induction of c-fos mRNA expression by semiquantitative polymerase chain reaction. Control experiments were performed with phenyl-N-acetyl-alpha-D-galactosaminide or the tyrosine kinase inhibitor tyrphostin A25. RESULTS: Phytohemagglutinin (0.1 microg/ml) significantly stimulated proliferation in all three cell lines after 48-72 h. MAPK activation was detected after 15-30 min, and an induction of c-fos mRNA expression after 15- 30 min of stimulation. Mitogenic effects were blocked by preincubation with phenyl-N-acetyl-alpha-D-galactosaminide or tyrphostin A25. CONCLUSION: Phytohemagglutinin stimulated proliferation, MAPK activation and induction of c-fos mRNA expression. The lectin may contribute to intestinal mucosal growth and regeneration thereby preventing gut atrophy.

Targeting and release of phytohemagglutinin from the roots of bean seedlings.

Phytohemagglutinin (PHA), an abundant vacuolar seed protein of the common bean (Phaseolus vulgaris), is a tetramer of two homologous polypeptides, PHA-E and PHA-L. The roots of bean seedlings release into the culture medium a cross-reacting lectin that is most closely related to PHA-E. Reverse-transcriptase polymerase chain reaction with root mRNA as template was used to identify PHA transcripts in the roots of bean seedlings. Roots were found to contain mRNA for PHA-E but not for PHA-L. Indirect immunocytochemical detection with colloidal gold and antibodies to deglycosylated PHA showed that in the meristem of the primary root, PHA accumulates in vacuoles. However, in elongated root cells PHA was found only in the cell walls, indicating targeting to an alternate location. These results are discussed in relation to the various mechanisms that may account for the release of a normally vacuolar protein by roots.

Red kidney beans--to eat or not to eat?

The importation of dry red kidney beans (a variety of the species Phaseolus vulgaris) for cultivation or consumption in South Africa is prohibited because of their potential toxicity to humans. It has been established that the haemagglutinating lectins (e.g. phytohaemagglutinin (PHA)) in kidney beans are responsible for this toxicity. Dry bean varieties available on the South African market for human consumption as well as locally produced (for this study) and imported dry red kidney beans and imported canned red kidney beans were compared. The PHA activity and the effect of heat thereon were measured, before and after overnight soaking. The PHA activity in extracts of uncooked and incompletely cooked red kidney beans was not higher than the levels measured in 50% of the other bean varieties included in the study. These findings indicate that the toxic potentials and health risks associated with red kidney beans are similar to those of other dry beans already commercially available to South Africans.

Isolation of the lectin and an L4 isolectin from Phaseolus vulgaris by affinity chromatography on insoluble ovomucoid.

Lectins from extracts of Phaseolus vulgaris seeds have potent cell-agglutinating and lymphocyte-stimulating activity. An affinity adsorbent for lectins with specificity for the oligosaccharide structure was prepared by transforming ovomucoid, an oligosaccharide-rich glycoprotein, into an insoluble and stable gel. The ovomucoid was made insoluble by boiling a 20% solution (200 mg/ml) in 0.1 M Tris-HCl, pH 8.9, for 20 min. This insoluble gel was desialylated by treatment with 50 mN sulfuric acid for 1 h at 90 degrees C and fixed with 1% glutaraldehyde, pH 7.4, for 10 min. The Phaseolus lectin and the L4 isolectin could be isolated essentially in a single-step procedure, using different eluting conditions: 50 mM sodium formate buffer, pH 3.0, was used for PHA elution; a different column was eluted with 15 mM sodium tetraborate, pH 8.0, for desorbed L4 isolectin. Polyacrylamide gel electrophoresis of the lectin showed five distinct bands, whereas the L4 isolectin only presented one band. From 250 mg of saturated column, 8.25 mg of PHA was isolated. This adsorbent could be used several times with little change in binding capacity or selectivity.

Isolation of the lectin and an L4 isolectin from Phaseolus vulgaris by affinity chromatography on insoluble ovomucoid

Lectins from extracts of Phaseolus vulgaris seeds have potent cell-agglutinating and lymphocyte-stimulating activity. An affinity adsorbent for lectins with specificity for the oligosaccharide structure was prpared by transforming ovomucoid, an oligosaccharide-rich glycoprotein, into an insoluble and stable gel. The ovomucoid was made insoluble by boiling a 20 per cent solution (200 mg/ml) in 0.1 M Tris-HCl, pH 8.9, for 20 min. This insoluble gel was desialylated by treatment with 50 mN sulfuric acid for 1h at 90ºC and fixed with 1 per cent glutaraldehyde, pH 7,4, for 10 min. The Phaseólus lectin and the L4 isolectin could be isolated essentially in a single-step procedure, using different eluting conditions: 50 mM sodium formate buffer, pH 3,0, was used for PHA elutions; a different column was eluted with 15 mM sodium tetraborate, pH 8.0, for desorbed L4 isolectin. Polyacrylamide gel electrophoresis of the lectin showed five distinct bands, whereas the L4 isolectin only presented one band. From 250 mg of saturated column, 8.25 mg of PHA was isolated. This adsorbent could be used several times with little change in binding capacity or selectivity

Isolation of an immunosuppressive lectin from Phaseolus vulgaris L. cv Cacahuate using stroma.

An immunosuppressive lectin was isolated from seed of Phaseolus vulgaris cv Cacahuate using physically entrapped stroma. The lectin was found to be a 94 kDa tetrameric protein. When 50 micrograms, of this lectin were administered intraperitoneally 2 days before the immunization with sheep red blood cells, humoral response against the immunogen was completely inhibited. Other properties of the protein are discussed.

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry.

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.

Phaseolus vulgaris L. var. HUR 15--a potential indigenous source for commercial PHA preparation.

Mitogenic potential of the partially purified lectin from P. vulgaris isolated by ammonium sulphate precipitation was assessed by lymphocyte transformation test (LTT) and was compared with three commercially available phytohemagglutinins (PHA). The blast inducing capacity and mitotic index analysis revealed that this preparation has potential to be used in the indigenous commercial production of PHA which is routinely used for human chromosomal studies from the peripheral blood culture at, at present, is imported.

IgA binding lectins isolated from distinct Artocarpus species demonstrate differential specificity.

The discovery of jacalin, a group of lectins from jackfruit seeds (Artocarpus heterophyllus), has attracted considerable attention due to its numerous interesting immunological properties as well as its usefulness in the isolation of various serum proteins. We have further identified a similar lectin from the seeds of Champedak (Artocarpus integer) which we refer to as lectin-C and performed comparative studies with two types of jacalin isolated from different batches of the Malaysian jackfruit seeds (jacalin-M1 and jacalin-M2). The three purified lectins demonstrated equivalent apparent Mr of about 52,500, each of which comprised of a combination of two types of non-covalently-linked subunits with apparent Mr of approximately 13,300 and 16,000. The lectins demonstrated equal haemagglutinating activity against human erythrocytes of blood groups A, B, AB and O. Our data also demonstrated that lectin-C, jacalin-M1 and jacalin-M2 are similar by selectively precipitating human serum IgA1 and colostral sIgA but not IgA2, IgD, IgG and IgM. When immunoelectrophoresis was performed on normal human sera and reacted with the lectins, single precipitin arcs corresponding to IgA immunoprecipitates were detected with lectin-C and jacalin-MI. Jacalin-M2, however, exhibited two closely associated precipitin arcs. The binding of these lectins with IgA was pronouncedly inhibited in the presence of p-nitrophenyl-beta-D-galactopyranoside, 1-o-methyl-alpha-D-galactopyranoside, D-melibiose, N-acetyl-D-galactosamine and D-galactose. The data therefore provide evidence on the differential specificity of IgA binding lectins isolated from seeds of similar as well as distinct Artocarpus species.