Isolation and Characterization of a Novel Mannose- and Fructose-Binding Lectin from the Edible Wild Mushroom Hygrophorus russula (Fr.) Quél.

Abstract

A novel lectin, with a molecular mass of 18.6 kDa and a unique N-terminal amino acid sequence, was purified from dried fruiting bodies of the mushroom Hygrophorus russula (Fr.) Quél.. The purification procedure encompassed successive ion exchange chromatography on CM-cellulose, DEAE-cellulose, Q-Sepharose, SP-Sepharose and Mono S. The hemagglutinating activity of the lectin (NHRL) was stable at temperatures below 50°C and in the presence of 6 mM NaOH or 25 mM HCl. The activity was activated by Cu²⁺, Al³⁺, Fe³⁺, and Pb²⁺ ions, and inhibited by Hg²⁺ ions. Among the sugars tested, only mannose and fructose inhibited its hemagglutinating activity.

Introduction

Lectins are glycoproteins or proteins which can agglutinate cells or precipitate glycoconjugates (Goldstein et al., 1980). According to their carbohydrate specificities, lectins can be divided into six groups: (1) d-glucose- or d-mannose-specific, (2) N-acetylglucosamine-specific, (3) N-acetyl-galactosamine-specific, (4) d-galactose-specific, (5) l-fucose-specific, and (6) N-acetylneuraminic acid-specific (Sharon and Lis, 1989). Lectins display a wide spectrum of biological activities encompassing antiproliferative, antitumor, antifungal, antiviral, anti-insect and immunomodulatory activities (De Mejia and Prisecaru 2005, Etzler, 1985; Feng et al., 2006; Ng, 2004; Peumans and Vandamme, 1995; Sumisa et al., 2004; Wang and Ng, 2003).

Lectins from mushrooms have captured the attention of more and more researchers. Lectins have been examined from a variety of mushrooms such as Agaricus arvensis (Zhao et al., 2011), Boletus edulis (Zheng et al., 2007), Hericium erinaceum (Kawagishi et al., 1994; Li et al., 2010), Pleurotus ostreatus (Kawagishi et al., 2000; Wang et al., 2000), and Inocybe umbrinella (Zhao et al., 2009).

In a preliminary experiment hemagglutinating activity was observed in an extract of dried fruiting bodies of Hygrophorus russula which is an edible wild mushroom distributed in mixed forests 2700 – 3800 m above the sea level. However, the examination of a lectin from Hygrophorus russula for various potentially exploitable biological activities such as ribonuclease, antiproliferative and HIV-1 reverse transcriptase inhibiting activities has not been attempted. Thus we set out to isolate a lectin from this mushroom and to look for the aforementioned activities.

Materials and Methods

Purification of lectin    Dried fruiting bodies of the mushroom Hygrophorus russula (75 g) were collected form Sichuan Province in China. The fruiting bodies (50 g) were soaked in 750 mL of 0.15 M NaCl and then blended using a Waring blender. The homogenate was left at 4°C for 8 h. The supernatant obtained after centrifugation (9000 × g, 4°C, 10 min) was then dialyzed overnight prior to ion exchange chromatography on a CM-cellulose (Sigma) column (2.5 × 20 cm) in 10 mM HAc-NaAc buffer (pH 4.6). After removal of unadsorbed proteins in fraction C1, adsorbed proteins were desorbed sequentially with 50 mM NaCl, 300 mM NaCl, and 1 M NaCl in the starting buffer. The adsorbed fraction (C3) eluted with 300 mM NaCl was dialyzed, before ion exchange chromatography on DEAE-cellulose (Sigma) (2.5 × 10 cm) in 10 mM Tris-HCl buffer (pH 8.6). After elution of the unadsorbed fraction (D1), the column was eluted with 200 mM NaCl, and 1 M NaCl in the starting buffer. The second adsorbed fraction (D2) eluted with 200 mM NaCl was dialyzed before loading on a 2.5 × 4 cm column of Q-Sepharose (GE Healthcare) in 10 mM Tris-HCl buffer (pH 7.2). Almost all of the proteins were bound. The column was eluted with a linear 0 – 1 M NaCl gradient in the Tris-HCl buffer. The first adsorbed fraction (Q1) was dialyzed and loaded on an SP-Sepharose (GE Healthcare) (1.5 × 10 cm) column in 10 mM HAc-NaAc buffer (pH 4.6). After removal of unadsorbed proteins in fraction SP1, the column was eluted with a linear 0 – 1 M NaCl gradient in the 10 mM HAc-NaAc buffer (pH 4.6). The adsorbed fraction (SP2) was lyophilized, prior to ion exchange chromatography on a Mono S TM 4.6/100 PE column. The bound fraction (M2) exhibited lectin (hemagglutinating) activity.

Determination of molecular mass and N-terminal sequence    In accordance with the procedure of Laemmli and Favre (1973), the purified lectin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) employing a 12% resolving gel and a 5% stacking gel. After electrophoresis the gel was stained with Coomassie brilliant blue. The molecular mass of the purified lectin was determined by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) (Oleschuk et al., 2000). The N-terminal amino acid sequence of the lectin was carried out using an HP-G1000A Edman degradation unit and an HP 1000 HPLC system (Ng and Wang, 2004).

Assay for lectin (hemagglutinating) activity    A serial twofold dilution of the lectin solution in a 96-well culture plate prepared using physiological saline was mixed with 25 µL of a 2% suspension of rabbit red blood cells in physiological saline at room temperature. The results were read after about 0.5 h when the blank control 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 (Wang et al., 2000).

Test of inhibition of lectin-induced hemagglutination by various carbohydrates    The initial concentration of carbohydrates used was 400 mM. Serial twofold dilutions of a sugar solution in physiological saline were prepared, and then an equal volume (25 µL) of lectin solution with 16 hemagglutination units was added. After 30 min at room temperature, the mixture was mixed with 50 µL of a 2% rabbit erythrocyte suspension. The results were read after about 1 h when the blank control had fully sedimented. The minimum concentration of the sugar which completely inhibited 32 hemagglutination units of the lectin preparation was calculated.

The effects of temperature, NaOH solution, HCl solution and solutions of metal chlorides on hemagglutinating activity of the lectin were examined as previously described (Wang et al., 1998).

Effect of lectin on tumor cell proliferation    The antiproliferative activity of the purified lectin was determined as follows (Wang et al., 1995). The hepatoma HepG2 cell line and breast cancer MCF7 cell line were purchased from American Type Culture Collection. The cell lines were maintained in Dulbecco modified Eagles' Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/L streptomycin and 100 IU/mL penicillin, at 37° in a humidified atmosphere of 5% CO₂. Cells (1 × 10⁴) in their exponential growth phase were seeded into wells of a 96 microtiter plate (Nunc, Denmark) and incubated for 3 h before addition of the lectin. Incubation was carried out for another 48 h. Radioactive precursor [³H-methyl] thymidine (1 µCi, from GE Healthcare), was then added to each well and incubated for 6 h. The cultures were then harvested by using a cell harvester. Incorporation of radionuclide to tumor cells was measured by using a liquid scintillation counter (Feng et al., 2006).

Assay of HIV-1 reverse transcriptase (HIV-1 RT) inhibitory activity    The assay was conducted using an HIV-1 RT ELISA kit, following the instructions of the manufacturer (Germany) (Wang and Ng, 2001). The assay takes advantage of the ability of HIV-1 RT to synthesize DNA, commencing from the template/primer hybrid poly(A) oligo(dT)15. The digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same DNA molecule, which is freshly synthesized by the reverse transcriptase. The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol. The absorbance of the samples at 405 nm can be determined using a microtiter plate (ELISA) reader and is directly correlated to the level of RT activity. A fixed amount (4 – 6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of the test protein was calculated as percentage inhibition compared with the control without any protein added. Pinto bean lectin with anti HIV-1 RT activity was chosen as a positive control (Lam and Ng, 2001).

Assay for RNase activity    The activity of the purified lectin toward yeast tRNA (Sigma) was determined by measuring the formation of acid-soluble, UV-absorbing species with the method of Wang and Ng (1999). The lectin was incubated with 200 µg tRNA in 150 µL of 100 mM MES buffer (pH 6.0) at 37° for 15 min. The reaction was stopped by addition of 350 µL of ice-cold 3.5% perchloric acid and leaving the reaction mixture on ice for 15 min. After centrifugation (15,000 g, 15 min) at 4°C, OD260 of the supernatant was determined after appropriate dilution. One unit of enzymatic activity is defined as the amount of enzyme that brings about an increase in OD260 of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified condition.

Results and Discussion

Isolation of lectin    The fraction of the fruiting body extract unadsorbed on CM-cellulose (C1) contained RNase activity while the absorbed fraction C3 contained hemagglutinating activity. C3 was subsequently divided into an unbound fraction D1 and two bound fraction D2 and D3. Activity was found in fraction D2 (Table 1). When fraction D2 was loaded on Q-Sepharose, it was resolved into a tiny fraction Q1 and a large fraction Q2. Fraction Q1 was separated on SP-Sepharose into an unadsorbed fraction SP1 and an adsorbed fraction SP2 (Fig. 1). Hemagglutinating activity resided in fraction SP2 which was split into an unadsorbed faction M1 and an adsorbed fraction M2 on Mono S (Fig. 2). Hemagglutinating activity was detected in fraction M2 (Table 1). More than 100-fold purification of H. russula lectin from the fruiting body extract was achieved in the present study.

Fig. 1.

Ion exchange chromatography on SP-Sepharose of a fruiting body extract of H. russula, previously adsorbed successively on CM-cellulose, DEAE-cellulose and Q-Sepharose. Peak SP1 was eluted with 10 mM HAc-NaAc buffer (pH 4.6). Peak SP2 was eluted with a linear NaCl concentration gradient (0 – 1 M) in the HAc-NaAc buffer. Pooled fraction was indicated by an asterisk. The dotted line represents the NaCl concentration gradient.

Fig. 2.

Ion exchange chromatography of fraction SP2 from SP-Sepharose column on a Mono S TM 4.6/100 PE column by FPLC on an AKTA purifier. Peak M1 was eluted with 10 mM HAc-NaAc buffer (pH 4.2). Peak M2 was eluted with a linear NaCl concentration gradient (0 – 1 M) in the HAc-NaAc buffer. Flow rate: 0.7 mL/min. fraction size: 1.4 mL. Pooled fraction was indicated by an asterisk. The dotted line represents the NaCl concentration gradient.

Table 1. Yields and hemagglutinating activities of various chromatographic fractions derived from 75 g dried H. russula fruiting bodies.

Fraction	Yield (mg)	Specific activity (U/mg)	Total activity (U×103)	Recovery of activity (%)	Purification fold
Extract	11742	155	1824	100	1
C3	279.8	2058	576	31.6	13.3
D2	68.4	6175	422.4	23.2	39.8
Q1	29.6	11730	347.2	19.0	75.5
SP2	8.8	15784	138.9	7.6	101.6
M2	1.1	19727	21.7	1.2	127

In SDS-PAGE, the novel H. russula lectin (NHRL) manifested a single band with a molecular mass of 18.6 kDa (Fig. 3) which was similar to the molecular mass determined by using MALDI-TOF-MS. Like most of the reported mushroom lectins, NHRL has a subunit molecular mass around 20 kDa (Wang et al., 1998). The sequence of the first 30 N-terminal amino acid residues of NHRL was TIGNA KPVLV QQEIV GGRRI AFDDA REVCA which demonstrated slight similarity to other mushroom lectins (Fig. 4 and 5). It displayed some similarity to B. edulis lectin and slight resemblance to the 41 kDa subunit of P. ostreatus lectin, 16.1 kDa subunit of A. cylindracea lectin and A. arvensis lectin (Wang et al., 2000; Wang et al., 2002; Zhao et al., 2011; Zheng et al., 2007). A comparison of the sequence of the first thirty N-terminal residues in NHRL with that of the H. russula lectin reported in the literature (Suzuki et al., 2012) disclosed some differences (Fig. 5).

Fig. 3.

SDS-PAGE results.

Left lane: purified lectin. Right lane: molecular mass marker proteins, from top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and α-lactalbumin (14.4 kDa).

Fig. 4.

Comparison of N-terminal sequences of lectins from H. russula in this study and other mushrooms/fungi. Identical residues are indicated in gray.

Fig. 5.

Comparison of N-terminal sequences of NHRL (H. russula isolated in this study) and HRL (H. russula isolated in study of Suzuki et al. (2012)). Eleven different amino acid residues (shown in italics) are observed at corresponding positions in the sequence of the first thirty residues.

Characterization of isolated lectin    The isolated lectin NHRL showed specificity toward d(+)-mannose and d(−)-fructose (Table 2). Although, several plant lectins with mannose specificity have been discovered (Bourne et al., 1999; Chu and Ng, 2006), only a very small number of mushroom lectins with mannose specificity have been demonstrated (Suzuki et al., 2012).

Table 2. Effects of various carbohydrates on hemagglutination induced by H. russula lectin.

Sugar	Concentration (mM)
	200	100	50	25	12.5	6.25	3.12	1.56	0.78	control
inulin	+	+	+	+	+	+	+	+	+	+
d-melibiose	+	+	+	+	+	+	+	+	+	+
d-(−)-fructose	−	−	−	+	+	+	+	+	+	+
l-(+)-arabinose	+	+	+	+	+	+	+	+	+	+
l-rhamnose	+	+	+	+	+	+	+	+	+	+
d-xylose	+	+	+	+	+	+	+	+	+	+
l-(−)-sorbose	+	+	+	+	+	+	+	+	+	+
inositol	+	+	+	+	+	+	+	+	+	+
α-lactose	+	+	+	+	+	+	+	+	+	+
d-(+)-galactose	+	+	+	+	+	+	+	+	+	+
cellobiose	+	+	+	+	+	+	+	+	+	+
d-glucose	+	+	+	+	+	+	+	+	+	+
d-(+)-mannose	−	−	−	−	−	+	+	+	+	+
raffinose	+	+	+	+	+	+	+	+	+	+
maltose	+	+	+	+	+	+	+	+	+	+
O-Nitrophenyl-β-d-galactopyranananoside	+	+	+	+	+	+	+	+	+	+

“+” represents presence of hemagglutinating activity;

“−” represents absence of hemagglutinating activity;

Initial hemagglutinating activity before addition of carbohydrate: 16 U (hemagglutination units)

NHRL was more stable in an acidic environment. It was stable in the presence of 6 – 25 mM HCl solutions and 6 mM NaOH solution. The hemagglutinating activity was halved in 12.5 mM NaOH and 50 mM HCl solutions, and undetectable in 100 mM HCl and 25 – 100 mM NaOH solutions (Table 3).

Table 3. Effects of NaOH and HCl solutions on hemagglutinating activity of H. russula lectin.

HCl (mM)	6	12.5	25	50	100
Activity (U)	64	64	64	32	0
NaOH (mM)	6	12.5	25	50	100
Activity (U)	64	32	0	0	0

Initial hemagglutinating activity before addition of NaOH and HCl solutions: 64 U (hemagglutination units)

The hemagglutinating activity of NHRL was unaffected in the presence of a variety of metallic chlorides including CaCl₂, KCl, MnCl₂, ZnCl₂, MgCl₂, CdCl₂, and FeCl₂. The activity underwent a progressive increase in the presence of 1 10 mM CuCl₂. It was doubled (to 128 units), quadrupled (to 256 units), and increased eight-fold (to 512 units) in the presence of 2.5 mM, 5 mM, and 10 mM CuCl₂. The activity was doubled in the presence of 1.25 mM and 2.5 mM AlCl₃, 10 mM PbCl₂, and 5 mM and 10 mM FeCl₃. The activity was inhibited only by HgCl₂. In the presence of 5 mM and 10 mM HgCl₂, the activity was reduced to 25% (Table 4). By comparison, the activities of R. lepida lectin and P. citrinopileatus lectin were also inhibited by Hg²⁺ ions. It is remarkable that Cu²⁺ ions dose-dependently augmented the hemagglutinating activity of NHRL. In contrast, D. biflorus lectin binds Cu²⁺ ions which, however, do not affect its sugar binding activity (Borrebaeck et al., 1981).

Table 4. Effects of cations on hemagglutinating activity of H. russula lectin.

Ion	10 mM	5 mM	2.5 mM	1.25 mM
Ca2+	64	64	64	64
K+	64	64	64	64
Mn2+	64	64	64	64
Zn2+	64	64	64	64
Mg2+	64	64	64	64
Cd2+	64	64	64	64
Hg2+	16	16	64	64
Al3+	256	256	128	128
Pb2+	128	64	64	64
Cu2+	512	256	128	64
Fe3+	128	128	64	64
Fe2+	64	64	64	64

Initial hemagglutinating activity before addition of cations: 64 U (hemagglutination units)

The hemagglutinating activity of NHRL was stable up to 50°C for 30 min. It started to decline at 60°C. Only half of the activity remained at 60°C and 70°C. Even at 90°C, a quarter of the activity remains (Table 5). By comparison, X. hypoxylon lectin lost all hemagglutinating activity at 55°C (Liu et al., 2006), and P. ostreatus lectin was stable only below 35°C (Wang et al., 2000). The thermostability of NHRL was similar to that of P. adiposa lectin which was also stable below 50°C (Zhang et al., 2009).

Table 5. Effect of temperature on hemagglutinating activity of H. russula lectin.

Temperature (°C)	10	20	30	40	50	60	70	80	90	100
Activity (U)	32	32	32	32	32	16	16	8	8	0

Initial hemagglutinating activity before exposure to various temperatures: 32 U (hemagglutination units)

In contrast to lectins that displayed antiproliferative effect on HepG2 and MCF7 tumor cell lines (Wang et al., 2000; Zhang et al., 2010; Zhang et al., 2009; Zhao et al., 2009), The lectin did not inhibit proliferation of the HepG2 and MCF7 tumor cell lines when tested up to 200 µM (data not shown). This finding demonstrated that the hemagglutinating and antiproliferative activities may be separate and unrelated in some lectins. At 3.2 µM, 16 µM, 80 µM, and 400 µM, the lectin inhibited HIV-1 reverse transcriptase by 17.89%, 46.32%, 49.33%, and 77.31%, respectively. The IC₅₀ was 81 µM. The ability of H. russula lectin to inhibit HIV-1 reverse transcriptase is consistent with the reports of the HIV-1 reverse transcriptase inhibitory activity of some plant and mushroom lectins (Wang et al., 2001). The mechanism of inhibition probably involves protein-protein interaction, similar to what has been shown for the inhibition of HIV-1 reverse transcriptase by HIV-1 protease (Bottcher and Grosse, 1997).

After we had commenced the present study on NHRL, a report appeared on the isolation of a lectin designated as H. russula lectin (HRL) and composed of four identical 18.5 kDa subunits from the mushroom H. russula (Suzuki et al., 2012). Both HRL and NHRL were adsorbed on cationic (S and SP) exchangers. A step of chromatography on Sephadex G50 using mannose to elute adsorbed proteins was employed in the isolation of HRL. The specific hemagglutinating activity (32000 U/mg) and yield (2.7 mg/100 g mushroom) of HRL was higher than those of NHRL (19727 U/mg and 1.5 mg/100 g mushroom, respectively). HRL and NHRL were similar in subunit molecular size and carbohydrate specificity. When the sequence of the first thirty N-terminal residues in NHRL is compared with that of the H. russula lectin reported in the literature (Suzuki et al., 2012), eleven different amino acid residues (shown in italics) are observed at corresponding positions in the sequences, indicating a 63.3% identity (Fig. 5). Most of the differences are not caused by replacements with similar residues like substitution of arginine by lysine, glutamine by asparagine, or leucine by isoleucine.

The hemagglutinating activity of HRL was stable at temperatures under 60°C for 30 min, and over a wide pH range of 2.0 to 9.5. The hemagglutinating activity of NHRL was stable up to 50°C for 30 min and started to decline at 60°C and when exposed to 12.5 mM NaOH and 50 mM HCl. Thus HRL seemed to have higher thermal and pH stability. Al³⁺, Fe³⁺, Pb²⁺ and Cu²⁺ ions potentiated while Hg²⁺ ions undermined the hemagglutinating activity of NHRL in the present study. These ions had not been tested by Suzuki et al. (2012). In both the present study and the investigation of Suzuki et al. (2012), Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions were shown to be without effect on the hemagglutinating activity of lectin from H. russula.

Unlike NHRL, HRL has not been tested for antiproliferative activity toward tumor cells, inhibitory activity toward HIV-1 reverse transcriptase and ribonuclease activity toward yeast transfer RNA. The observation that NHRL did not inhibit tumor cell proliferation signified that hemagglutinating and antiproliferative activities may be separate and unrelated in some lectins. NHRL was devoid of ribonuclease activity. In fact, a ribonuclease has previously been isolated and characterized from H. russula by us (Zhu et al., 2013). A lectin from the mushroom Russula lepida also lacked ribonuclease activity (Zhang et al., 2010). In this aspect, NHRL was different from amphibian lectins which demonstrated ribonuclease and antitumor activities (Benito et al., 2005; Tatsuta et al., 2013).

In view of the various aforementioned differences between HRL and NHRL, it is likely that they represent similar and related but not identical proteins. The mushrooms utilized by Suzuki et al. (2012) and those employed in the present work might be from two different cultivars/strains. It has been shown that even lectins in fruiting bodies and mycelia of the same mushroom Tricholoma mongolicum displayed different characteristics (Wang et al., 1998).

In summary, a lectin with a distinctive N-terminal sequence, specificity toward mannose and fructose, hemagglutinating activity with relatively high thermostability and susceptibility to inhibition by some metallic chlorides and potentiation by other metallic chlorides, was isolated from H. russula fruiting bodies. This report represents an addition to the existing information on mushroom lectins. It provides additional information to and/or complement what has been reported by Suzuki et al. (2012).

Acknowledgement    This work was financially supported by National Grants of China (2010CB732202)

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