Article pubs.acs.org/JAFC
Isolation of a Hemagglutinin with Potent Antiproliferative Activity and a Large Antifungal Defensin from Phaseolus vulgaris cv. Hokkaido Large Pinto Beans Cuiming Yin, Jack Ho Wong,* and Tzi Bun Ng* School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China S Supporting Information *
ABSTRACT: Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 μM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali. KEYWORDS: hemagglutinin, antiproliferative, antifungal, defensin
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60 g of beans was homogenized in a Waring blender in 500 mL of 20 mM Tris-HCl buffer (pH 7.6). The homogenate was centrifuged at 16000 rpm for 30 min at 4 °C. The supernatant was filtered by using a piece of filter paper before loading onto an Affi-gel blue gel (Bio-Rad) column (18 cm × 5 cm) that had been pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.6). Then, the column was washed with 1 M NaCl in 20 mM Tris-HCl buffer (pH 7.6) to elute the adsorbed proteins. The adsorbed fraction was dialyzed against double-distilled water overnight at 4 °C and lyophilized into powder form. The powder was resuspended (20 mg/mL buffer) in 20 mM NH4HCO3 buffer (pH 9.6) before FPLC-anion exchange chromatography on a Mono Q column (GE Healthcare) using an AKTA Purifier (GE Healthcare). Adsorbed proteins were eluted using a 0−1 M NaCl gradient in 20 mM NH4HCO3 buffer (pH 9.6). The first peak containing adsorbed materials was collected, dialyzed, lyophilized, resuspended in 20 mM Tris-HCl buffer (pH 7.6) (5 mg/mL), and then subjected to FPLC-gel filtration on a Superdex 75 HR 10/300 column (GE Healthcare). The first major absorbance peak contained purified Hokkaido large pinto bean lectin. The three chromatographic steps used for purifying defensin from Hokkaido large pinto beans were the same as described above for the purification of hemagglutinin. In the Mono Q purification step, the unbound fraction was collected and dialyzed against double-distilled water, and buffer was added, resulting in a protein solution in 20 mM Tris-HCl buffer (pH 7.6). Then the sample was subjected to FPLC-gel filtration on a Superdex 75 HR 10/300 column (GE Healthcare). The last major absorbance peak contained purified Hokkaido large pinto bean defensin. Molecular Mass Determination. Sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a 15% separating gel and a 5% stacking gel. The protein sample was
INTRODUCTION From ancient times to the present, pathogen invasion is a health hazard to most of living organisms. During the process of evolution, organisms have developed various defense mechanisms to eliminate these potential life-threatening risks. Lectins and defensins are two types of defense proteins that plants secrete for self-protection.1 Lectins (hemagglutinins) are sugar-binding proteins with high carbohydrate selectivity, which exist ubiquitously in diverse species of plants, animals, viruses, bacteria, and fungi.2 Along with a defensive nature, some of them, such as lectins from edible plants, demonstrate a strong capability of inhibiting cancer cell proliferation. For example, concanavalin A (Con A), a mannoseand glucose-binding lectin isolated from jack bean, was found to have considerable potential in anticancer applications.3 However, apart from it, currently available lectins may have their own limitations for further development and application. Thus, the discovery of more potential targets is of great significance and a worthwhile undertaking. Phaseolus vulgaris is a leguminous species that produces common edible beans. Different P. vulgaris cultivars share the same characteristic; that is, most of them contain a lectin or a phytohemagglutinin as a storage protein. Even though lectins from various cultivars have similar molecular sizes, they may possess different biological activities and vary in the potency of a particular activity. Antifungal proteins such as defensin may or may not be present in a cultivar. Hence, we set out to isolate a lectin and an antifungal protein from P. vulgaris cultivar Hokkaido large pinto bean.
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MATERIALS AND METHODS
Received: Revised: Accepted: Published:
Purification of Hemagglutinin from Hokkaido Large Pinto Bean. Hokkaido large pinto beans were purchased from a Japanese food supermarket in Hong Kong. After soaking in distilled water overnight, © 2015 American Chemical Society
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January 26, 2015 May 7, 2015 May 12, 2015 May 12, 2015 DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry diluted with loading buffer containing β-mercaptoethanol (10% glycerol, 0.4% SDS, 0.005% bromophenol blue, and 20 mM EDTA in 0.5 M Tris-HCl, pH 7.5, 5% β-mercaptoethanol) to reduce disulfide bonds in the proteins. The samples were boiled in water for 5 min. Then, electrophoresis was performed at a constant current of 10 mA and a constant voltage of 120 V for about 75 min. After electrophoresis, the gel was stained with Coomassie brilliant blue, and it was put on a shaker to shake for 1 h and then destained with 10% acetic acid overnight.4 Determination of N-Terminal Amino Acid. N-Terminal amino acid sequence analysis was carried out using an HP 1000A Edman degradation unit and an HP 1000 HPLC system (Agilent Technologies, Santa Clara, CA, USA). Mass Spectrum Analysis. The molecular mass of the antifungal protein was verified by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), using an Applied Biosystems 4700 Proteomics Analyzer. Matrix-assisted laser desorption/ionization time-of-flight time-offlight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) was utilized to fragment sample proteins by MS/MS, and the fragments were identified by top-down proteomic analysis. The defense protein was identified by analysis of MS and MS/MS data using NCBInr and SwissProt databases (Greenplant).5 Assay of Hemagglutinating Activity. A 50 μL test sample was subjected to a 2-fold serial dilution with phosphate-buffered saline (PBS) (pH 7.2) in a 96-well microtiter U-plate. Afterward, 50 μL of a rabbit red blood cell suspension (2%) in PBS was added to each diluted sample. PBS (50 μL) mixed with 50 μL rabbit red blood cell was used as a negative control. The plate was incubated at room temperature until the red blood cells in the negative control formed a red dot at the bottom of the well, which means it had fully sedimented. The hemagglutinating activity of each of the test samples was then recorded. One hemagglutination unit or hemagglutination titer is defined as the reciprocal of the highest dilution of the lectin sample that induces hemagglutination. Specific hemagglutinating activity is the number of hemagglutination units per milligram of protein.6 Determination of pH Stability and Temperature Stability of Hemagglutinating Activity. To ascertain the effect of pH, solutions/ buffers with different pH values from 0 to 14 were prepared as follows: pH 0−1, HCl; pH 2−5, NH4OAc; pH 6−10, Tris-HCl; pH 11−12, NaHCO3; and pH 13−14, NaOH. The protein sample was mixed with an equal volume (50 μL) of a solution at a specified pH and incubated at room temperature for 30 min. Then, the mixtures were neutralized and assay of hemagglutinating activity was carried out. The hemagglutinating activity remaining was determined.7 To test the effect of temperature, the protein sample was heated at different temperatures from 20 to 100 °C, at 10 °C intervals for 30 min. The sample was immediately cooled on ice for another 30 min to terminate the incubation and then warmed up to room temperature. The assay of hemagglutinating assay was carried out as described above. The hemagglutinating activity of the lectin following incubation at room temperature was considered as 100% activity. Effects of Carbohydrates and Metal Ions on Hemagglutinating Activity. To investigate the carbohydrate specificity of the purified sample, 200 mM solutions of various kinds of sugars including N-acetylglucosamine, L-arabinose, D-fructose, D-fucose, D-galactose, D-glucose, lactose, maltose, D-mannose, raffinose, L-rhamnose, and D-xylose were used to dissolve the lyophilized sample to 0.1 mg/mL. Assay of hemagglutinating activity was carried out using the carbohydrate solutions at their particular concentrations for 2-fold serial dilution instead of PBS. The percentage of hemagglutinating activity remaining was calculated.8 On the other hand, the effects of metallic chlorides encompassing calcium chloride, manganese chloride, ferrous chloride, magnesium chloride, copper chloride, and zinc chloride on the hemagglutinating activity of Hokkaido large pinto bean hemagglutinin were also tested. First, the hemagglutinating activity of the original hemagglutinin solution was assessed and the fold number of 2-fold serial dilution that caused the test sample solution to begin to lose its hemagglutining activity was recorded. Then the sample solution was diluted with the recorded fold. Then, 25 μL of a 200 mM solution of a specified metallic
salt (in 0.9% NaCl) was subjected to a 2-fold serial dilution with 0.9% NaCl in a 96-well plate. Afterward, 25 μL of diluted sample solution was added to each well, and the mixture was incubated at room temperature for 1 h. In the control group, 25 μL of NaCl was used for 2-fold serial dilution instead of the metallic salt. Fifty microliters of a 2% rabbit erythrocyte suspension were then added to each well for another 2 h incubation at room temperature. Hemagglutination was estimated afterward. A second control experiment containing only the metal salt and red blood cells was run concurrently to ascertain the effect of each metal salt on the red blood cells in the assay. Assay of Antifungal Activity. Antifungal activity assay was carried out for investigating the antifungal potential of Hokkaido large pinto bean hemagglutinin. Different phytopathogenic fungi including Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, Fusarium oxysporum, and Valsa mali were grown in Petri dishes (90 mm × 15 mm) containing 10 mL of potato dextrose agar. A small amount of each fungal species was planted in the middle of the dish and then incubated at room temperature for a few days until it developed into a mycelial colony. Test samples (2 and 1 mg/mL) were added onto sterile blank round paper disks (0.625 cm in diameter), which were placed 0.5 cm away from the edge of the mycelial colony. Sterile buffer was added as control. After that the plates were incubated at room temperature until the mycelia had grown near the disk of the control group. If the mycelium could not grow near the disk and a clear invagination without mycelium was developed, it indicated that the test sample possessed antifungal activity.9 For testing the antifungal activity of the purified proteins against Candida albicans, 1 × 107 cells were treated with 2 mg of protein and incubated in Sabouraud dextrose broth (pH 5.5, Sigma). In the control, the protein sample was replaced by autoclaved broth. All eppendorf tubes were placed in an orbital shaker at 37 °C with shaking at a speed of 200 rpm and incubated for 6 h. Ten-fold serial dilution of 50 μL of the mixture containing the test protein and cells was performed in broth. Fifty microliters of the fourth and fifth 10-fold diluted solution were spread on agar plates. Cell counting was implemented after incubation of the plates at 37 °C for 24 h. The method for testing the antifungal activity of Hokkaido large pinto bean defensin was as previously described. Six species of fungi were tested including M. arachidicola, S. turcica, B. maydis, F. oxysporum, V. mali, and C. albicans. For the assay of antifungal activity toward C. albicans, the concentration of defensin used was 0.1 mg/mL. Assay of Antiproliferative Activity. Nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7 and MDA-MB-231), hepatoma (HepG2) cells, and an immortalized normal nasopharyngeal epithelial cell line NP 69 and human embryo liver (WRL 68) cells, from American Type Culture Collection, were suspended in RPMI medium at a cell density of 5 × 104 cells/mL. A 100 μL aliquot of these cell suspensions was seeded and incubated in the 96-well plates overnight. Then, the protein samples at concentrations of 0.49−125 μM (0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, and 125 μM) in 100 μL of RPMI medium were added to the wells, followed by incubation in a humidified atmosphere of 5% CO2 at 37 °C. After incubation for 24 and 48 h, the medium was discarded, and the wells were washed with PBS. Then, 25 μL of a 5 mg/mL solution of 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) in PBS was added into each well, and the plates were incubated at 37 °C for 4 h. After incubation, the plates were centrifuged at 2500 rpm for 5 min. The supernatant was carefully removed, and 150 μL of dimethyl sulfoxide (DMSO) was added into the wells to dissolve the MTT (formazan). OD 590 nm was measured within 10 min by using a microplate reader. Percentage inhibition of the MCF7, HepG2, and CNE2 cells was calculated by using the following formula: [(OD 590 nm of the control (without test protein) − OD 590 nm of the well with test protein at a particular concentration)/OD 590 nm of the control] × 100%.10 Results were expressed as means ± standard deviation (SD). The median inhibition concentration (IC50) was determined using SPSS 11.0.1 statistical software (SPSS Inc., Chicago, IL, USA). For betweengroup comparisons, one-way ANOVA was used. Differences with p values of ≤0.05 were considered to be statistically significant. 5440
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry
Figure 1. Purification of Hokkaido large pinto bean hemagglutinin: (A) Affi-gel blue gel; (B) Mono Q elution of fraction I; (C) Superdex 75 gel filtration of fraction II. The regions framed with dotted lines signify target fractions with hemagglutinating activity, which were collected and selected for the next step of purification. Peaks A and B represent fractions eluted at the 9th and 9.5th mL, respectively.
Figure 2. (A) Affi-gel blue gel eluate. Crude, crude extract; unbound, unbound fraction; bound, fraction I. (B) Mono Q eluate: 12, 13, 14, 15, and16, fractions collected at 12th to 16th mL. (C) Fractions A and B eluted from Superdex 75 eluted at the 9th and 9.5th mL, which were labeled in Figure 1C.
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RESULTS Purification of Hemagglutinin from Hokkaido Large Pinto Bean. Three chromatographic steps were utilized for the purification of hemagglutinin from Hokkaido large pinto bean. Hemagglutinins possess at least one noncatalytic domain that reversibly binds to specific carbohydrate structure. This property enables them to aggregate red blood cells. By using the test for hemagglutinating activity to monitor all of the chromatographic fractions, the target chromatographic fraction possessing
hemagglutinating activity was identified and selected for further purification The first chromatographic step on Affi-gel blue gel separated proteins into an unbound fraction that contained most of the proteins of Hokkaido large pinto bean, as shown in Figures 1A and 2A, and a bound fraction, which appeared as a sharper peak. The bound fraction I was obtained by elution with 1 M NaCl solution in the starting buffer. Both unbound and bound fractions were tested by using the test for hemagglutinating activity. 5441
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry
For investigation of carbohydrate selectivity, none of the carbohydrates at 200 mM concentration interacted with Hokkaido large pinto bean hemagglutinin including D-galactose, D-fructose, D-xylose, D-glucose, D-mannose, L-rhamnose, L-fucose, L-arabinose, xylitol, glucosamine, glucuronic acid, galactonic acid, maltose, α-lactose, and raffinose, as the hemagglutining activity was not attenuated after the addition of various kinds of carbohydrates. Testing the effect of metal ions on the hemagglutinating activity of Hokkaido large pinto bean hemagglutinin disclosed that 48.8 μM Cu2+ or Fe2+ ions, 97.7 μM Mn2+ ions, 195.3 μM Zn2+ ions, and 781.1 μM Ca2+ ions enabled the diluted protein sample solution to regain its hemagglutinating activity, indicating an augmenting effect. Assay of Antifungal Activity. The mycelia of different phytopathogenic fungi all successfully grew near the disks in the presence of Hokkaido large pinto bean hemagglutinin, as in the case of the negative control, and there was no difference in the cell number of C. albicans between the hemagglutinin-treated group and the control group. The data suggest that Hokkaido large pinto bean hemagglutinin had no antifungal effect. Assay for Antiproliferative Activity. The antiproliferative activity of Hokkaido large pinto bean hemagglutinin against HepG2, MDA-MB-231, MCF7, and CNE2 cancer cells was assessed by MTT assay. Results of the assay disclosed that treatment with Hokkaido large pinto bean hemagglutinin for 24 h did not achieve its maximum inhibitory effect (Figure 3A). At concentrations up to 125 μM, the hemagglutinin did not achieve 50% inhibition of each type of cancer cells after 24 h of treatment. The inhibitory effect against MDA-MB 231 metastatic breast cancer cells was minimal. The lectin achieved only 21% inhibition at the highest tested concentration (125 μM), compared with 43, 45, and 47% inhibition of proliferation of HepG2, MCF7, and CNE2 cancer cells, respectively. However, after uninterrupted incubation with Hokkaido large pinto bean hemagglutinin for 48 h, the inhibitory effect against each type of cancer cells was stronger than that observed after 24 h of treatment, with IC50 values of 8.135, 6.067, and 7.492 μM toward HepG2, MCF7, and CNE2 cancer cells, respectively. The IC50 value for MCF7 cells was smaller than that for CNE2 cancer cells. A further decline in proliferation rate was obtained in CNE2 cells after treatment with 125 μM hemagglutinin, which was 74% compared to 67%. At 125 μM concentration, Hokkaido large pinto bean hemagglutinin inhibited the growth of MDA-MB-231 cancer cells by 28% (Figure 3B). The hemagglutinin did not affect the viability of normal liver cell line WRL68 at 24 h, but
The bound fraction I was identified as the target chromatographic fraction containing hemagglutinating activity. From the SDS-PAGE gel photo shown in Figure 2A, proteins with a size of 30 kDa constituted the bulk of the adsorbed fraction. The second step was FPLC-cation exchange chromatography of bound fraction I from the Affi-gel blue gel column on Mono Q, which yielded one unadsorbed fraction and two major adsorbed peaks (Figure 1B). Hemagglutinating activity was enriched in fraction II. In the third step, fraction II was loaded to an FPLC-gel filtration Superdex 75 column for the purpose of further purification. A major peak was obtained including fractions A and B, which were collected at 9 and 9.5 mL, respectively (Figure 1C). Totally a yield of 190 mg of protein from fractions A and B was obtained from 60 g of Hokkaido large pinto bean (Table 1). Table 1. Purification of Hemagglutinin from Hokkaido Large Pinto Bean (60 g) chromatographic fraction
specific hemagglutinating activity (titer mg−1)
total protein (mg)
recovery (%)
crude extract Affi-gel blue gel Mono Q Superdex 75
34854 51200 63212 102400
3294 699 399 190
100.00 31.21 22.00 16.95
From the SDS-PAGE gel photo shown in Figure 2C, a single band with a molecular weight around 30 kDa was observed in fractions A and B, indicating that the target hemagglutinin was eluted in the ninth milliliter. On the basis of the standard calibration curve for the Superdex 75 column (data not shown), the molecular mass of Hokkaido large pinto bean hemagglutinin was about 60 kDa. This indicated that the hemagglutinin is a protein with a molecular mass of about 60 kDa and composed of two subunits. Determination of Temperature Stability, pH Stability, Carbohydrate Selectivity, and Metal Ion Effect. Hokkaido large pinto bean hemagglutinin exhibited moderate thermostability. Its hemagglutinating activity was maintained at temperatures up to 60 °C but dropped dramatically at 70 °C, when only half of the hemagglutinating activity remained. At 80 °C only 0.39% residual activity was detected, and the activity was completely abolished at 90 °C. Testing for pH stability revealed that it retained full activity in the pH range of pH 2−12, indicating fairly high pH stability. The hemagglutinating activity was halved at both pH 1 and 13 and vanished at pH 0 and 14.
Figure 3. Results of MTT assay for Hokkaido large pinto bean hemagglutinin: MTT assay results after treatment of HepG2, MDA-MB-231, MCF7, and CNE2 cancer cells with different concentrations of Hokkaido large pinto bean hemagglutinin for (A) 24 h and (B) 48 h. Results represent the mean ± SD (n = 3). 5442
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Figure 4. Purification of Hokkaido large pinto bean defensin: (A) affinity chromatography on Affi-gel blue gel; (B) FPLC of Affi-gel blue gel fraction I on Mono Q; (C) gel filtration of Mono Q fraction II on Superdex 75 by FPLC. Fractions a, b, c, and d were collected at elution volumes of 10.5, 12.5, 17, and 17.5 mL, respectively. Regions encircled with dotted lines signify target fractions with antifungal activity, which were collected and selected for the next step of purification.
Figure 5. SDS-PAGE results for isolation of Hokkaido large pinto bean defensin: (A) Affi-gel blue gel eluate (crude, crude extract; unbound, unbound fraction; bound, fraction I); (B) Mono Q eluate (M, molecular weight marker; 3 and 4, fraction collected at the 3rd and 4th mL, respectively); (C) M, molecular mass marker; a, b, c, d, fractions collected at elution volumes of 10.5, 12.5, 17, and 17.5 mL, respectively.
lectins/hemagglutinins from various P. vulgaris cultivars is presented in Table 4. Purification of Hokkaido Large Pinto Bean Defensin. Hokkaido large pinto bean defensin was discovered during the purification of Hokkaido pinto bean hemagglutinin. By
caused inhibition when the treatment was lengthened to 48 h (IC50 value > 80 μM), and gave rise to an IC50 value of 45 μM after treatment for 72 h (Table 3). Yet the effect was much weaker compared with the aforementioned tumor cell lines (IC50 values < 10 μM). A comparison of the characteristics of 5443
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry
Figure 6. Antifungal activity of Hokkaido large pinto bean defensin toward Mycosphaerella arachidicola, Fusarium oxysporum, Setosphaeria turcica, and Bipdaris maydis. (−), negative control (water); a, b, and c refer to fractions a, b, and c, which were collected at elution volumes of 10.5, 12.5, and 17 mL, respectively, during gel filtration on Superdex 75. Antifungal activity was detected only in fraction c.
monitoring the antifungal activity of chromatographic fraction obtained at each purification step with the assay of antifungal activity, it was observed that the crude extract and sample obtained after purification on Affi-gel blue gel (relevant results are shown in Figures 4A and 5A) contained antifungal protein(s), as they both exerted antifungal effect toward M. arachidicola (data not shown). In the second chromatographic step using FPLC-cation exchange on Mono Q, the unadsorbed fraction referred to as fraction II in Figure 4B exhibited antifungal activity. The proteins in this fraction (third and fourth milliliter eluted from Mono Q) were checked by SDS-PAGE, as shown in Figure 5B. After further purification of Mono Q fraction II by FPLC-gel filtration on a Superdex 75 column, several main peaks were obtained (Figure 4C). Fractions a, b, c, and d were collected at elution volumes of 10.5th, 12.5th, 17th, and 17.5th mL, respectively. The molecular size and purity of the proteins in the fractions are shown in Figure 5C. After assay of antifungal activity of each fraction, both fractions c and d were found to be effective (Figure 6). Collection of fractions c and d resulted in totally a yield of only 3.2 mg of protein from 60 g of Hokkaido large pinto beans (Table 2). Mass Spectrum Analysis. The molecular weight of the protein was confirmed by MALDI-TOF MS to be 10888 Da (Supporting Information). An analysis of MS and MS/MS data using NCBInr and SwissProt databases revealed 40% similarity with defensin D1 of P. vulgaris protein accession gi|312982410. Hence, the defense protein from fraction c can be regarded as a member of the defensin group.
Table 2. Purification of Defensin from Hokkaido Large Pinto Beans (60 g) chromatographic fraction
yield (mg)
yield (%)
crude extract Affi-gel blue gel fraction I Mono Q fraction II Superdex 75 fractions c and d
3294 699 20.1 3.2
100.0 21.22 0.61 0.097
Table 3. Percentage Reduction of Viability of WRL68 Normal Liver Cells (Mean ± SD, n = 3) Caused by Hemagglutinin from Hokkaido Large Pinto Beana hemagglutinin concn (μM)
24 h of incubation
48 h of incubation
72 h of incubation
2.5 5 10 20 40 80
6.28 ± 5.35a 3.63 ± 1.65a 1.84 ± 1.21a 4.57 ± 4.22a 3.12 ± 2.98a 7.03 ± 2.91a
6.96 ± 5.88a 2.31 ± 1.92a 1.22 ± 0.93a 17.67 ± 7.82b 26.54 ± 3.77b 44.71 ± 5.32b
14.43 ± 7.38b 11.46 ± 8.85b 22.55 ± 12.34b 40.26 ± 2.76c 46.33 ± 7.89c 65.51 ± 1.23c
a Different letters (a, b, c) next to the data indicate statistically significant difference. The same letter represents no statistically significant difference.
Antifungal Activity of Hokkaido Large Pinto Bean Defensin. Five fungal species were utilized in the assay of antifungal activity, comprising M. arachidicola, S. turcica, B. 5444
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry maydis, F. oxysporum, and V. mali. Distilled water was used instead of protein sample as negative control. The antifungal activity of fractions a, b, and c was tested. It was found that fraction b had no antifungal effect on all five types of fungal species, as the mycelial growth extended to the paper disks. Fraction a had only a weak antifungal effect on M. arachidicola. Fraction c was capable of inhibiting the growth of M. arachidicola, S. turcica, B. maydis, and F. oxysporum, as distinct crescent-like invaginations could be seen around the paper disks. Yet it could not inhibit the growth of the pathogenic fungus V. mali (data not shown). Protein in fraction d had a molecular size identical to that of the protein in fraction c, and it also possessed antifungal activity toward the same types of fungi, suggesting they are the same protein (data not shown). Hokkaido large pinto bean defensin exerted strong activities toward M. arachidicola and B.maydis and a weaker activity toward S. turcica and F. oxysporum. The defensin had no antifungal effect on C. albicans at the concentration of 0.1 mg/mL (data not shown).
also be taken into consideration when assessing the utility/value of a protein. With respect to structure and molecular size, Hokkaido large pinto bean hemagglutinin shared common characteristics with many lectins of P. vulgaris, which are dimeric proteins with a molecular mass of about 60 kDa.21 Yet there are exceptions, for example, a 94 kDa tetrameric lectin purified from the seeds of the Cacahuate cultivar of P. vulgaris.26 Despite these similar characteristics, hemagglutinins or lectins from different cultivars may perform distinctively regarding many biological activities, which may be due to the differences in their sequences. The N-terminal sequence is important for identifying a protein. By examining the N-terminal sequence, a number of P. vulgaris lectins have been identified.23,27−30 However, in this study no peaks were detected after seven sequencing cycles, indicating that the hemagglutinin most likely possessed a blocked N-terminus. This is not a unique case, as lectins of Castanea crenata,31 Schizophyllum commune,32 and Sclerotium rolfsii33 also had a blocked N-terminus. Lectins or hemagglutinins from P. vulgaris possess different thermal and pH stabilities. For instance, lectin from jade bean had modest pH and heat stability: it was stable only at pH 4.5− 9.4 and 30−70 °C,21 whereas lectins purified from Anasazi bean and the Indian cultivar of French bean manifested high pH and heat stability. They were stable at 80 and 90 °C, respectively, and retained their hemagglutinating activity after treatment with solutions at pH 1−14 and 1−13, respectively.19,27 The high thermal stability may be due to the absence of disulfide linkages, which become unstable at elevated temperatures or attributed to the absence of heat-sensitive tryptophan residues.34 Some lectins had poor thermostability and pH stability, such as French bean cultivar no. 35, which retained its hemagglutinating activity at an ambient temperature below 50 °C or at a pH within the range of 6−8.35 Hemagglutinin purified from Hokkaido large pinto bean was characterized by a modest thermostability and pH stability; it was stable at 30−60 °C and at pH values from 2 to 12. Considering its potential for application, it may be utilized as an agent for injection as it is stable at physiological temperature and pH in plasma. As the pH of gastric fluid ranges from 1 to 3, it still has a potential to be employed as a drug for oral administration even though its activity would be halved at pH 1. With regard to carbohydrate selectivity, selected simple saccharides did not exert any inhibitory effect on the hemagglutinating activity of Hokkaido large pinto bean hemagglutinin. A number of lectins isolated from other P. vulgaris cultivars including cultivar 12, cultivar 35, Indian cultivar of French beans, Hokkaido red bean, northeast red bean, etc., also exhibited this common phenomenon.19,23,34,36 On the other hand, the sugarbinding ability of lectins of some other P. vulgaris cultivars has been discovered. For instance, lectin from cultivar no. 1 of French beans bound specifically to glucuronic acid,22 and lectins from both pinto beans and Extralong Autumn purple beans were galactose-specific.28 In addition, polysaccharides can also be the binding target of some lectins. For example, lectin isolated from Blue Tiger King bean was polygalacturonic acid-binding specific;28 meanwhile, the hemagglutinating activity of lectin from red kidney bean was reduced by several glycoproteins.29 Lectins are found to be divalent cation-dependent proteins.20,37 During purification, they may lose ions or become desaturated with ions, which provides a rationale to the assay of the effect of metal ions in this study. Results displayed that metal ions including Ca2+, Zn2+, Mn2+, Cu2+, or Fe2+ ions enhanced the hemagglutinating activity of Hokkaido large pinto bean
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DISCUSSION Nowadays, many lectins from edible plants with potent antitumor activity have been discovered. For instance, legume lectin Con A induced apoptosis in human melanoma A375 cells.3 Momordica charantia lectin exerted an inhibitory action on nasopharyngeal carcinoma cells.11 Banana lectin demonstrated a suppressive effect on leukemia (L1210) and HepG2 cells.12 Broccolini lectin13 and taro lectin14, etc., are all potential candidates as anticancer agents. P. vulgaris merits research efforts because of its relatively inexpensive price, multiple cultivar sources, and high potential in anticancer activity. As Hokkaido large pinto bean has not been investigated before, we selected it for the present study. By applying affinity chromatography on Affi-gel blue gel and Blue Sepharose, ion exchange chromatography on SP-Sepharose, Mono Q, and Q Sepharose, and FPLC-gel filtration on Superdex 75 and Superdex 200, etc., a variety of lectins with protential were successfully purified from different organisms, such as lectins from jelly fish Nemopilema nomurai,15 bighead carp Aristichthys nobilis,16 coral Acropora millepora,17 Annona muricata seeds,18 and various P. vulgaris cultivars including French bean Indian cultivar,19 Blue Tiger King,20 jade bean,21 northeast red bean,22 and so on. The purification methods adopted in the present study can be used to obtain the target proteins in a fast and simple way. By using fewer purification steps, the loss of the target protein could be minimized. The purification efficiency and the percentage recovery of lectin, which is calculated using the formula percentage recovery = (total hemagglutinating titer of purified sample/total hemagglutinating titer in crude extract) × 100%, showed the yield of Hokkaido large pinto bean hemagglutinin was noteworthy in that 190 mg of hemagglutinin could be purified from 60 g of beans with 17% recovery. It may be due to the abundant occurrence of this storage protein in Hokkaido large pinto beans and the few steps of purification employed. Many lectins isolated from other cultivars of P. vulgaris may not have the same advantages. For instance, the yield of lectin from 100 g of French bean cultivar no. 12 was only 4.8 mg with 18.1% recovery;23 the corresponding values for Anasazi beans were 26 mg and 14% recovery;24 and the values for French beans (cultivar no. 1) were 96.25 mg and 18.73% recovery.7 Although larger amounts of lectin from other cultivars have been reported, for example, 428 mg of brown kidney bean lectin from 100 g of beans with about 57% recovery,25 other advantages such as performance in assays of various biological activities should 5445
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
Article
Journal of Agricultural and Food Chemistry Table 4. Summary of Properties of some Phaseolus vulgaris Lectins mol wt (kDa)
thermostability (°C)
ion effect ≤100 μM
IC50 (μM) anticancer activity, 48 h
2−12
up to 60 up to 50
HepG2 (8.1), MCF7 (6.07), and CNE2 (7.49) HepG2 (7.9 ± 0.5)
ND
3−10
Cu2+, Fe2+, Mn2+ Fe3+,Ca2+
4.5−9.4 6−8
30−70 0−50
Ca2+ N/A
MCF7 (169) HepG2 (100), MCF7 (2)
ND Valsa mali
ND
2−12
up to 65
N/A
N/A
glucosamine
7−8
up to 60
Mg2+
CNE1 (1.63), MCF7 (11.66), CNE2 (35) (24 h) nasopharyngeal carcinoma (HONE-1) cells (17.3)
cultivar
yield
Hokkaido large pinto bean Blue Tiger King53
190 mg/60 g
60
NDa
81 mg/350 g
60
162 mg/50 g 1.1 g/100 g
60.8 64
polygalacturonic acid ND ND
292 mg/100 g
64
110 mg/90 g
58
jade bean54 French bean no. 3535 northeast red bean55 Chinese pinto bean56 a
sugar binding
pH stability
antifungal activity
N/Ab
Valsa mali
ND, not determined. bN/A, not applicable.
similar antitumor activity, such as lectin from Lactarius flavidulus (IC50 = 8.9 μM and 6.81 μM for HepG2 and leukemic L1210 cells, respectively);10 Nagaimo lectin significantly reduced the growth of MCF7, HepG2, and CNE2 cells after 24 h of treatment, with IC50 values of 3.17, 7.12, and 19.79 μM, respectively.46 With the advantages of pH stability and strong anticancer acitivity, Hokkaido large pinto bean hemagglutinin has the potential to be developed as an antineoplastic agent. A summary of the properties of some P. vulgaris lectins (Table 4) revealed that Hokkaido large pinto bean hemagglutinin manifested the advantageous features of a potent antiproliferative effect toward different types of cancer cells, a wider pH range over which it was stable, and a considerably higher yield, which demonstrate its potential as an anticancer candidate. Three types of chromatography including affinity chromatography, ion exchange chromatography, and FLPC-gel filtration were utilized for purification of defensin from Hokkaido large pinto beans. The protocol resembles purification procedures for some other defensins.47,48 It has been reported that plant defensins have been discovered to possess an isoelectric point around 8−9.49 This facilitates isolation of plant defensins by means of ion exchange chromatography: adsorption on cation exchangers and lack of binding to anion exchangers in neutral buffers (e.g., Tris-HCl, pH 7.6) or acidic buffers (e.g., NH4OAc, pH 4.6). The yield of the 10888 Da Hokkaido large pinto bean defensin was only 3.2 mg from 60 g of beans. In other studies on purification of defensin, such as the purification of northeast red bean antifungal peptide, 8 mg of antifungal peptide was extracted from 100 g of beans. Higher yields were observed in the purification of defensin from Legumi secchi bean and ground bean, which were 43 and 14 mg from 100 g of beans, respectively. However, a lower yield was also encountered in the purification of cloud bean defensin, which was 1.12 mg per 100 g of seeds. With regard to its biological activity, Hokkaido large bean defensin displayed antifungal activity on different types of fungi including M. arachidicola, S. turcica, F. oxysporum, and B. maydis. However, fungi such as V. mali and C. albicans were resistant to Hokkaido large pinto bean defensin. In addition, most of the previously reported plant defensins were small proteins with a molecular size ranging from 5 to 9 kDa.49 With a molecular size of 10.888 kDa, Hokkaido large pinto bean defensin is a big defensin. Defensins with a large size also exist in animals. For instance, a 79-residue defensin, which is responsible for host defense, was found in the Japanese horseshoe crab.50 To recapitulate, the pinto bean is an edible bean common in the United States and the Far East. The pH-stable and thermostable hemagglutinin, isolated with a remarkable yield
hemagglutinin at different concentrations, whereas Mg2+ ions failed to strengthen the diluted hemagglutinin to regain its hemagglutining capacity. Cu2+ or Fe2+ ions had the most obvious effect at the minimum concentration, followed by Mn2+ and Zn2+ ions, whereas Ca2+ ions were the least effective. It should be remembered that the effect of metal ion was uncertain. It was demonstrated that Ca2+, Zn2+, Mn2+, and Mg2+ had no effect on the hemagglutinating activity of Grifola frondosa lectin,38 whereas they restrained the lectin of Schizophyllum commune.39 This may be due to the change of protein structure in the presence of metal ions.40 For the P. vulgaris species, the effect on lectins varies among different cultivars. The activity of Con A, which is a wellknown lectin isolated from jack beans, could be enhanced in the presence of Ca2+, Mg2+, and Mn2+ ions.21 The potentiating effect of Ca2+ ions was not very commonly found and has been reported only for a few Phaseolus lectins, including P. coccineus lectin,41 P. lunatus lectin,42 and P. acutifolius lectin,43 which was probably because Ca2+ ions enable the lectins to resist tryptic proteolysis. For antifungal activity, Hokkaido large pinto bean hemagglutinin did not affect the growth of the tested fungi. To date, it has been found that some lectins possess antifungal activity. For example, Aleuria aurantia lectin inhibited the growth of Mucor racemosus.44 French bean no. 35 lectin exhibited antifungal activity against V. mali, F. oxysporum, and Rhizoctonia solani.35 However, a number of lectins without antifungal effect have been reported, such as lectin from jade bean,21 Extralong Autumn purple bean,28 and ground bean45 etc. The proliferation of several kinds of cancer cells encompassing hepatoma (HepG2) cells, breast cancer (MCF7) cells, and nasopharyngeal cancer (CNE2) cells was reduced by Hokkaido large pinto bean hemagglutinin in a time- and dose-dependent manner, whereas its antiproliferative effect on MDA-MB-231 breast cancer cell was only meager. The range of tumors cells found to be sensitive to Hokkaido large pinto bean hemagglutinin was large, compared to that of lectin purified from Blue Tiger King bean, which was specific toward HepG2 cells,20 as well as lectin from French bean cultivar no. 35, which was active toward MCF7 cells but not toward HepG2 cancer cells.35 The anticancer effect of Hokkaido large pinto bean hemagglutinin in vitro was potent among many lectins that have been discovered to date. For instance, its IC50 values for HepG2, MCF7, and CNE2 cells after a 48 h exposure were 8.14, 6.07, and 7.49 μM, respectively, which were much smaller in magnitude than that of acaconin from seeds of Acacia conf usa (IC50 = 128 ± 9 μM for MCF7 cells);10 jade bean lectin (IC50 = 169 μM for MCF7 cells);21 and Chinook salmon roe lectin (IC50 = 68 μM for HepG2 cells), etc. Some lectins showed a 5446
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
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Journal of Agricultural and Food Chemistry
(11) Fang, E. F.; Zhang, C. Z.; Ng, T. B.; Wong, J. H.; Pan, W. L.; Ye, X. J.; Chan, Y. S.; Fong, W. P. Momordica charantia lectin, a type II ribosome inactivating protein, exhibits antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo. Cancer Prev. Res. 2012, 5 (1), 109−121. (12) Cheung, A. H.; Wong, J. H.; Ng, T. B. Musa acuminata (Del Monte banana) lectin is a fructose-binding lectin with cytokine-inducing activity. Phytomedicine 2009, 16 (6−7), 594−600. (13) Xu, P.; Zhang, T.; Guo, X.; Ma, C.; Zhang, X. Purification, characterization, and biological activities of broccolini lectin. Biotechnol. Prog. 2015, March 3. DOI: 10.1002/btpr.2070 [Epub ahead of print]. (14) Chan, Y. S.; Wong, J. H.; Ng, T. B. A cytokine-inducing hemagglutinin from small taros. Protein Pept. Lett. 2010, 17 (7), 823− 830. (15) Imamichi, Y.; Yokoyama, Y. Purification, characterization and cDNA cloning of a novel lectin from the jellyfish Nemopilema nomurai. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2010, 156 (1), 12− 18. (16) Pan, S.; Tang, J.; Gu, X. Isolation and characterization of a novel fucose-binding lectin from the gill of bighead carp (Aristichthys nobilis). Vet. Immunol. Immunopathol. 2010, 133 (2−4), 154−164. (17) Kvennefors, E. C. E.; Leggat, W.; Hoegh-Guldberg, O.; Degnan, B. M.; Barnes, A. C. An ancient and variable mannose-binding lectin from the coral Acropora millepora binds both pathogens and symbionts. Dev. Comp. Immunol. 2008, 32 (12), 1582−1592. (18) Damico, D. C. S.; Freire, M. G. M.; Gomes, V. M.; Toyama, M. H.; Marangoni, S.; Novello, J. C.; Macedo, M. L. R. Isolation and characterization of a lectin from Annona muricata seeds. J. Protein Chem. 2003, 22 (7−8), 655−661. (19) Sharma, A.; Wong, J. H.; Lin, P.; Chan, Y. S.; Ng, T. B. Purification and characterization of a lectin from the Indian cultivar of French bean seeds. Protein Pept. Lett. 2010, 17 (2), 221−227. (20) Fang, E. F.; Pan, W. L.; Wong, J. H.; Chan, Y. S.; Ye, X. J.; Ng, T. B. A new Phaseolus vulgaris lectin induces selective toxicity on human liver carcinoma Hep G2 cells. Arch. Toxicol. 2011, 85 (12), 1551−1563. (21) Cheung, R. C. F.; Leung, H. H.; Pan, W. L.; Ng, T. B. A calcium ion-dependent dimeric bean lectin with antiproliferative activity toward human breast cancer MCF-7 cells. Protein J. 2013, 32 (3), 208−215. (22) Chan, Y. S.; Wong, J. H.; Fang, E. F.; Pan, W. L.; Ng, T. B. A hemagglutinin from Northeast red beans with immunomodulatory activity and anti-proliferative and apoptosis-inducing activities toward tumor cells. Protein Pept. Lett. 2013, 20 (10), 1159−1169. (23) Leung, E. H. W.; Wong, J. H.; Ng, T. B. Concurrent purification of two defense proteins from French bean seeds: a defensin-like antifungal peptide and a hemagglutinin. J. Pept. Sci. 2008, 14 (3), 349−353. (24) Ang, A. S. W.; Cheung, R. C. F.; Dan, X. L.; Chan, Y. S.; Pan, W. L.; Ng, T. B. Purification and characterization of a glucosamine-binding antifungal lectin from Phaseolus vulgaris cv. Chinese pinto beans with antiproliferative activity towards nasopharyngeal carcinoma cells. Appl. Biochem. Biotechnol. 2014, 172 (2), 672−686. (25) Chan, Y. S.; Zhang, Y. B.; Ng, T. B. Brown kidney bean BowmanBirk trypsin inhibitor is heat and pH stable and exhibits anti-proliferative activity. Appl. Biochem. Biotechnol. 2013, 169 (4), 1306−1314. (26) Vargasalbores, F.; Hernandez, J.; Cordoba, F.; Zenteno, E. Isolation of an immunosuppressive lectin from Phaseolus vulgaris L cv Cacahuate using stroma. Prep. Biochem. 1993, 23 (4), 473−483. (27) Sharma, A.; Ng, T. B.; Wong, J. H.; Lin, P. Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans). J. Biomed. Biotechnol. 2009, 2009, 929568. (28) Fang, E. F.; Lin, P.; Wong, J. H.; Tsao, S. W.; Ng, T. B. A lectin with anti-HIV-1 reverse transcriptase, antitumor, and nitric oxide inducing activities from seeds of Phaseolus vulgaris cv. Extralong Autumn purple bean. J. Agric. Food Chem. 2010, 58 (4), 2221−2229. (29) Ye, X. Y.; Ng, T. B.; Tsang, P. W. K.; Wang, J. Isolation of a homodimeric lectin with antifungal and antiviral activities from red kidney bean (Phaseolus vulgaris) seeds. J. Protein Chem. 2001, 20 (5), 367−375.
(190 mg/60 g), has potent antiproliferative activity toward various tumor cells including nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7 cells), and hepatoma (HepG2) cells and thus noteworthy health-promoting activity. Currently there are not many publications reporting the action of lectins against nasopharyngeal cancer cells. The isolated defensin has a larger molecular weight than previously reported defensins and antifungal activity toward various phytopathogenic fungi and could be used in the biological control of agriculturally important fungi. It deserves mention that by comparison some antifungal proteins have activity against only one or two fungal species.51,52
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ASSOCIATED CONTENT
* Supporting Information S
Supplementary Figure 1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jafc.5b00475.
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AUTHOR INFORMATION
Corresponding Authors
*(J.H.W.) E-mail:
[email protected]. Phone: 852-39438031. Fax: 852-26035123. *(T.B.N.) E-mail:
[email protected]. Phone: 852-39436872. Fax: 852-26035123. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Wong, J. H.; Ng, T. B.; Cheung, R. C.; Ye, X. J.; Wang, H. X.; Lam, S. K.; Lin, P.; Chan, Y. S.; Fang, E. F.; Ngai, P. H.; Xia, L. X.; Ye, X. Y.; Jiang, Y.; Liu, F. Proteins with antifungal properties and other medicinal applications from plants and mushrooms. Appl. Microbiol. Biotechnol. 2010, 87 (4), 1221−1235. (2) Li, Y. R.; Liu, Q. H.; Wang, H. X.; Ng, T. B. A novel lectin with potent antitumor, mitogenic and HIV-1 reverse transcriptase inhibitory activities from the edible mushroom Pleurotus citrinopileatus. Biochim. Biophys. Acta, Gen. Subj. 2008, 1780 (1), 51−57. (3) Liu, B.; Li, C. Y.; Bian, H. J.; Min, M. W.; Chen, L. F.; Bao, J. K. Antiproliferative activity and apoptosis-inducing mechanism of voncanavalin A on human melanoma A375 cells. Arch. Biochem. Biophys. 2009, 482 (1−2), 1−6. (4) Laemmli, U. K.; Johnson, R. A. Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles. J. Mol. Biol. 1973, 80 (4), 601−611. (5) Fagerquist, C. K.; Sultan, O. Top-down proteomic identification of furin-cleaved α-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS. J. Biomed. Biotechnol. 2010, 2010, 123460. (6) Wong, J. H.; Ng, T. B. Isolation and characterization of a glucose/ mannose-specific lectin with stimulatory effect on nitric oxide production by macrophages from the emperor banana. Int. J. Biochem. Cell Biol. 2006, 38 (2), 234−243. (7) Chan, Y. S.; Wong, J. H.; Ng, T. B. A glucuronic acid binding leguminous lectin with mitogenic activity toward mouse splenocytes. Protein Pept. Lett. 2011, 18 (2), 194−202. (8) Koike, T.; Beppu, H.; Kuzuya, H.; Maruta, K.; Shimpo, K.; Suzuki, M.; Titani, K.; Fujita, K. A 35 kDa mannose-binding lectin with hemagglutinating and mitogenic activities from “Kidachi Aloe” (Aloe arborescens Miller var. natalensis Berger). J. Biochem. 1995, 118 (6), 1205−1210. (9) Ye, X. Y.; Wang, H. X.; Ng, T. B. Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab). Biochem. Biophys. Res. Commun. 2000, 269 (1), 155−159. (10) Lam, S. K.; Ng, T. B. Acaconin, a chitinase-like antifungal protein with cytotoxic and anti-HIV-1 reverse transcriptase activities from Acacia confusa seeds. Acta Biochim. Polym. 2010, 57 (3), 299−304. 5447
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
Article
Journal of Agricultural and Food Chemistry
(50) Kouno, T.; Mizuguchi, M.; Aizawa, T.; Shinoda, H.; Demura, M.; Kawabata, S.; Kawano, K. A novel β-defensin structure: big defensin changes its N-terminal structure to associate with the target membrane. Biochemistry 2009, 48 (32), 7629−7635. (51) Wang, H.; Ng, T. B. Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus of f icinalis seeds. Biochem. Biophys. Res. Commun. 2001, 289 (1), 120−124. (52) Wang, H. X.; Ng, T. B. Ascalin, a new anti-fungal peptide with human immunodeficiency virus type 1 reverse transcriptase-inhibiting activity from shallot bulbs. Peptides 2002, 23 (6), 1025−1029. (53) Fang, E. F.; Pan, W. L.; Wong, J. H.; Chan, Y. S.; Ye, X. J.; Ng, T. B. A new Phaseolus vulgaris lectin induces selective toxicity on human liver carcinoma Hep G2 cells. Arch. Toxicol. 2011, 85 (12), 1551−1563. (54) Cheung, R. C.; Leung, H. H.; Pan, W. L.; Ng, T. B. A calcium iondependent dimeric bean lectin with antiproliferative activity toward human breast cancer MCF-7 cells. Protein J. 2013, 32 (3), 208−215. (55) Chan, Y. S.; Wong, J. H.; Fang, E. F.; Pan, W.; Ng, T. B. A hemagglutinin from northeast red beans with immunomodulatory activity and anti-proliferative and apoptosis-inducing activities toward tumor cells. Protein Pept. Lett. 2013, 20 (10), 1159−1169. (56) Ang, A. S.; Cheung, R. C.; Dan, X.; Chan, Y. S.; Pan, W.; Ng, T. B. Purification and characterization of a glucosamine-binding antifungal lectin from Phaseolus vulgaris cv. Chinese pinto beans with antiproliferative activity towards nasopharyngeal carcinoma cells. Appl. Biochem. Biotechnol. 2014, 172 (2), 672−686.
(30) Xia, L. X.; Ng, T. B. A hemagglutinin with mitogenic activity from dark red kidney beans. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2006, 844 (2), 213−216. (31) Nomura, K.; Ashida, H.; Uemura, N.; Kushibe, S.; Ozaki, T.; Yoshida, M. Purification and characterization of a mannose/glucosespecific lectin from Castanea crenata. Phytochemistry 1998, 49 (3), 667− 673. (32) Chumkhunthod, P.; Rodtong, S.; Lambert, S. J.; FordhamSkelton, A. P.; Rizkallah, P. J.; Wilkinson, M. C.; Reynolds, C. D. Purification and characterization of an N-acetyl-D-galactosaminespecific lectin from the edible mushroom Schizophyllum commune. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760 (3), 326−332. (33) Sathisha, G. J.; Prakash, Y. K. S.; Chachadi, V. B.; Nagaraja, N. N.; Inamdar, S. R.; Leonidas, D. D.; Savithri, H. S.; Swamy, B. M. X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry. Amino Acids 2008, 35 (2), 309−320. (34) Wong, J. H.; Wan, C. T.; Ng, T. B. Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean). J. Sci. Food Agric. 2010, 90 (1), 70−77. (35) Lam, S. K.; Ng, T. B. Isolation and characterization of a French bean hemagglutinin with antitumor, antifungal, and anti-HIV-1 reverse transcriptase activities and an exceptionally high yield. Phytomedicine 2010, 17 (6), 457−462. (36) Chan, Y. S.; Ng, T. B. Northeast red beans produce a thermostable and pH-stable defensin-like peptide with potent antifungal activity. Cell Biochem. Biophys. 2013, 66 (3), 637−648. (37) Neth, O.; Jack, D. L.; Dodds, A. W.; Holzel, H.; Klein, N. J.; Turner, M. W. Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect. Immun. 2000, 68 (2), 688−693. (38) Kawagishi, H.; Nomura, A.; Mizuno, T.; Kimura, A.; Chiba, S. Isolation and characterization of a lectin from Grifola f rondosa fruiting bodies. Biochim. Biophys. Acta, Gen. Subj. 1990, 1034 (3), 247−252. (39) Han, C. H.; Liu, Q. H.; Ng, T. B.; Wang, H. X. A novel homodimeric lactose-binding lectin from the edible split gill medicinal mushroom Schizophyllum commune. Biochem. Biophys. Res. Commun. 2005, 336 (1), 252−257. (40) Sureshkumar, T.; Priya, S. Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization. Appl. Biochem. Biotechnol. 2012, 168 (8), 2257−2267. (41) Feria, M.; PerezSantiago, A.; Cuevas, D.; Martinez, M.; Cordoba, F. Purification and partial characterization of a new anti-A(1) lectin of Phaseolus coccineus collected in Oaxaca, Mexico. Prep. Biochem. Biotechnol. 1996, 26 (1), 31−46. (42) Roberts, D. D.; Goldstein, I. J. Effect of carbohydrate and metalion binding on the reactivity of the essential thiol-groups of lima bean lectin. J. Biol. Chem. 1984, 259 (2), 903−908. (43) Vargasalbores, F.; Delafuente, G.; Agundis, C.; Cordoba, F. Purification and characterization of a lectin from Phaseolus acutifolius var Latifolius. Prep. Biochem. 1987, 17 (4), 379−396. (44) Amano, K.; Katayama, H.; Saito, A.; Ando, A.; Nagata, Y. Aleuria aurantia lectin exhibits antifungal activity against Mucor racemosus. Biosci., Biotechnol., Biochem. 2012, 76 (5), 967−970. (45) Wong, J. H.; Ng, T. B. Purification of a trypsin-stable lectin with antiproliferative and HIV-1 reverse transcriptase inhibitory activity. Biochem. Biophys. Res. Commun. 2003, 301 (2), 545−550. (46) Chan, Y. S.; Ng, T. B. A lectin with highly potent inhibitory activity toward breast cancer cells from edible tubers of Dioscorea opposita cv. nagaimo. PLoS One 2013, 8 (1), No. e54212. (47) Wu, X.; Sun, J.; Zhang, G.; Wang, H.; Ng, T. B. An antifungal defensin from Phaseolus vulgaris cv. ‘Cloud Bean’. Phytomedicine 2011, 18 (2−3), 104−109. (48) Lam, S. K.; Ng, T. B. Purification and characterization of an antifungal peptide with potent antifungal activity but devoid of antiproliferative and HIV reverse transcriptase activities from Legumi secchi beans. Appl. Biochem. Biotechnol. 2013, 169 (7), 2165−2174. (49) Carvalho Ade, O.; Gomes, V. M. Plant defensins − prospects for the biological functions and biotechnological properties. Peptides 2009, 30 (5), 1007−1020. 5448
DOI: 10.1021/acs.jafc.5b00475 J. Agric. Food Chem. 2015, 63, 5439−5448
