Purification and Characterization of a Novel Lectin from Chinese Leek

Article pubs.acs.org/JAFC

Purification and Characterization of a Novel Lectin from Chinese Leek Seeds Jing Hong, Tao-tao Chen, Lei Hu, Jie Yang, Pei Hu, and Shao-yun Wang* College of Biological Science and Technology, Fuzhou University, 2 Xue Yuan Road, University Town, Fuzhou, Fujian 350108, China S Supporting Information *

ABSTRACT: A novel lectin, CLSL, was purified from Chinese leek seeds by ion exchange chromatography on SP Sephadex C-25 and gel filtration chromatography on Sephadex G50. The lectin had a molecular weight of 23.6 kDa and was composed of two identical subunits linked by disulfide bonds, a conclusion based on SDS-PAGE under reducing and nonreducing conditions. CLSL was a glycoprotein with a carbohydrate content of 3.6%. It exerted potent agglutinating activity against rat red blood cells at a concentration of 8.9 μg/mL. Hemagglutination of rat erythrocytes was inhibited by D-fructose, mannitol, and sorbose at the concentration of 20 mM. The hemagglutinating activity of CLSL was maintained at 100 °C for 60 min and under acidic pH conditions but was lost at neutral and alkaline pH conditions. The hemagglutinating activity was stimulated by Ca2+, Fe2+, and Cu2+ but inactivated by Ba2+ at a concentration of 10 mM. Ba2+-mediated inactivation of CLSL was caused by CLSL conformational change induced by barium ions, according to the results of circular dichroism and fluorescence spectroscopy. Deconvolution of the CLSL circular dichroism indicated that it was an α-helical lectin with α-helix and β-fold contents of 35.8% and 8.6%, respectively. CLSL could also selectively inhibit cell proliferation. KEYWORDS: Chinese leek seeds, lectin, purification, fluorescence spectroscopy, circular dichroism

■

INTRODUCTION Lectins constitute a class of carbohydrate-binding proteins of nonimmune origin that are ubiquitously found in microorganisms, plants, and animals.1 Lectins play an important role in cell signaling and defense.2 Plant lectins have been found in seeds, flowers, leaves, and roots, depending on the species.2,3 They commonly serve as defense proteins and play multiple physiological roles. For instance, plant lectins with antiviral,4 antimicrobial,5,6 anti-inflammatory,7 anticancer,8 and antiinsect5,9 activity have been found. The carbohydrate moieties determine the biological potentials of lectins and enable the selective binding of lectins to sugars on cell surfaces.10 Plant lectins are divided into several subgroups that bind to specific monosaccharides, such as D-mannose/D-glucose, D-galactose/ N-acetyl-D-galactosamine, L-fucose, and N-acetylneuraminc acid.11 Chinese leek is a monocotyledon plant belonging to the Allium genus with plants like onion and garlic. It is widely distributed in most regions (e.g., Asia, Europe, America, etc.) and serves as important cooking material. Seeds of the Chinese leek, namely Allium tuberosum Rottler, are collected after maturation and dried. Chinese leek seed extract exerts multiple physiological functions, such as antimicrobial,12,13 anticancer,14 and aphrodisiac effects.15 Thus far, lectins from the genus Allium have been isolated and characterized from garlic,16 ramsons,17 shallots,18 etc. Furthermore, active biomolecules such as steroidal saponin,19 steroidal oligoglycosides,20 and oils21 have been isolated from Chinese leek seeds and identified. However, to the best of our knowledge, research on lectins from Chinese leek seeds has not yet been reported. In this article, we focused on purification as well as the structural and biochemical characterization of a novel lectin, designated CLSL, from Chinese leek seeds. The effects of © 2015 American Chemical Society

CLSL on blood clotting parameters were studied. Furthermore, the mechanism of the structural interaction between Ba2+ and CLSL was deduced.

■

MATERIALS AND METHODS

Materials. Dried Chinese leek seeds were purchased from the local seed market (Fuzhou, China). Sephadex G50 and SP Sephadex C25 were purchased from GE Healthcare (Gothenburg, Sweden). All other chemicals and reagents were of analytical grade from Sinopharm Chemical Reagent Co., Ltd. (Fuzhou, China). Extraction of Chinese Leek Seeds Protein. Chinese leek seed powder (10.0 g) was homogenized (1:20 w/v) in 0.2 mol/L PBS buffer (pH 7.0) for 10 h at 4 °C after ultrasonic-assisted extraction. The homogenate was centrifuged at 14 300 g for 15 min at 4 °C. The supernatant was the crude extract of Chinese leek seed protein, designated as CLSP. Purification of CLSP. CLSP was dialyzed against 20 mmol/L acetate buffer (pH 5.0) for 24 h and subjected afterward to a SPSephadex column (1.5 × 20 cm) pre-equilibrated in 20 mmol/L acetate buffer (pH 5.0). The unabsorbed fraction was eluted with the equilibration buffer, and the absorbed fraction was eluted with 20 mmol/L acetate buffer (pH 5.0) containing a linear gradient of 0−1.0 M NaCl. The elution rate was 0.5 mL/min, and the absorbance was monitored at 280 nm. The fraction showing higher hemagglutinating activity was concentrated and loaded on a Sephadex G50 column (1.5 × 120 cm) pre-equilibrated with deionized water after filtering through a 0.22 μm membrane. The elution rate was 0.3 mL/min, and the absorbance was determined at 280 nm. The hemagglutinating activity was determined for all of the chromatographic fractions. Received: Revised: Accepted: Published: 1488

September 23, 2014 December 23, 2014 January 8, 2015 January 8, 2015 DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry

sample after being maintained for 24 h. Incubation was carried out for another 24 h, and the cell viability of each well was measured by MTT assay.27 Statistical Analysis. All experiments were carried out in triplicate (n = 3), and all data were expressed as means ± standard deviation. Analysis of variance (ANOVA) was performed using SPSS 19.0 (SPSS, Chicago, IL, USA). The significance in differences was determined by Duncan’s multiple range test (P < 0.05).

The purified fraction with the highest hemagglutinating activity was Chinese leek seed lectin, designated as CLSL. Assay of Hemagglutinating Activity. Hemagglutinating activity was performed using the method reported by Silva et al.22 with modifications. Two-fold serial dilutions of the sample (25 μL) were mixed with erythrocyte buffer solution (25 μL) and 2% (v/v) supernatant of red blood cells (50 μL) at 25 °C in the 96-well microtiter plate. Saline was used as the blank control, and the results were recorded after incubation at 25 °C for 1 h. The maximum number of a serial two-fold dilution of the sample showing hemagglutinating activity was defined as the hemagglutinating titer (U). Gel Electrophoresis. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a 15.0% separating gel and 5.0% stacking gel under nonreducing and reducing conditions according to the modified method of Zhu et al.23 The molecular weight of CLSL was determined by the migration rate with reference to the standard molecular weight marker. Analysis of N-Terminal Amino Acid Sequence. The purified CLSL was loaded to SDS-PAGE on 4−12% gradient NuPAGE Bis− Tris gel and then mobilized onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. N-Terminal sequencing by Edman degradation was performed by using an Applied Biosystems model 471A protein sequenator (Applied Biosystems, Shanghai, China). Carbohydrate-Binding Specificity. To confirm the specificity of CLSL for sugars, the inhibition of hemagglutination was performed using a method described by Takahashi et al. with modifications.24 The 2-fold serial dilution samples (25 μL) were incubated with 25 μL sugar solutions (20 mmol/L) for 30 min at room temperature, and hemagglutination was analyzed as described above. The beginning protein concentration was 0.5 mg/mL, and its hemagglutinating activity was 128 U. Measurement of Carbohydrate Content. The total sugar content of purified CLSL was measured by the anthrone−sulfuric acid method with reference to glucose, according to the modified method of Alberto Leyva et al.25 Effect of pH, Temperature, and Metal Ions. The pH stability of CLSL was measured by incubating 50 μL of CLSL (1.0 mg/mL) in buffers with different pH values (pH 2.0−11.0, 50 μL) for 1 h at room temperature. The temperature stability of CLSL was determined by incubating 50 μL of CLSL at different temperature (20−100 °C) for 60 min. The effect of metallic compounds (BaCl2, CaCl2, LiCl, CuCl2, FeCl2, and K2Cr3O4) was measured by incubating 50 μL of CLSL in equivalent volume of metallic compounds buffer. Circular Dichroism. Circular dichroism (CD) is an effective physical technique for evaluating structures and structural changes of proteins.26 For the analysis of the secondary structure of purified CLSL, the CD spectra were measured in 2.0 mM acetic acid−sodium acetate buffer (pH 5.0), using a J-810 spectropolarimeter at the room temperature in a wavelength range of 190−260 nm. The spectrum was recorded with the response time of 1 s and scan speed of 100 nm/min. Each data point was an average of three accumulations. Fluorescence Spectroscopy. The purified CLSL (20 mmol) dissolved in 2.0 mL of deionic water was analyzed using a 970 CRT spectrofluorometer (Shanghai, China) at room temperature, with both the excitation and the emission band-pass filters set at 5 nm. Samples were irradiated at excitation wavelength of 295 nm, and emission spectra were recorded in the range 290−400 nm. Quenching of the protein intrinsic fluorescence was performed by addition of microaliquots of the quenchers (10 mmol/L BaCl2 solutions) to the protein. The synchronous fluorescence spectra, focused on investigating the influence of Ba2+ on conformation of CLSL, was carried out at the wavelength interval (Δλ) of 15 and 60 nm between the excitation and emission wavelengths, respectively, with both the excitation and the emission band-pass filters set at 5 nm. Antiproliferative Activity. The cell lines HepG2, LO2, and Caco-2 were cultivated in 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 mg/L of streptomycin, and 100 U/mL of penicillin at 37 °C in a humidified atmosphere of 5% CO2. Cells (1.5 × 104/well) were seeded into a 96-well culture plate in their exponential growth phase and added to different concentrations of

■

RESULTS Purification of CLSL. Chinese leek seed extract was purified with two chromatographic steps, namely ion exchange chromatography with a SP Sephadex C-25 column and size exclusion chromatography with a Sephadex G-50 column. As shown in Figure 1A, the extract was resolved into seven peaks

Figure 1. Purification of lectin (CLSL) from Chinese leek seeds. (A) Purification of CLSL using ion exchange chromatography on SP-Sephadex C25 column and fraction A7 showed the higher hemagglutinating activity. (B) Purity of fraction A7 from SP-Sephadex C25 column identified by SDS-PAGE. (C) Further purification of fraction A7 from SP-Sephadex C25 column using gel filtration chromatography on a Sephadex G50 column and fraction G1 exerted the higher agglutinating activity. (D) Purity of fraction G1 from Sephadex G50 column determined by RP-HPLC.

after the SP Sephadex C-25 column, but only fraction A7 showed stronger hemagglutinating activity (Table S1, Supporting Information). Purification of fraction A7 was examined with SDS-PAGE, and two bands were observed (Figure 1B). Subsequently, fraction A7 was concentrated and applied onto a Sephadex G-50 column for further purification. As shown in Figure 1C, fraction A7 was resolved into two peaks (G1 and G2), 1489

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry

Figure 2. Molecular weight of CLSL calculated by SDS-PAGE and its standard curve. (A) Lanes: Marker, molecular weight marker; 1 and 2, experiment performed under reduced and nonreduced condition stained by Coomassie brilliant blue; 3, CLSL stained by the periodic acid Schiff’s reagent. (B) Calculated standard curve of CLSL. The position of CLSL on the regression curve was represented as a red dot.

Table 1. Summary of Purification Process for CLSL and Its Hemagglutinating Activity fractions

total protein (mg)

extraction yield (%)

HAa (U)

purification fold

crude extractionb SP Sephadex C-25 Sephadex G-50

554.00 ± 0.15 215.68 ± 0.35 23.04 ± 0.10

11.08 ± 0.03 4.31 ± 0.02 0.46 ± 0.02

64 128

1.0 18.7

a

HA: hemagglutinating activity. The tested concentration of sample was 1.0 mg/mL. bCrude extraction was gained from 5.0 g Chinese leek seed powder. SD: standard deviation. All the values were mean ± SD.

and only fraction G1 was identified as the desired fraction. Purification of fraction G1 was confirmed by the elution of single peak from reverse phase-high performance liquid chromatography (RP-HPLC) and a single band on SDS-PAGE (Figures 1D and 2A). The above process resulted in an 18.7-fold purification (Table 1). Structural Characterization. CLSL appeared as a single band on SDS-PAGE. Molecular weights of the bands under reducing and nonreducing conditions were 11.7 and 23.6 kDa, respectively, suggesting that CLSL was a dimer consisting of two identical subunits connected by disulfide bonds (Figure 2A). A brick-red band was observed after PAS staining, indicating CLSL was a glycoprotein (Figure 2A). The sugar content of CLSL was 3.6 ± 0.25%, measured by anthrone− sulfuric acid method. The CD spectrum of CLSL was characterized by one positive peak near 195 nm, two negative peaks near 210 and 223 nm, respectively, and a positive to negative crossover at 200 nm (Figure 3), which was characteristic of an α-helical protein. The secondary structure content estimated by Yang’s method28 was 35.8% α-helixes, 8.6% β-sheets, 18.3% β-turns, and 37.3% random coils. N-Terminal Amino Acid Sequences of CLSL. N-Terminal amino acid sequences of CLSL were determined by Edman degradation. The results showed that the seven N-terminal residues of CLSL were Cys-Val-Ile-Ala-Pro-Val-Ala (CVIAPVA). Characterization of Hemagglutinating Activity. The hemagglutinating activity of CLSL was partly inhibited by D-fructose, sorbose, and mannitol at the concentration of 20 mmol/L with the inhibition ratios of 75%, 25%, and 75%,

Figure 3. CD spectrum of CLSL in acetate buffer (20 mM, pH 5.0). The concentration of CLSL was 0.4 mg/mL. The positive peak near 195 nm and two negative peaks near 210 and 223 nm, respectively, indicated an α-helical structure of CLSL.

Table 2. Effect of Various Carbohydrates on CLSL (Initial Hemagglutination Activity: 128 U) carbohydrate (20 mM)

residual activity (titer/U)

raffinose lactose D-glucose D-fructose sucrose D-xylose D-mannose D-galactose maltose sorbose mannitol fucose

128 128 128 32 128 128 128 128 128 64 32 128

respectively. Other sugars studied did not affect the hemagglutinating activity of CLSL (Table 2), indicating complex sugar specificity of CLSL.29 As Table 3 shows, temperatures in the range 20−100 °C did not have significant effects on the hemagglutinating activity of CLSL, implying that CLSL possessed strong thermostability 1490

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry Table 3. Effect of Temperature on CLSL (Initial Hemagglutination Activity: 128 U) temperature (°C) residual activity (titer/U)

20 128

30 128

40 128

50 128

60 128

70 128

80 256

90 128

100 128

Table 4. Effect of pH on CLSL (Initial Hemagglutination Activity: 128 U) pH residual activity (titer/U)

2 128

3 128

4 128

5 128

6 128

7 32

8 0

9 0

10 0

11 0

Table 5. Effect of Metal Ions on CLSL (Initial Hemagglutination Activity: 128 U) metal ions (10 mmol/L) residual activity (titer/U)

CuCl2 212

CaCl2 212

BaCl2 0

like a lectin from Moringa oleifera.30,31 The hemagglutinating activity was retained at acidic pH values ranging from 2.0 to 6.0. However, the activity began to decrease at neutral pH and was completely lost in the alkaline pH range 8.0−11.0 (Table 4). The influence of metal ions on the hemagglutinating activity was studied. The hemagglutinating activity of CLSL was completely inhibited by Ba2+ but was greatly enhanced in the present of Ca2+, Fe2+, and Cu2+ at a concentration of 10 mmol/L (Table 5). Antiproliferative Activity. Inhibition of proliferations of HepG2 cells was observed in the presence of CLSL, and its IC50 value was 30 μM. In contrast, proliferation of Caco-2 cells and the normal cells LO2 were only slightly affected by up to 40 μM CLSL (Figure 4), suggesting that the antiproliferative potential of CLSL was selective.

K2Cr3O4 8

LiCl 128

FeCl2 212

decreased with increased Ba2+ concentrations. A slight blue shift of the maximum emission wavelength was observed at the wavelength interval of 15 nm, and it did not show significant shift at the wavelength interval of 60 nm (Figure 5B,C). To further investigate the influence of Ba2+ on the CLSL conformation, CD spectroscopy of CLSL was carried out with the addition of Ba2+ (Figure 6). A positive peak at 195 nm and two negative peaks near 210 and 225 nm shifted toward the zero graduation with the increase of Ba2+ concentrations. Moreover, an apparent change of its ellipticity was observed at the Ba2+ concentration of 5.0 μmol/L, and no significant difference was observed at higher Ba2+ concentrations, suggesting that the reaction was under saturation conditions.

■

DISCUSSION In the present work, a novel lectin (CLSL) was purified from Chinese leek seeds and displayed activity against rat erythrocytes. The purity of CLSL was confirmed by SDS-PAGE and RP-HPLC. CLSL was a dimeric glycoprotein consisting of two identical subunits linked by disulfide bonds with a molecular weight of 23.6 kDa. It produced a smear on SDS-PAGE, similar to that of some reported lectins.22,29 The smear was probably because glycoproteins possessed the heterogeneous glycosylation pattern and stronger hydration in water solution due to the presence of carbohydrate units and disulfide bonds.33,34 Similar to CLSL, a mannose-binding protein (ATL) from Chinese Allium tuberosum was also a dimeric protein.35,36 ATL had an N-terminal sequence of RNVLLNGEGLYAGQS, different from that of CLSL (CVIAPVA). The seven residue sequence of CLSL was submitted to a blast search in the NCBI database. Although some similarities to the sequences of other reported lectins were observed, they were far away from the N-terminus (Table 6), which inferred that CLSL was a novel

Figure 4. Inhibitory effect of CLSL on proliferation of HepG2, LO2, and Caco-2. Proliferation of HepG2 cells was inhibited with an IC50 of 30 μM. Cell proliferation was determined by MTT assay (data represent means ± SD, n = 3).

Table 6. N-Terminal Amino Acid Sequence Alignment of CLSL in NCBI Database

Effect of Ba2+ on the Hemagglutinating Activity of CLSL. To investigate the mechanism of Ba2+-mediated inhibition of hemagglutinating activity, the fluorescence spectra of CLSL quenched by Ba2+ at different concentrations were analyzed (Figure 5A). Quenching of the intrinsic fluorescence of CLSL and a slight blue shift of the maximum emission fluorescence peak (about 2 nm) were observed. The synchronous fluorescence spectra of CLSL in the presence of Ba2+ were also shown in Figure 5. The synchronous fluorescence was characteristic of tyrosine residues (Tyr) and tryptophan residues (Trp) at the wavelength interval (Δλ) of 15 and 60 nm between the excitation and emission wavelengths, respectively.32 It was obvious that the maximum emission peak intensities of both tyrosine and tryptophan

lectin. CLSL was identified to be an α-helix protein with CD spectroscopy. Compared with CLSL, different lectins possessed different contents of secondary structures (Table 7). For example, 1491

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry

Figure 5. Fluorescence spectra of CLSL and fluorescence quenching spectra of CLSL quenched by barium ions. (A) Synchronous fluorescence spectra of CLSL at different concentrations of barium ions in acetate buffer (20 mM, pH 5.0). (B) and (C) CLSL concentration (20 μM) and final concentrations of barium ions (0, 5, 10, 25, 50, 75, and 100 nM, separately).

Figure 6. Superimposition of far-UV (A) and near-UV (B) CD spectra of CLSL recorded at presence of different barium ions concentrations in acetate buffer (20 mM, pH 5.0).

an α/β lectin contains 46% α-helixes, 12% β-sheets, 17% β-turns, and 25% unordered structures, as reported by Luz et al.31 A cotyledonary lectin from Luetzelburgia auriculata is composed of 6% α-helixes, 46% β-sheets, 17% β-turns, and 31% unordered structures.37 CLSL exerted strong thermostability similar to lectins reported by Oliveira et al.,38 Ngai et al.,39 and Santos et al.30 The hemagglutinating activity of CLSL was stable at 100 °C for 60 min. The intramolecular disulfide bonds may be responsible for its thermostability.40,41 However, CLSL was only active in acidic conditions and rapidly inactivated in neutral and

Table 7. Secondary Structure Contents of CLSL Compared with Other Lectins secondary structure

CLSL LAA cMoL BfL MoL

α-helixes (%)

β-sheets (%)

β-turns (%)

random coils (%)

reference

35.8 6 46 19 28

8.6 46 12 27 23

18.3 17 17 22 20

37.3 31 25 32 28

the present 37 31 22 41 1492

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry basic conditions. This might be because the hydroxyl ions increased with pH, induced changes of ionization state on the lectin surface, and affected the interaction between lectin and the cell membrane.42−44 A similar phenomenon was observed with an Archidendron jiringa Nielsen-derived lectin.6 Lectins are a class of highly specific carbohydrate-binding proteins. Their multiple function (e.g., recognition and defense against pathogens) is closely related to the types of carbohydratebinding.45 The activities of lectins are often inhibited by monosaccharide and oligosaccharides.46 In the present study, CLSL exhibited complex carbohydrate specificity, and the activity could be inhibited by carbohydrates including D-fructose, sorbitol, and mannitol, the binding of which likely prevented attachment of lectins to cell membranes.46 To the best of our knowledge, there has been no evidence demonstrating that hemagglutinating activity is related to antiproliferative activity. Despite that, both hemagglutinating and antiproliferative activities are related to the high binding specificity of lectins to oligosaccharides, and it is well-known that malignant transformation is accompanied by aberrant glycosylation of surface structures that may affect tumor invasion and metastasis.47 Lectins could recognize the specific conformation of a cell surface due to specific binding of oligosaccharides and affect proliferation of cells. In the present paper, CLSL showed a selective antiproliferation activity against HepG2 cells but not Caco-2 and LO2 cells. The reason for the selectivity might be the discrepant oligosaccharides on HepG2, Caco-2, and LO2 cell surfaces. Divalent metal compounds (e.g., CuCl2, CaCl2, and FeCl2) could dramatically improve the activity of CLSL, one reason for which might be that some divalent metal ions (e.g., Ca2+ and Mn2+) contribute to maintaining the conformation of lectin and stabilize amino acid residues in specific sugar-side-chain-binding sites.42,43 However, inhibition of the hemagglutinating activity of CLSL by Ba2+ was noteworthy. The slight blue shift on fluorescence spectra demonstrated that Ba2+ interacted with CLSL and quenched its intrinsic fluorescence.48 Moreover, synchronous fluorescence spectra also showed that not only was fluorescence intensities of tyrosine and tryptophan residues decreased but there was also a slight blue shift of tyrosine residues, suggesting the increase in hydrophobicity around tyrosine residues.32,48 The most likely reason for the above changes was that conformation of CLSL had changed. Notably, we also found that the α-helix content of CLSL gradually increased, accompanied by an equal decrease of its β-fold content when the Ba2+ concentration increased (Figure S1, Supporting Information), which hinted that the interaction between CLSL and Ba2+ resulted in transformation of its β-fold to α-helical conformations, destroying the conformation of its glycosylation active domains. Therefore, pathways of Ba2+-mediated inactivation of CLSL speculated that the binding of Ba2+ changed the conformation of CLSL, leading to conformational changes of glycosylation domains and inhibition of activity. It was also likely that by directly binding to glycosylation domains, Ba2+ hindered the recognition and interaction between CLSL and sugar side chains on cell surface (Figure 7). In conclusion, we reported purification and characterization of a novel lectin, CLSL from Chinese leek seeds, and its interaction with Ba2+. The blood clotting parameters and biological functions of CLSL as a potential anticoagulant and platelet antiaggregation protein were examined in detail. The possible mechanism of interaction between CLSL and Ba2+ was deduced.

Figure 7. Sketch of the mechanism of interaction between CLSL and barium ions. Pathway A: Barium ions directly bound to carbohydratebinding sites and caused the overall structural shrinkage of CLSL. Pathway B: The binding of barium ions caused the critical conformation change of CLSL and blocked the active sites. Both of the above pathways could destroy the conformation of its glycosylation domain, leading to the inactivation of CLSL.

Although further experiments should be conducted to elucidate the mechanism of CLSL interaction with sugars and explore its applications in the pharmaceutical industry, the present study provided a valuable basis for future research.

■

ASSOCIATED CONTENT

* Supporting Information S

Secondary structural content of CLSL titrated with different concentrations of Ba2+ and hemagglutinating activity of the different fractions from SP Sephadex C-25 column. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*S.W. telephone: +86-591-22866375. Fax: +86-591-22866278. E-mail: [email protected]. Funding

This research was supported by grants from the National Natural Science Foundation of China (No. 31000335) and the Fujian Province Natural Science Foundation (No. 2013J05050). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED CLSP, Chinese leek seed protein; CLSL, Chinese leek seed lectin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CD, circular dichroism; RP-HPLC, reverse phase-high performance liquid chromatography 1493

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry

■

(20) Ikeda, T.; Tsumagari, H.; Nohara, T. Steroidal Oligoglycosides from the Seeds of Allium tuberosum. Chem. Pharm. Bull. 2000, 48, 362−365. (21) Hu, G. H.; Lu, Y. H.; Wei, D. Z. Fatty acid composition of the seed oil of Allium tuberosum. Bioresour. Technol. 2005, 96, 1630− 1632. (22) Silva, M. C. C.; Santana, L. A.; Mentele, R.; Ferreira, R. S.; De Miranda, A.; Silva-Lucca, R. A.; Sampaio, M. U.; Correia, M. T. S.; Oliva, M. L. V. Purification, primary structure and potential functions of a novel lectin from Bauhinia forficata seeds. Process Biochem.(Oxford, U. K.) 2012, 47, 1049−1059. (23) Zhu, Z. C.; Chen, Y.; Ackerman, M. S.; Wang, B.; Wu, W.; Li, B.; Obenauer-Kutner, L.; Zhao, R.; Tao, L.; Ihnat, P. M.; Liu, J.; Gandhi, R. B.; Qiu, B. Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE. J. Pharm. Biomed. Anal. 2013, 83, 89−95. (24) Takahashi, K. G.; Kuroda, T.; Muroga, K. Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2008, 150, 45−52. (25) Leyva, A.; Quintana, A.; Sánchez, M.; Rodríguez, E. N.; Cremata, J.; Sánchez, J. C. Rapid and sensitive anthrone−sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: Method development and validation. Biologicals 2008, 36, 134−141. (26) Li, R.; Nagai, Y.; Nagai, M. Changes of tyrosine and tryptophan residues in human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated chains and recombined hemoglobin. J. Inorg. Biochem. 2000, 82, 93−101. (27) Twentyman, P. R.; Luscombe, M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 1987, 56, 279−285. (28) Yang, J. T.; Wu, C. C.; Martinez, H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986, 130, 208−269. (29) 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, 326−332. (30) Santos, A. F. S.; Luz, L. A.; Argolo, A. C. C.; Teixeira, J. A.; Paiva, P. M. G.; Coelho, L. C. B. B. Isolation of a seed coagulant Moringa oleifera lectin. Process Biochem.(Oxford, U. K.) 2009, 44, 504− 508. (31) Luz, L. D. A.; Silva, M. C. C.; Ferreira, R. d. S.; Santana, L. A.; Silva-Lucca, R. A.; Mentele, R.; Oliva, M. L. V.; Paiva, P. M. G.; Coelho, L. C. B. B. Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters. Int. J. Biol. Macromol. 2013, 58, 31−36. (32) Zhang, H. X.; Huang, X.; Mei, P.; Gao, S. Interaction between Glyoxal-bis-(2-hydroxyanil) and Bovine Serum Albumin in Solution. J. Solution Chem. 2008, 37, 631−640. (33) Geyer, H.; Geyer, R. Strategies for analysis of glycoprotein glycosylation. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 1853−1869. (34) Zhu, K.; Zhou, H. Purification and characterization of a novel glycoprotein from wheat germ water-soluble extracts. Process Biochem. (Oxford, U. K.) 2005, 40, 1469−1474. (35) Hu, G. H.; Lu, Y. H.; Wei, D. Z. Chemical characterization of Chinese chive seed. Food Chem. 2006, 99, 693−697. (36) Ooi, L. S. M.; Yu, H.; Chen, C. M.; Sun, S. S. M.; Ooi, V. E. C. Isolation and Characterization of a Bioactive Mannose-Binding Protein from the Chinese Chive Allium tuberosum. J. Agric. Food Chem. 2002, 50, 696−700. (37) Oliveira, J. T.A.; Melo, V. M. M.; Camara, M. F. L.; Vasconcelos, I. M.; Beltramini, L. M.; Machado, O. L. T.; Gomes, V. M.; Pereira, S. P.; Fernandes, C. F.; Nunes, E. P.; Capistrano, G. G. G.; MonteiroMoreira, A. C. O. Purification and physicochemical characterization of

REFERENCES

(1) Van Damme, E. J.; Peumans, W. J.; Pusztai, A.; Bardocz, S. Handbook of Plant Lectins: Properties and Biomedical Applications; Wiley: West Sussex, England, 1998. (2) Darville, L. N. F.; Merchant, M. E.; Maccha, V.; Siddavarapu, V. R.; Hasan, A.; Murray, K. K. Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis). Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2012, 161, 161−169. (3) Peumans, W. J.; Damme, E. J. M. Lectins as Plant Defense Proteins. Plant Physiol. 1995, 109, 347−352. (4) Ding, J.; Bao, J.; Zhu, D.; Zhang, Y.; Wang, D. C. Crystal structures of a novel anti-HIV mannose-binding lectin from Polygonatum cyrtonema Hua with unique ligand-binding property and super-structure. J. Struct. Biol. 2010, 171, 309−317. (5) Santi-Gadelha, T.; Rocha, B. A. M.; Gadelha, C. A. A.; Silva, H. C.; Castellon, R. E. R.; Gonçalves, F. J. T.; Toyama, D. O.; Toyama, M. H.; De Souza, A. J. F.; Beriam, L. O. S.; Martins, J. L.; Joazeiro, P. P.; Cavada, B. S. Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode. Pestic. Biochem. Phys. 2012, 103, 15−22. (6) Charungchitrak, S.; Petsom, A.; Sangvanich, P.; Karnchanatat, A. Antifungal and antibacterial activities of lectin from the seeds of Archidendron jiringa Nielsen. Food Chem. 2011, 126, 1025−1032. (7) Alencar, V. B. M.; Alencar, N. M. N.; Assreuy, A. M. S.; Mota, M. L.; Brito, G. A. C.; Aragão, K. S.; Bittencourt, F. S.; Pinto, V. P. T.; Debray, H.; Ribeiro, R. A.; Cavada, B. S. Pro-inflammatory effect of Arum maculatum lectin and role of resident cells. Int. J. Biochem. Cell Biol. 2005, 37, 1805−1814. (8) Wu, Y.; Wang, H.; Ng, T. B. Purification and characterization of a lectin with antiproliferative activity toward cancer cells from the dried fruit bodies of Lactarius flavidulus. Carbohydr. Res. 2011, 346, 2576− 2581. (9) Ota, E.; Tsuchiya, W.; Yamazaki, T.; Nakamura, M.; Hirayama, C.; Konno, K. Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida. Phytochemistry 2013, 89, 15−25. (10) Lis, H.; Sharon, N. Lectins Carbohydrate-Specific Proteins That Mediate Cellular Recognition. Chem. Rev. 1998, 98, 637−674. (11) Vasconcelos, I. M.; Oliveira, J. T. A. Antinutritional properties of plant lectins. Toxicon 2004, 44, 385−403. (12) Lee, C. F.; Han, C. K.; Tsau, J. L. In vitro inhibitory activity of Chinese leek extract against Campylobacter species. Int. J. Food Microbiol. 2004, 94, 169−174. (13) Mau, J. L.; Chen, C. P.; Hsieh, P. C. Antimicrobial effect of extracts from Chinese Chive, Cinnamon, and Corni Fructus. J. Agric. Food Chem. 2001, 49, 183−188. (14) Shao, J.; Dai, J.; Ma, J. K. H. A pilot study on anticancer activities of Chinese leek. J. Altern. Complement. Med. 2001, 7, 517− 522. (15) Hu, G. H.; Lu, Y. H.; Mao, R. G.; Wei, D. Z.; Ma, Z. Z.; Zhang, H. Aphrodisiac properties of Allium tuberosum seeds extract. J. Ethnopharmacol. 2009, 122, 579−582. (16) Clement, F.; Pramod, S. N.; Venkatesh, Y. P. Identity of the immunomodulatory proteins from garlic (Allium sativum) with the major garlic lectins or agglutinins. Int. Immunopharmacol. 2010, 10, 316−324. (17) Smeets, K.; Van Damme, E. J.; Van Leuven, F.; Peumans, W. J. Isolation, characterization and molecular cloning of a leaf-specific lectin from ramsons (Allium ursinum L.). Plant Mol. Biol. 1997, 35, 531−535. (18) Mo, H.; Vandamme, E.; Peumans, W.; Goldstein, I. Purification and Characterization of a Mannose-Specific Lectin from Shallot (Allium ascalonicum) Bulbs. Arch. Biochem. Biophys. 1993, 306, 431− 438. (19) Hu, G. H.; Mao, R. G.; Ma, Z. Z. A new steroidal saponin from the seeds of Allium tuberosum. Food Chem. 2009, 113, 1066−1068. 1494

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495

Article

Journal of Agricultural and Food Chemistry a cotyledonary lectin from Luetzelburgia auriculata. Phytochemistry 2002, 61, 301−310. (38) Oliveira, M. D. L.; Andrade, C. A. S.; Santos-Magalhães, N. S.; Coelho, L. C. B. B.; Teixeira, J. A.; Carneiro-da-Cunha, M. G.; Correia, M. T. S. Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity. Lett. Appl. Microbiol. 2008, 46, 371− 376. (39) Ngai, P. H. K.; Ng, T. B. A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells. Biochem. Biophys. Res. Commun. 2004, 314, 988−993. (40) Liu, Q.; Wang, H.; Ng, T. B. Isolation and characterization of a novel lectin from the wild mushroom Xerocomus spadiceus. Peptides 2004, 25, 7−10. (41) Katre, U. V.; Suresh, C. G.; Khan, M. I.; Gaikwad, S. M. Structure−activity relationship of a hemagglutinin from Moringa oleifera seeds. Int. J. Biol. Macromol. 2008, 42, 203−207. (42) Eretan, K.; Eretan, O. B. Purification and Partial Characterization of a Lectin from the Fresh Leaves of Kalanchoe crenata (Andr.) Haw. J. Biochem. Mol. Biol. 2004, 37, 229−233. (43) Ahmad, S.; Khan, R. H.; Ahmad, A. Physicochemical characterization of Cajanus cajan lectin: effect of pH and metal ions on lectin carbohydrate interaction. Biochim. Biophys. Acta 1999, 1427, 378−384. (44) Adolph, L.; Lorenz, R. Enzyme Diagnosis in Diseases of the Heart, Liver, and Pancreas; Karger: Basel, Switzerland, New York, 1982. (45) Francis, F.; Jaber, K.; Colinet, F.; Portetelle, D.; Haubruge, E. Purification of a new fungal mannose-specific lectin from Penicillium chrysogenum and its aphicidal properties. Fungal Biol. 2011, 115, 1093−1099. (46) Shao, B.; Wang, S.; Zhou, J.; Ke, L.; Rao, P. A novel lectin from fresh rhizome of Alisma orientale (Sam.) Juzep. Process Biochem. (Oxford, U. K.) 2011, 46, 1554−1559. (47) Hakomori, S. Glycosylation defining cancer malignancy: New wine in an old bottle. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 10231− 10233. (48) Zhang, J.; Chen, L.; Zeng, B.; Kang, Q.; Dai, L. Study on the binding of chloroamphenicol with bovine serum albumin by fluorescence and UV−vis spectroscopy. Spectrochim. Acta, Part A 2013, 105, 74−79. (49) Chen, L.; Yue, Q.; Zhang, M.; Wang, C.; Li, S.; Che, Y.; OrtizLopez, F. J.; Bills, G. F.; Liu, X.; An, Z. Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis. BMC Genomics 2013, 14, 339−339. (50) Jia, J.; Zhao, S.; Kong, X.; He, W.; Appels, R.; Pfeifer, M.; Tao, Y.; Zhang, C.; Ma, Y.; Gao, L.; Gao, C.; Spannagl, M.; Mayer, K. F. X.; Li, D.; Pan, S.; Zheng, F.; Hu, Q.; Xia, X.; Liang, Q.; Chen, J.; Wicker, T.; Gou, C.; Kuang, H.; He, G.; Luo, Y.; Keller, B.; Xia, Q.; Lu, P.; Wang, J.; Zou, H.; Zhang, R.; Xu, J.; Gao, J.; Middleton, C.; Quan, Z.; Liu, G.; Wang, J.; Yang, H.; Liu, X.; He, Z.; Mao, L.; Wang, J. Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation. Nature 2013, 496, 91−95.

1495

DOI: 10.1021/jf5046014 J. Agric. Food Chem. 2015, 63, 1488−1495