Isolation and characterization of a novel lectin from the mushroom Armillaria luteo-virens

BBRC Biochemical and Biophysical Research Communications 345 (2006) 1573–1578 www.elsevier.com/locate/ybbrc

Isolation and characterization of a novel lectin from the mushroom Armillaria luteo-virens K. Feng a

a,b

, Q.H. Liu a, T.B. Ng c, H.Z. Liu a, J.Q. Li d, G. Chen d, H.Y. Sheng d, Z.L. Xie d, H.X. Wang a,*

State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100094, China b College of Food Science, Heilongjiang August First Land Reclamation University, Daqing 163319, China c Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong, China d Agriculture and Animal Husbandry College, Qinghai University, Xining 810003, China Received 12 April 2006 Available online 17 May 2006

Abstract From the dried fruiting bodies of the mushroom Armillaria luteo-virens, a dimeric lectin with a molecular mass of 29.4 kDa has been isolated. The purification procedure involved (NH4)2SO4 precipitation, ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin could not be inhibited by simple sugars but was inhibited by the polysaccharide inulin. The activity was stable up to 70 C but was acidand alkali-labile. Salts including FeCl3, AlCl3, and ZnCl2 inhibited the activity whereas MgCl2, MnCl2, and CaCl2 did not. The lectin stimulated mitogenic response of mouse splenocytes with the maximal response achieved by 1 lM lectin. Proliferation of tumor cells including MBL2 cells, HeLa cells, and L1210 cells was inhibited by the lectin with an IC50 of 2.5, 5, and 10 lM, respectively. However, proliferation of HepG2 cells was not affected. The novel aspects of the isolated lectin include a novel N-terminal sequence, fair thermostability, acid stability, and alkali stability, together with potent mitogenic activity toward spleen cells and antiproliferative activity toward tumor cells.  2006 Elsevier Inc. All rights reserved. Keyword: Lectin

Lectins, well known for their interaction with carbohydrates and glycoproteins, have been isolated from animals [1,2], plants [3–6], fungi [7–20], and bacteria [21]. Some of them have been shown to inhibit tumor cells [7,9,11,15], fungi [22–24], bacteria [25], and viruses [26], and stimulate immune cells [10,14,15]. Hence they have drawn the attention of many investigators. Mushrooms are rich in proteins, one of which is lectin. Lectins from different mushroom species display a variety of molecular masses, subunit numbers, carbohydrate specificity, and amino acid sequences [20]. Some mushroom lectins display antiproliferative activity toward tumor cells *

Corresponding author. E-mail address: [email protected] (H.X. Wang).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.061

[11,15], antitumor activity against tumors [9], mitogenic activity toward spleen cells [10,15], and inhibitory activity toward HIV-1 reverse transcriptase [27]. The objective of the present study was to isolate a lectin from the mushroom Armillaria luteo-virens and find out if it has some distinctive characteristics. Materials and methods Isolation of lectin. Dried fruiting bodies of Armillaria luteo-virens (100 g) were soaked for 1 h in 2500 ml of 0.15 M NaCl, homogenized, and then left to stand overnight (4 C). The homogenate was centrifuged at 10000g for 20 min. Proteins were precipitated with 80% (NH4)2SO4. The precipitate was dissolved in and dialyzed against distilled water. Ion exchange chromatography on a DEAE-cellulose (Sigma) column (2.5 · 20 cm) in 10 mM NH4HCO3 buffer (pH 9.2) was then carried out. After the flowthrough fraction (D1) had come off the column, the column

K. Feng et al. / Biochemical and Biophysical Research Communications 345 (2006) 1573–1578 sphere of 5% CO2. Cells (1 · 104) in their exponential growth phase were seeded into each well of a 96-well culture plate (Nunc, Denmark) and incubated for 3 h before addition of the lectin. Incubation was carried out for another 48 h. Radioactive precursor, 1 lCi, ([3H-methyl]-thymidine, from Amersham Biosciences) was then added to each well and incubated for 6 h. The cultures were then harvested by a cell harvester. The incorporated radioactivity was determined by liquid scintillation counting.

Results The re-dissolved (NH4)2SO4-precipitated fraction of the fruiting body extract was fractionated on DEAE-cellulose into a flowthrough fraction D1 with hemagglutinating (lectin) activity and three adsorbed fractions D2, D3, and D4 without activity. D1 was subsequently fractionated on CMcellulose into an unadsorbed fraction CM1, and three adsorbed fractions CM2, CM3, and CM4. Hemagglutinating activity resided only in fraction CM2 eluted with 50 mM NaCl (Data not shown). When fraction CM2 was loaded on Q-Sepharose, it was resolved into a flowthrough fraction Q1 and two adsorbed fractions Q2 and Q3. Hemagglutinating activity was found in the most strongly adsorbed fraction Q3 which was also the major fraction (Fig. 1). Fraction Q3 was separated on CM-cellulose into an unadsorbed fraction CM1 and an adsorbed fraction CM2. Fraction CM2 represented purified Armillaria luteo-virens lectin (ALL) that gave a single 29.4-kDa peak in gel filtration on Superdex 75 (data not shown) (Fig. 2). In SDS–PAGE, ALL appeared as a single band indicating that it is a homodimer, and that the MW of the subunits was 14.7 kDa (Fig. 3). When subjected to glycoprotein staining with periodic acid-Schiff (PAS) reagent, the protein did not stain with the reagent. About 25-old purification was achieved and 24 mg purified lectin was obtained from 100 g dried fruiting bodies (Table 1). The lectin did not bear sequence resemblance to mushroom lectins but there was slight similarity to some non-lectin proteins (Table 2). The hemagglutinating activity of ALL was stable up to 70 C. No activity was detectable at 80 C and 90 C (Table 3). The activity was stable in 1

0.5 Q3

0.8

0.4

0.6

0.3

0.4

0.2

0.2

NaCl (M)

was eluted sequentially with 50 mM NaCl, 150 mM NaCl, and 1 M NaCl in 10 mM NH4HCO3 buffer (pH 9.2). Fraction D1 was then subjected to ion exchange chromatography, on a column of CM-cellulose (Sigma) (1.5 · 20 cm) in 10 mM NH4OAc buffer (pH4.6). After the flowthrough fraction (CM1) had passed through the column, the column was eluted successively with 50 mM NaCl, 150 mM NaCl, and 1 M NaCl in 10 mM NH4OAc buffer (pH 4.6) to yield fractions CM2, CM3, and CM4, respectively. Fraction CM2 was then loaded on a column of Q-Sepharose (Amersham Biosciences) (1.5 · 20 cm) in 10 mM NH4HCO3 (pH 8.8). After removal of unadsorbed fraction Q1, adsorbed proteins were desorbed with a gradient of 0–0.5 M NaCl in 10 mM NH4HCO3 buffer (pH 8.8). Fraction Q3 was rechromatographed on a 1.5 · 20 cm column of CM-cellulose (Sigma). Unadsorbed protein was eluted in fraction CM1 while the adsorbed fraction CM2 was desorbed with a linear gradient of 0–0.5 M NaCl. Fraction CM2 was then subjected to gel filtration by fast protein liquid chromatography on a Superdex 75 HR 10/30 column (Amersham Biosciences) in 0.2 M NH4HCO3 buffer (pH 8.5). Determination of molecular mass and N-terminal sequence. The purified lectin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) for molecular mass determination in accordance with the procedure of Laemmli and Favre [28]. Gel filtration on an FPLC-Superdex 75 column, which had been calibrated with molecular mass markers (Amersham Biosciences), was conducted to determine the molecular mass of the lectin. The N-terminal sequence of the lectin was determined by using a Hewlett-Packard HP G1000A Edman degradation unit and an HP 1000 HPLC System [6]. Assay for lectin (hemagglutinating) activity. A serial twofold dilution of the lectin solution in microtiter U-plates (50 ll) was mixed with 50 ll of a 2% suspension of rabbit red blood cells in phosphate-buffered saline (pH 7.2) at 20 C. The results were read after about 1 h when the blank had fully sedimented. The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting hemagglutination, was reckoned as one hemagglutination unit. Specific activity is the number of hemagglutination units per mg protein [9]. The hemagglutinating inhibition tests to investigate inhibition of lectin-induced hemagglutination by various carbohydrates were performed in a manner analogous to the hemagglutination test. Serial twofold dilutions of sugar samples were prepared in phosphate-buffered saline. All of the dilutions were mixed with an equal volume (25 ll) of a solution of the lectin with 32 hemagglutination units. The mixture was allowed to stand for 30 min at room temperature and then mixed with 50 ll of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar in the final reaction mixture, which completely inhibited 32 hemagglutination units of the lectin preparation, was calculated [9]. The effects of temperature, NaOH solution, HCl solution, and solutions of metallic chlorides on hemagglutinating activity of the lectin were examined as previously described [9,11]. Assay of mitogenic activity of lectin toward mouse splenocytes. The assay of mitogenic activity was performed as described by Wang et al. [10]. Splenocytes were isolated from BALB/c mice. The cells were diluted with RPMI medium containing 10% fetal bovine serum and then seeded (2 · 106 cells/0.2 ml/well) in 96-well microplates. The lectin was then added at various concentrations. Cells cultured in the absence of the lectin served as control. The cells were incubated at 37 C in a humidified atmosphere of 5% carbon dioxide for 24 h. The cells were viable after 24 h. During the last 6 h, the cells in one well were pulsed with 0.5 lCi of [3Hmethyl]-thymidine (specific activity 5 lCi/mmol, Amersham Biosciences) in 10 ll and were then harvested onto a glass fiber filter using a cell harvester. The radioactivity was determined using a Beckman liquid scintillation counter. The proliferative (mitogenic) response was expressed as mean counts per min (c.p.m.). Assay of antiproliferative activity on tumor cell lines. The antiproliferative activity of the purified lectin was determined as follows [15]. The cell lines L1210 (leukemia), MBL2 (leukemia),HeLa (cervical),and HepG2 (hepatoma) were purchased from American Type Culture Collection. The various cell lines were maintained in Dulbecco modified Eagles’ Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 100 mg/l streptomycin, and 100 IU/ml penicillin at 37 C in a humidified atmo-

A280 (nm)

1574

0.1 Q2

Q1 0

0 0

50

100

150

200

250

Elution volume (ml) Fig. 1. Anion exchange chromatography of fraction CM2 on a Q-Sepharose column (1.5 · 20 cm). Starting buffer: 10 mM NH4HCO3 buffer (pH 8.8). Dotted line indicates linear NaCl concentration gradient used to elute adsorbed proteins.

K. Feng et al. / Biochemical and Biophysical Research Communications 345 (2006) 1573–1578

0.5 CM1

0.4 0.3

CM2 (ALL)

0.4

0.2

NaCl (M)

A 280 (nm)

0.6

0.2 0.1 0

0 0

100

200

1575

12.5–50 mM NaOH and 25 mM HCl, and reduced to 25% in 50 and 100 mM HCl. Activity was indiscernible in 02 M NaOH/HCl (Table 4). The activity was not affected by CaCl2, MgCl2, and MnCl2, but was inhibited by ZnCl2, FeCl2 and AlCl3, with FeCl3 being the most potent inhibitor (Table 5). Inulin was the only among the carbohydrates tested that was capable of inhibiting the hemagglutinating activity of Armillaria luteo-virens lectin (Table 6). Of the four tumor cell lines tested, the lectin was able to inhibit the proliferation of three of them (Table 7). ALL at a concentration of 1 lM was able to evoke maximal mitogenic response from mouse splenocytes (Fig. 4).

Elution volume (ml) Fig. 2. Cation exchange chromatography of fraction Q3 on a CMcellulose column (1.5 · 20 cm). Starting buffer: 10 M NH4OAc buffer (pH 5.2). Dotted line indicates linear NaCl concentration gradient used to elute adsorbed proteins.

Fig. 3. SDS–PAGE results. Left lane: purified lectin. Right lane: molecular mass marker from Amersham Biosciences.

Discussion The lectin isolated from Armillaria luteo-virens (ALL) has an N-terminal amino acid sequence not found in previously reported mushroom lectins and other mushroom proteins. Its resemblance to a glucosyltransferase and two transport proteins is restricted to only small segments of the interior of the molecules. Hence ALL is a novel mushroom lectin. ALL is stable in the presence of 0.0125– 0.05 M NaOH solutions and 0.0125 M HCl solution. Hemagglutinating activity is detectable in 0.1 M NaOH and 0.1 M HCl solution. Peziza sylvestris lectin loses all of its hemagglutinating activity in 0.1 M NaOH and 0.1 M HCl solution [29]. ALL is more stable in this aspect than some other mushroom lectins and plant lectins [3]. The hemagglutinating activity of ALL is completely preserved up to 70 C. By comparison, P. sylvestris lectin loses all of its hemagglutinating activity at 55 C [28] and Pseudostellaria heterophylla lectin retains only 50% of its hemagglutinating activity at 40 C [3]. ALL is distinctive in that its hemagglutinating activity cannot be inhibited

Table 1 Yields and hemagglutinating activities of various chromatographic fractions (from 100 g dried Armillaria luteo-virens fruiting bodies) Fraction

Yield (mg)

Specific hemagglutinating activity(units/mg)

Total hemagglutinating activity (units)

Recovery of hemagglutinating activity (%)

Folds of purification

Crude extract D1 D1CM2 D1CM2Q3 D1CM2Q3CM2 (purified lectin)

3346 374 84 44 24

574 3417 10221 13925 14333

1.9 · 106 1.3 · 106 8.6 · 105 6.1 · 105 3.4 · 105

100 66.7 44.4 31.2 17.7

1 6.0 17.8 24.3 25.0

Table 2 N-terminal sequence of Armillaria luteo-virens lectin (ALL) Armillaria luteo-virens lectin Mg2+ transporter, Cor-like (Arthuobcuter sp. FB24) (325–338) Nicotinamide mononucleotide transporter (Oenococeus oeni) (151–159) 4-Amino-4-deoxy-L-arabinose transferase and related glycosyltransferase from Crocosphaera watsonii (158–169) Identical corresponding amino acid residues are underlined. - - -: Space created to maximize similarity.

GVVFFAAFKQTKWIV GVILYAAFRHNKWI -VVFFAA- - - - -WIV - - -FFKSFKQPKWLV

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Table 3 Effect of temperature on hemagglutinating activity of Armillaria luteo-virens lectin Temp (C) Hemagglutination titer(U)

10 32

20 32

30 32

40 32

50 32

60 32

70 32

80 0

90 0

Table 4 Effects of NaOH and HCl on hemagglutinating activity of Armillaria luteo-virens lectin (initial hemagglutinating activity: 32 U) NaOH (M) Conc. Hemagglutination titer (U)

HCl (M)

0.0125 32

0.025 32

0.05 32

0.1 16

0.2 0

0.0125 32

0.025 16

0.05 8

0.1 8

0.2 0

Table 5 Effects of cations on hemagglutinating activity of Armillaria luteo-virens lectin (initial hemagglutinating activity: 32 U) Cation

Concentration (mM) 10

5

2.5

1.25

CaCl2 ZnCl2 MnCl2 MgCl2 FeCl3 AlCl3

32 16 32 32 2 8

32 16 32 32 2 8

32 32 32 32 4 16

32 32 32 32 4 16

Table 6 Effect of various carbohydrates on hemagglutination induced by Armillaria luteo-virens lectin (8 hemagglutinating units) Sugar (mM)

200

100

50

25

12.5

6.25

3.12

1.56

0.78

PBS

D-Mannose

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

D-Fructose D-Xylose Adonitol L-Arabinose Sorbose Inulin Raffinose L-Rhamnose D-Melezitose D-Melibiose Cellobiose D-Ribose Inositol D-Glucose Sucrose D-Galactose Galactitol O-Nitrophenyl -b-D-galacto-pyranoside 4-o-b-D-Galactopyranosyl-D-glucose

Note. +, hemagglutination activity;

,no hemagglutination activity; and PBS, phosphate-buffered saline.

Table 7 Antiproliferative activity of Armillaria lutes-virens lectin toward tumor cells IC50 (lM) against tumor cell lines Tumor cell line ALL

L1210 10

MBL2 2.5

HeLa 5

HepG2 No activity

incorporation (kCPM)

3

( H)thymidine

K. Feng et al. / Biochemical and Biophysical Research Communications 345 (2006) 1573–1578

30

20

10

0

16

8

4

2

1

0.5

0.25

0

Concentration of ALL (μM) Fig. 4. Mitogenic response of mouse splenocytes to Armillaria luteo-virens lectin (ALL).

by a variety of simple sugars but can be inhibited by inulin, FeCl3, and AlCl3. It manifests potent mitogenic activity toward murine spleen cells, producing a 10-fold stimulation at 1 lM concentration. ALL inhibits proliferation of different tumor cell lines with different potencies, with the highest potency toward MBL2 cells, intermediate potency toward HeLa cells, and the lowest potency toward L1210 cells. ALL is devoid of antiproliferative activity on HepG2 cells. The molecular size of ALL and its number of subunits fall within the range shown by other mushroom lectins [20]. ALL can be purified by using a protocol comprising anion and cation exchange chromatography and gel filtration, similar to the isolation procedures previously employed for other mushroom lectins [9,10,14,15,29]. However, ALL is unadsorbed on DEAE-cellulose, in contrast to other mushroom lectins [9,10,14]. In summary, ALL is a lectin with unique features. Acknowledgment We thank Ms. Fion Yung for excellent secretarial assistance. References [1] M. Engel, M. Bachmann, H.C. Schroeder, B. Rinkevich, Z. Kljajic, G. Uhlenbruck, W.E.G. Mueller, A novel galactose and arabinose specific lectin from the sponge Pellina semitubulosa. Isolation, characterization and immunological properties, Biochimie 74 (1992) 527–537. [2] T.B. Ng, Y.W. Lam, N.Y.S. Woo, The immunostimulatory activity and stability of grass carp (Ctenopharyngodon idellus) roe lectin, Vet. Immunol. Immunopathol. 94 (2003) 105–112. [3] J.H. Wong, T.B. Ng, A homotetrameric agglutinin with antiproliferative and mitogenic activities from haricot beans, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2005). [4] T.B. Ng, Y.L. Yu, Isolation of a novel heterodimeric agglutinin from rhizomes of Smilax glabra, the Chinese medicinal material tufuling, Int. J. Biochem. Cell Biol. 33 (2001) 269–277. [5] J.H. Wong, T.B. Ng, Isolation and characterization of a glucose/ mannose/rhamnose-specific lectin from the knife bean Canavalia glacdiata, Arch. Biochem. Biophys. 439 (2005) 91–98.

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