A sialic acid-binding lectin from the legume Maackia fauriei: comparison with lectins from M. amurensis

Plant Science 167 (2004) 1315–1321 www.elsevier.com/locate/plantsci

A sialic acid-binding lectin from the legume Maackia fauriei: comparison with lectins from M. amurensis Bum Soo Kima, Kyung Taik Oha,1, Due Hyeon Choa, Yun Jung Kima, Wan Mo Kooa, Kwang Hoon Kongb, HaHyung Kima,* a

Physical Pharmacy Laboratory, College of Pharmacy, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul 156-756, South Korea b Department of Chemistry, College of Natural Science, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul 156-756, South Korea Received 20 October 2003; received in revised form 20 April 2004; accepted 29 June 2004 Available online 22 July 2004

Abstract A lectin that exhibits hemagglutination activity and cytotoxicity against human cancer cell lines has been purified from the legume Maackia fauriei. This lectin, designated M. fauriei agglutinin (MFA), is a tetramer of 115.6 kDa consisting of 30 kDa subunits with a pI of 4.9. The hemagglutination activity of MFA was inhibited by N-acetylneuraminic acid, Neu5Aca2–3Galb1–4GlcNAc, and sialoglycoproteins. MFA was stable at pH values from 4.0 to 8.5, and at temperatures below 50 8C, and its activity was affected by demetalization with EDTA. MFA has a high homology with lectins from M. amurensis—which is the only legume source of lectins that bind to specific carbohydrate chains containing sialic acid—in its N-terminal 20 amino acid sequence. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sialic acid; Lectin; Bark; Maackia fauriei; Legume

1. Introduction Lectins are carbohydrate-binding proteins that are able to induce cell agglutination or the precipitation of glycoconjugates [1]. They have been found in viruses, bacteria, fungi, animals, and plants. Plant lectins can be purified from seed, bark, leaves, roots, tubers, and fruits [2], and are suggested to have an important role in plant defense against pathogens [3]. Lectins with a sialic acid-binding specificity are potenAbbreviations: BSM, bovine submaxillary mucin; EDTA, ethylenediaminetetraacetic acid; IEF, isoelectric focusing; MAH, Maackia amurensis hemagglutinin; MAHb, M. amurensis bark hemagglutinin; MAL, M. amurensis leukoagglutinin; MALb, M. amurensis bark leukoagglutinin; MALDI– TOF, matrix-assisted laser desorption/ionization time-of-flight; MFA, M. fauriei agglutinin; Neu5Ac, N-acetylnueraminic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin. * Corresponding author. Tel.: +82 2 8205612; fax: +82 2 8235612 E-mail address: [email protected] (H. Kim). 1 Current address: Department of Pharmaceutical Sciences, Nebraska Medical Center, Omaha, NE 68198-6025, USA.

tially useful proteins in glycobiology or cancer research such as detection, localization, and isolation of sialoglycoconjugates [4–6]. Most of the sialic acid-specific lectins have been purified from invertebrates, and only a few plant-derived lectins that specifically bind to sialic acid have been studied, such as those from Triticum vulgaris (wheat germ agglutinin, WGA) [7,8], Sambucus species [9,10], and Maackia amurensis [11]. Although many types of lectin have been isolated from legume, M. amurensis is the only such source of lectins that bind to carbohydrate chain containing sialic acid. M. amurensis hemagglutinin (MAH) and M. amurensis leukoagglutinin (MAL) have been isolated from seeds [12,13]. It also has been reported that M. amurensis bark hemagglutinin (MAHb) and M. amurensis bark leukoagglutinin (MALb) have been isolated from bark [14]. According to the International Legume Database and Information Service (http://www.biodiversity.soton.ac.uk/ Legumeweb), there are nine species of Maackia genera, comprising four species of M. amurensis, M. buergeri, M. fauriei, two species of M. floribunda, and M. tashiroi. Among these,

0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.06.029

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M. fauriei, which is known as Solbinamu in Korea [15,16], is taxonomically synonymous to two species of M. floribunda; few research reports have described this plant. In this paper, we report the purification of a new lectin with a sialic acid-binding specificity obtained from the bark of M. fauriei. We also compare its molecular characterization and carbohydratebinding specificity with those of lectins from M. amurensis.

2. Materials and methods 2.1. Purification Bark (600 g) of M. fauriei collected at Mt. Halla, Jeju, South Korea, was homogenized and extracted overnight in 0.15 M NaCl at 4 8C. The homogenate was filtered with a 0.45 mm membrane filter and concentrated using a protein concentrator (Stirred Cell, Amicon). The concentrated crude protein extract was applied onto a Sepharose CL-6B (Pharmacia) column (1.6 cm  17 cm), which had been equilibrated with 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl. The active fractions were further purified on a fetuinaffinity column (1 cm  8 cm), which was made with immobilized fetuin on CNBr-activated Sepharose 4B according to the manufacturer’s instructions (Pharmacia). The column was equilibrated with 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl. After standing for 1 h at 4 8C, the column was washed with the same buffer, and 100 mM Na2HPO4–NaOH buffer (pH 11.0) was used for elution. The eluted fractions were immediately neutralized with 1 M HCl, and dialyzed against 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl at 4 8C. 2.2. Hemagglutination and hemagglutination inhibition assay The hemagglutinating activity was assessed using 6.25% (v/v) human ABO, rat, and mouse erythrocytes. Lectin (50 ml) was serially diluted two-fold in 0.15 M NaCl, and an equal volume of erythrocyte suspension was added to the microtiter plates. The plates were gently shaken and allowed to stand for 1 h at room temperature. The minimum concentration of purified lectin that caused agglutination was defined as one unit, and the hemagglutination titer was defined as the reciprocal of the highest dilution exhibiting agglutination of the erythrocytes. For hemagglutination inhibition assay, one unit of lectin (50 ml) was incubated for 1 h at room temperature with

Table 2 Inhibition of hemagglutination activity of MFA Inhibitorsa

Minimum inhibitory concentration

N-Acetylneuraminic acid (mM) Neu5Aca2–3Galb1–4GlcNAc (mM) Neu5Aca2–6Galb1–4GlcNAc (mM) Fetuin (mg/ml) BSM (mg/ml) Thyroglobulin (mg/ml) Asialofetuin (mg/ml) Asilao-BSM (mg/ml) Asialothyroglobulin (mg/ml)

10 0.027 >1.4 0.04 0.16 0.48 >1.0 >1.0 >1.0

a b-D-Glucose, D-galactose, L-fucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, methyl-a-D-glucopyranoside, a-lactose, b-lactose, D-lactose, b-gentiobiose, maltose, D-cellobiose, and D-raffinose exhibited no inhibition at the concentrations up to 100 mM.

various concentrations of inhibitors (50 ml) (see Table 2) dissolved in 0.15 M NaCl. A 6.25% (v/v) suspension (50 ml) of human A erythrocytes was then added to the mixture. After standing for 1 h at room temperature, the minimum concentration of inhibitor that completely inhibited hemagglutination was determined. All inhibitors (except asialoglycoproteins) used in this study were purchased from Sigma. Asialoglycoproteins were prepared according to the method of Spiro [17]. 2.3. Electrophoretic analyses Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of Laemmli [18] using a 12% acrylamide slab gel in the presence and absence of 2-mercaptoethanol. The gel was stained with Coomassie blue R-250. Isoelectric focusing (IEF) on a gel was estimated by using a 2% ampholyte IEFready gel (pH 3–10) according to the manufacturer’s instructions (Bio-Rad). 2.4. Protein concentration Protein concentration was determined by the method of Bradford [19], using bovine serum albumin as a standard. 2.5. Mass spectrometry analysis Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrum was obtained using a Voyager-RP mass spectrometer (PerSeptive Biosystems) according to the method of Woo et al. [20].

Table 1 Purification of MFA from the bark of M. fauriei Fraction

Hemagglutination titer

Total protein (mg)

Specific activitya

Purification fold

Recovery (%)

Crude extract Sepharose CL-6B column Fetuin-affinity column

256 512 1024

525.0 147.0 25.5

0.5 3.5 40.2

1 7 80.4

100.0 28.0 4.9

a

Specific activity is expressed as the ratio of hemagglutination titer/total protein.

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2.6. Gel-filtration chromatography

3. Results and discussion

Gel-filtration chromatography was performed using a Superdex-200 HR column (Pharmacia) equilibrated with 10 mM phosphate buffer, 0.15 M NaCl (pH 7.4) at a flow rate of 0.5 ml min 1, and monitored at 280 nm. The molecular weight markers used for calibration are b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa) (Sigma).

3.1. Hemagglutination activity and purification of M. fauriei agglutinin

2.7. pH and temperature stability Lectins dissolved in neutral buffer were substituted for various pH buffers (pH 3–12) containing 0.15 M NaCl through centrifugation at 600  g for 2 h at 4 8C using Microcon YM-10 (Millipore), and incubated for 1 h at room temperature. The buffers used were: 20 mM citric acid–NaOH (pH 3–6), 20 mM Tris–HCl (pH 7–9), 20 mM sodium bicarbonate–NaOH (pH 10), and 50 mM Na2HPO4–NaOH (pH 11–12) containing 0.15 M NaCl. The hemagglutination titer was then measured as described in Section 2.2. In the neutral condition, the hemagglutination titer against human A erythrocytes was measured after treatment of the lectin at 4–80 8C in 5 8C increments for 1 h. 2.8. Effect of cations on the activity Lectin was dialyzed overnight against 100 mM ethylenediaminetetraacetic acid (EDTA) solution, after which it was dialyzed against 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl at 4 8C. The hemagglutination titer was then measured in the absence or presence of 0–50 mM CaCl2 and MnCl2.

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Crude protein was extracted from the bark of M. fauriei and agglutinate human ABO erythrocytes at a titer of 256, and rat and mouse erythrocytes at a titer of 1024 (data not shown). The crude protein extract was then concentrated using a protein concentrator and stored at 4 8C. The colored contaminants of the concentrated crude protein extract were removed using Sepharose CL-6B column chromatography eluted with 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl (data not shown). The hemagglutination activity of crude protein was inhibited by fetuin (data not shown), thus the immobilized form of fetuin was used as an affinity adsorbent for the purification of the lectin. The Sepharose CL-6B column fractions with hemagglutination activity were slowly loaded onto a fetuin-affinity column pre-equilibrated with 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl. After extensive washing with the same buffer, the bound fraction in the affinity column was eluted by 100 mM Na2HPO4–NaOH buffer (pH 11.0), and fractions with hemagglutination activity were successfully eluted (Fig. 1 ). The eluted fractions were immediately neutralized with 1 M HCl, and dialyzed against 10 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl. The eluted fractions with hemagglutination activity were designated M. fauriei agglutinin, MFA. The conditions result in the loss of the activity of MFA (see Section 3.4); for example, 0.1 M EDTA (or buffers adjusted to pH 2–4 and pH 8–10) were applied for elution, but using 100 mM Na2HPO4–NaOH buffer (pH 11.0) remained the optimal method for eluting the MFA from a fetuin-affinity column. A typical purification method could produce at least 25.5 mg of pure MFA with a specific activity of 40.2 and a recovery yield of no less than 4.9% (Table 1).

2.9. N-terminal amino acid sequence analysis 3.2. Molecular mass and subunits The N-terminal amino acid sequence of lectin was analyzed using an automatic protein sequencer (model 476A01-120, Applied Biosystems). 2.10. Cytotocicity Human breast cancer MCF-7, human melanoma G-361, human hepatoma SNU-449 and human colorectal cancer SNU-C1 cell lines were purchased from Korean Cell Line Bank. MAL and WGA were purchased from Sigma. Cells (1  104 cells/0.1 ml/well) were incubated with 100 ml of lectins in 96-well culture plates (Costar) for 72 h in a humidified atmosphere of 5% CO2 at 37 8C. The percentage decrease in viability of cancer cells was determined by 3[4,5-dimethyldiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay according to a modified Mosmann’s method [21,22]. The percentage decrease in cancer cell viability was calculated in a previous report [23].

Upon SDS–PAGE, with or without 2-mercaptoethanol, the purified MFA gave a single band corresponding to a molecular mass of approximately 30 kDa (Fig. 2a). Similarly, MALDI–TOF mass spectrum showed that the molecular mass of MFA was 29132.2 Da (Fig. 3). In the IEF gel, MFA was focused as one band with an estimated pI of 4.9 (Fig. 2b). These results indicate that MFA was successfully purified from crude protein extract and has no intermolecular disulfide bonds. MFA also appeared as a single band on IEF, confirming that no isolectin contamination occurred during the purification procedure. The gel-filtration chromatography results indicate that MFA has a molecular mass of 115.6 kDa for the native molecule (data not shown). Legume lectins generally consist of two or four subunits with relative molecular mass of 30 kDa [24]. It has been reported that MAH is a tetrameric protein with a molecular mass of 33 kDa as a subunit [11], and MAL is a dimer of

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Fig. 1. Elution profile of MFA on a fetuin-affinity column. Arrow indicates the point elution with 100 mM Na2HPO4–NaOH buffer (pH 11.0). (*) absorbance at 280 nm; (*) hemagglutination titer.

disulfide-containing subunits with a molecular mass of 70 kDa [25]. It also has been reported that M. amurensis bark lectins are tetrameric proteins with a molecular mass of 37 kDa (MALb) and 32 kDa (MAHb) [14]. The present results from electrophoretic analyses, mass spectrometry, and gel-filtration chromatography indicate that MFA is an acidic protein (or glycoprotein), and exists as a tetramer of 30 kDa subunits that are linked by non-covalent bonds. This is similar to those of other legume lectins including lectins from M. amurensis, except MAL. 3.3. Carbohydrate-binding specificity In order to identify the carbohydrate-binding specificity of MFA, we performed a competitive inhibition assay using various inhibitors. As indicated in Table 2, the hemagglutination activity of one unit of MFA was inhibited by 10 mM N-acetylneuraminic acid (Neu5Ac), with no inhibition evident using any mono- or di-saccharides despite the use of concentrations up to 100 mM. M. amurensis lectins [11] and

S. niger lectins [9] are more specific to sialoglycoconjugate than to free Neu5Ac. In particular, the hemagglutination activity of MAL was not inhibited by Neu5Ac even at a concentration of 100 mM [26]. On the other hand, the hemagglutination activity of MFA was inhibited by free Neu5Ac. Experiments with M. amurensis lectins demonstrate that MAH binds to the Neu5Aca2–3Galb1–3(Neu5Aca2–6)GalNAc sequence [12], and MAL binds to the Neu5Aca2– 3Galb1–4Glc/GlcNAc sequence [13,26]. MAHb and MALb bind to fetuin [14], and their specificity to sialoglycoconjugate is not clear. In an attempt to identify the binding specificity of MFA to sialoglycoconjugate, we applied glycoproteins and sialylated trisaccharides. The present results show that the hemagglutination activity of MFA was inhibited by the sialoglycoproteins fetuin (0.04 mg/ml), which contains Neu5Aca2–3Galb1–4GlcNAc and Neu5Aca2–6Galb1– 4GlcNAc sequences as terminal components of oligosaccharide residues [27], bovine submaxillary mucin (BSM)

Fig. 2. Electrophoretic analyses of MFA. (a) SDS–PAGE of the purification steps. Lane 1, Molecular weight markers; lane 2, crude protein extract; lane 3, MFA without 2-mercaptoethanol; lane 4, MFA with 2-mercaptoethanol. (b) IEF. Lane 5, pI markers; lane 6, MFA.

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Fig. 3. MALDI–TOF mass spectrum of MFA.

(0.16 mg/ml), and thyroglobulin (0.48 mg/ml). Furthermore, the hemagglutination was potently inhibited by 0.027 mM Neu5Aca2–3Galb1–4GlcNAc, whilst no inhibition was observed with Neu5Aca2–6Galb1–4GlcNAc even at a concentration of 1.4 mM. The asialoglycoproteins (1.0 mg/ml) asialofetuin, asialo-BSM, and asialothyroglobulin exhibited no inhibitory activity. These results indicate that MFA binds with a high specificity to carbohydrate chains containing terminal Neu5Ac in an a2–3 linkage— and not in an a2–6 linkage—to Galb1–4GlcNAc. This specificity is similar to that of MAL. From these inhibition results, it seems reasonable to suggest that the specificity of MFA to sialoglycoproteins and Neu5Aca2–3Galb1–4GlcNAc is similar to that of MAL, but different to that of free Neu5Ac, even though the affinity of MFA to Neu5Ac is 370-fold (10 mM/0.027 mM) weaker than that to Neu5Aca2–3Galb1–4GlcNAc (Table 2). 3.4. Effects of pH, temperature, and cations on the activity

inactivated at pH values from 11.0 to 12.0, and at temperatures above 65 8C (data not shown). MFA lost its hemagglutination activity following dialysis with EDTA, but the activity was fully restored by the addition of 25 mM CaCl2 and 25 mM MnCl2 (data not shown). The binding of carbohydrates to legume lectins requires bound Ca2+ and Mn2+ ions on the carbohydrate-binding site [24]. MFA also needs metal cations for the activity, and it appears that these are already bound to native MFA, as is the case for other legume lectins. 3.5. N-terminal amino acid sequence analysis A single N-terminal 20-amino acid sequence of MFA, SDELSFNINNFVPNQADLLF, was determined on an automatic Edman degradation amino acid sequencer, and it exhibits a high homology with lectins from M. amurensis [14,25,28] (Fig. 4). In particular, MFA has 85% homology with MAHb and MAH, and 90% with MALb and MAL. 3.6. Cytotoxicity

The hemagglutination activity of MFA was stable at pH values from 4.0 to 8.5, and at temperatures below 50 8C; the activity was halved at pH 8.5–11.0; and it was completely

The cytotoxicity of MFA against cancer cells was compared with MAL and WGA. MFA (8 mM) exerted cytotoxic

Fig. 4. The N-terminal amino acid sequence of MFA in comparison with lectins from M. amurensis. The results for MAHb and MALb [14], MAH [28], and MAL [25] are taken from the references. Homologous amino acid sequences are indicated by black boxes.

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Table 3 Decrease in viability of cancer cells after exposure to MFA, MAL, and WGA Cancer cell line

Lectin dose (mM)

Decrease in cancer cell viability (%) MFA

MAL

WGA

MCF-7

8 0.8 0.08

54.1  3.9 36.4  5.9 12.6  2.9

29.5  2.6 11.4  6.2 NDa

38.6  4.3 10.2  5.2 NDa

G-361

8 0.8 0.08

33.2  8.7 23.6  2.6 3.3  1.3

38.9  6.7 13.2  3.1 NDa

41.5  1.8 25.7  7.3 5.1  1.4

SNU-449

8 0.8 0.08

35.6  1.7 17.5  1.0 7.2  0.7

38.5  1.5 16.7  3.0 13.4  2.5

49.9  3.0 42.6  1.1 32.2  1.0

SNU-C1

8 0.8 0.08

a

ND NDa NDa

a

ND NDa NDa

57.7  0.7 40.4  3.2 8.0  0.5

Data represent mean  S.E.M. (n = 3). a Effect undetectable.

binding specificity to free Neu5Ac. The sialic acid-binding specificity and cytotoxicity of MFA should make it a very useful reagent in glycoconjugate and cancer research. Further investigations into quantitative data such as the binding constant and binding on the surface of the cancer cells of MFA and its structural information including full sequencing of this purified MFA are important to differentiating the molecular characterizations of MFA and lectins from M. amurensis, and these are currently underway in our laboratory.

Acknowledgement This work was supported by grant No. R01-2000-00000131-0 from the Basic Research Program of the Korea Science & Engineering Foundation. References

effects on the human breast cancer MCF-7 (54.1  3.9%), human melanoma G-361 (33.2  8.7%) and human hepatoma SNU-449 (35.6  1.7%) cell lines but had no effect on the human colorectal cancer SNU-C1 cell line (Table 3). WGA (8 mM) exerted the strongest cytotoxicity against G361 (41.5  1.8%), SNU-449 (49.9  3.0%), and SNU-C1 (57.7  0.7%) cell lines, with MFA exhibiting a similar cytotoxic effect to MAL. Both MFA and MAL did not affect the viability of the SNU-C1 cell line. Many plant lectins are cytotoxic against cancer cells, with the effect differing with cell type; however, the mechanisms of action remain poorly understood [29]. Wang et al. [22] reported the cytotoxic effect of a number of lectins; their results demonstrated that WGA exhibits the most cytotoxic effect upon human hepatoma, human choriocarcinoma and human melanoma cells with Mamurensis lectin exhibiting weaker effects. These differing cytotoxicities are probably related to diversity—mainly due to the type and linkage on the terminal sialic acid—on the surface membrane expression of sialoglycoconjugate between cancer cells [30]. The present results demonstrate a comparable cytotoxic effect against the human melanoma and human hepatoma cell lines. However, MFA (8 mM) exhibited the most deleterious effect on the viability of the human breast cancer MCF-7 (54.1  3.9%) cell line compared to MAL (29.5  2.6%) and WGA (38.6  4.3%).

4. Conclusion In the present study, a new sialic acid-binding lectin, MFA, has been purified from the bark of the legume M. fauriei. The molecular mass and number of subunits of purified MFA are similar to those of MAH; however, the carbohydrate-binding specificity is more similar to that of MAL than to that of MAH, even though they have different

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