First isolation and characterization of a novel lectin with potent antitumor activity from a Russula mushroom.
To date only a ribonuclease and a protein with anti-HIV-1 reverse transcriptase activity have been isolated from mushrooms of the genus Russula. In this study a novel lectin, with a molecular weight of 32 kDa, and a unique N-terminal sequence different from other lectins, was isolated from the mushroom Russula lepida. It represents the first lectin isolated from Russula mushrooms. The purification scheme involved [([NH.sub.4]).sub.2][SO.sub.4] precipitation, ion exchange chromatography on diethylaminoethyl DEAE-cellulose and SP-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin (RLL) was inhibited by inulin and O-nitrophenyl-[beta]-D-galacto-pyranoside. The lectin was stable at temperatures up to 70 [degrees]C (half of the activity was preserved at 80 [degrees]C), and in the presence of NaOH or HC1 solutions up to a concentration of 12.5 mM. Its hemagglutinating activity was reduced in the presence of [Mn.sup.2+], [Co.sup.2+], and [Hg.sup.2+] ions, and enhanced by [Cu.sup.2+] ions. It exhibited antiproliferative activity toward hepatoma Hep G2 cells and human breast cancer MCF-7 cells with an [IC.sub.50] of 1.6 [micro]M and 0.9 [micro]M, respectively. Daily intraperitoneal injections of RLL (5.0 mg/kg body weight/day for 20 days) brought about 67.6% reduction in the weight of S-180 tumor. RLL was devoid of antifungal, ribonuclease, and HIV-1 reverse transcriptase inhibitory activities.
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Lectins are carbohydrate-binding proteins or glycoproteins of nonimmune origin that can agglutinate cells or precipitate glyco-conjugates (Goldstein et al., 1980). Although they were first identified in plants, they are now known to distribute widely throughout the nature, including plants, animals, fungi, bacteria and viruses. In mushrooms, lectins have been localized on the caps, stipes and the mycelia (Ng, 2004). Most plant lectins are storage proteins and defence proteins when the plant or the seed is assaulted by insects or fungi (Janzen et al., 1976; Mirelman et al., 1975). In mammals, lectins play a part in the migration of lymphocytes from the bloodstream into the lymphoid organs and in metastasis of cancer cells (Wang et al., 1998). In mushrooms, lectins may play crucial roles in dormancy, growth, morphogenesis, morphological changes and molecular recognition during the early stages of mycorrhization (Guillot and Konska, 1997; Ng, 2004). Mushroom lectins manifest exploitable biological activities such as antiproliferative (Wang et al., 1995), antitumor (Wang et al., 2000), immunomodulatory (She et al., 1998), and HIV-1 reverse transcriptase inhibiting (Wang and Ng, 2001) activities. Hence they have captured the attention of many investigators.
Russula lepida, which is also called red mushroom by people in northeast China, is a wild mushroom. It belongs to Phylum Basidiomycota, Order Agaricales, Family Russulaceae. A prolyl endopeptidase and a seco-ring-A cucurbitane triterpenoid were reported from R. lepida (Jian-Wen et al., 2002; Yoshimoto et al., 1988). Recently, some new terpenoids and ceramides have been reported from Russula sp. (Gao et al., 2000; Gao et al., 2001; Tan et al., 2001). However, there are few reports on its proteinaceous constituents. The objective of the present study was to isolate a lectin from the mushroom R. lepida and find out if it has any distinctive characteristics.
Materials and methods
Isolation of lectin
Dried fruiting bodies of the mushroom Russula lepida (100 g) were homogenized in 0.15 M NaCl at 4 [degrees]C and extracted overnight at 4 [degrees]C. Then the homogenate was centrifuged at 8000g for 15min. [([NH.sub.4]).sub.2][SO.sub.4] was added to the supernatant to 80% saturation and the mixture was stirred. Subsequently, the mixture was centrifuged at 8000g for 15 min. Then the precipitate was dissolved, and dialyzed to remove [([NH.sub.4]).sub.2][SO.sub.4] before applying to a DEAE-cellulose (Sigma) column (2.5 x 20 cm) which had previously been equilibrated with and was then eluted with 10 mM phosphate buffer (pH 7.9). After removal of the un-adsorbed fraction (D1) containing strong hemagglutinating activity, three adsorbed fractions (D2, D3 and D4) were eluted with 50 mM NaCl, 150mM NaCl and 1 M NaCl in the starting buffer, respectively. The active fraction (Dl) was applied to a column of SP-Sepharose (GE Healthacre, 1.5 x 10 cm) which had been equilibrated and then eluted with 10 mM [NH.sub.4]OAc buffer (pH 4.8). Unbound material was eluted with the starting buffer while bound material was desorbed by addition of 50 mM NaCl, 150 mM NaCl and 1 M NaCl in the starting buffer, respectively. The active peak (SP2) containing hemagglutinating activity was subsequently chromatographed on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.15 M [NH.sub.4][HCO.sub.3] buffer (pH 8.5) using an AKTA Purifier (GE Healthcare). The first peak (SU1) was the purified lectin (RLL).
Determination of molecular weight and N-terminal sequence
The active fraction (SU1) was subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination in accordance with the procedure of Laemmli and Favre (1970). The molecular weight of the purified protein was also determined by FPLC-gel filtration as described above. N-terminal sequencing of the protein was carried out using an HP G-1000A Edman degradation unit and an HP 1000 HPLC system (Lam and Ng, 2001).
Assay for lectin (hemagglutinating) activity
A serial two fold dilution of the lectin solution was mixed with 25 [micro]l of a 2% suspension of rabbit erythrocytes in phosphate-buffered saline (pH 7.2) at 20 [degrees]C in microtiter U-plates (25 [micro]l). The results were observed after approximately 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/mg protein (Han et al., 2005; Zheng et al., 2007).
The hemagglutinating inhibition tests to investigate the inhibition of lectin-induced hemagglutination by various carbohydrates were performed in a manner analogous to the hemagglutination test. Serial two-fold dilutions of sugar samples were prepared in phosphate-buffered saline. All of the dilutions were mixed with an equal volume (25 [micro]1) of a solution of the lectin with 8 hemagglutination units. The mixture was permitted to stand at room temperature for 30 min and then mixed with 50 [micro]l of a 2% rabbit erythrocyte suspension. The minimum concentration of the sugar in the final reaction mixture, which totally inhibited 8 hemagglutination units of the lectin preparation, was determined (Feng et al., 2006).
The effects of temperature, NaOH solution, HC1 solution, and solutions of metallic chlorides on hemagglutinating activity of the lectin were investigated as described earlier (Han et al., 2005).
Assay of antiproliferative activity on tumor cell lines
The assay was carried out since some lectins demonstrate this activity (Wang et al., 2000). The tumor cell lines human breast cancer (MCF-7) and hepatoma (HepG2) obtained from American Type Culture Collection (ATCC) were maintained in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 mg/l streptomycin and 100 IU/ml penicillin at 37 [degrees]C in a humidified atmosphere of 5% [CO.sub.2]. Cells (1 x [10.sup.4]) 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 then continued for another 48 h. Radioactive precursor, 1 [micro]Ci, ([[.sup.3]H-methyl] thymidine, from GE Healthcare) was then added to each well followed by incubation for 6 h. The cultures were then harvested by using a cell harvester. The incorporated radioactivity was determined by liquid scintillation counting (Feng et al., 2006).
Assay for antitumor activity in vivo
The assay was carried out since some lectins demonstrate this activity (Wang et al., 2000). Male white Kunming mice (weighing 21 [+ or -] 1.5 g), kept at a constant temperature (24-26 [degrees]C) and under a 14-h light 10-h dark cycle, were used in this assay. Laboratory rodent chow (No. 1527; China Institute of Laboratory Animal Sciences, Beijing, China) and water were provided ad libitum. The tumor cell line Sarcoma 180 (S-180) was purchased from ATCC and maintained in the peritoneal cavity of the mice. A mouse was sacrificed on the seventh day after inoculation of S-180 tumor cells and the ascitic fluid was collected. After the cells had been washed three times with phosphate-buffered saline, the cell suspension was diluted to 5 x [10.sup.7] cells/ml. A 0.1 ml dose of the suspension was inoculated subcutaneously into the inguinal area of the mice (Wang et al., 2000).
After inoculation, the mice were randomly assigned to two groups (six mice per group) as follows: 1) group one was the lectin-treated group, which received daily intraperitoneal injections of RLL at the dose of 5.0 mg/kg body weight/day for 20 days; 2) group two was the control group treated with the same dose of phosphate-buffered saline (pH 7.2) daily for 20 days. All mice were fed and received water ad libitum. On the twenty-first day after inoculation of tumor cells, the mice were killed and their solid tumors were excised and weighed. The tumor inhibition activity was calculated as
%Tumor inhibition = (C-T)/C x 100%,
where C was the average tumor weight of control group and T was the average tumor weight of RLL-treated group.
Assay for HIV-1 reverse transcriptase inhibitory activity
The assay for human immunodeficiency virus type 1 (HIV)-l reverse transcriptase (RT) inhibitory activity was conducted in view of the reports that some lectins exhibit such activity (Wang and Ng, 2001). The activity was determined with using an enzyme-linked immunosorbent assay (ELISA) kit from Boehringer Mannheim (Germany). The assay makes use of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(A) [oligo(dT).sub.15]. The digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same DNA molecule, which is newly produced by the RT. The detection and quantification of synthesized DNA as a parameter for RT activity are based on a sandwich ELISA protocol. Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. An antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD), subsequently binds to the digoxigenin-labeled DNA. The peroxidase substrate is added. The peroxidase enzyme catalyzes cleavage of the substrate and a colored reaction product is formed. The absorbance of the samples at 405 nm can be measured with 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 RT was used. The inhibitory activity of the lectin was calculated as percent inhibition as compared to a control without the protein (Wang and Ng, 2001). The mushroom ribosome-inactivating protein hypsin was used as a positive control (Lam and Ng, 2001).
Assay for ribonuclease activity
The assay was performed because some lectins display this activity (Liu et al., 2007). Ribonuclease activity of RLL toward yeast tRNA (Sigma) was assayed by determining the generation of acid-soluble, UV absorbing species with the method of Mock et al. (Mock et al., 1996). The lectin was incubated with 200 [micro]g of tRNA in 150 [micro]g 100 mM morpholino-ethanesulfonic acid buffer (pH 6.0) at 37 [degrees]C for 1 h. The reaction was terminated by adding 350 [micro]l of ice-cold 3.4% perchloric acid. After leaving on ice for 15 min, the sample was centrifuged (15,000 x g, 15 min) at 4 [degrees]C. The OD 260 of the supernatant was read after appropriate dilution. One unit of enzymatic activity was defined as the amount of enzyme that brings about an increase of one per minute in OD 260 nm in the acid-soluble supernatant (Mock et al., 1996). Bovine pancreatic RNase A (Sigma) was used as a positive control.
Assay for antifungal activity
The assay was carried out since some lectins demonstrate this activity (Ciopraga et al., 1999). The assay for antifungal activity toward Fusarium oxysporum, Rhizoctonia cerealis, Rhizoctonia solani, and Sclerotinia sclerotiorum was carried out in 100 x 15 mm petri dishes containing 10 ml of potato dextrose agar (PDA). After development of the mycelial colony, sterile blank paper disks (0.625 cm in diameter) were laid at a distance of 0.5 cm away from the rim of the mycelial colony. An aliquot (15 [micro]l) of the lectin was added to a disk. The dishes were incubated at 23 [degrees]C for 72 h until mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity (Wang and Ng, 2006; Ye et al., 2001). The mushroom ribosome-inactivating protein hypsin was used as a positive control (Lam and Ng, 2001).
Results and discussion
Purification of RLL
The fraction of the fruiting body extract unadsorbed on DEAE-cellulose (D1) but not the adsorbed fractions (D2,3,4) contained the hemagglutinating activity (Table 1). Fraction Dl was divided into two fractions, SP1 and SP2, upon ion exchange chromatography on a SP-Sepharose column (Fig. 1). Hemagglutinating activity resided in the adsorbed fraction SP2 (Table 1). Subsequently, fraction SP2 was resolved into a larger fraction SU1 and a tiny fraction SU2 upon gel filtration on a Superdex 75 HR 10/30 column by FPLC on an AKTA Purifier (GE Healthcare; Fig. 2). Hemagglutinating activity was enriched in SU1 (Table 1). The purified lectin appeared as a single band with a molecular weight of 16 kDa in SDS-PAGE (Fig. 3). According to the FPLC, the purified lectin RLL had a molecular weight of 32 kDa and was composed of two subunits, each with a molecular weight of 16 kDa. The N-terminal sequence of the lectin was VWYIVAIKTDVPRTT. It did not bear sequence resemblance to previously published mushroom lectins and other lectins (Table 2) reported earlier according to a BLAST search.
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[FIGURE 2 OMITTED]
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Table 1 Yields and hemagglutinating activities of various chromatographic fractions derived from 100g dried fruiting bodies. Fraction Yield (mg) Specific activity Total activity (units) (units/mg) Crude extract 6690 480 3.2 x [10.sup.6] D1 899 3029 2.7 x [10.sup.6] SP2 71 10307 7.3 x [10.sup.5] SU1 39 13469 5.3 x [10.sup.5] Fraction Recovery of activity (%) Fold of purification Crude extract 100 1 D1 84.4 6.3 SP2 22.8 21.5 SU1 16.6 28.1 Table 2 Comparison of N-terminal sequence of R. lepida lectin with sequences of other mushroom lectins. Mushroom lectin Sequence R. lepida lectin V W * Y * I V * A I * K T D * V * P * R T T Agaricus bisporus lectin E Y Y * G V * P I * R D Q A R E N Q (95-109) Agrocybe aegerita lectin P W * L V E Q R V S D * V * A N Q F (78-92) Flammulina velutipes A Y L V K K I * D F D * Y T P * N W G R G T lectin (9-27) Grifola frondosa lectin N W * P A E M M I D * L K H P I V E M R (29-46) Pholiota aurivella Y S V T T P N S V K G G T N Q G lectin (1-15) Identical amino acid residues are highlighted in boldface and underscored are indicated with *.
This is the first report of a lectin isolated from a Russula mushroom. Russula lepida lectin (RLL) has a unique N-terminal amino acid sequence not found in previously reported mushroom lectins and non-mushroom lectins, indicating that it is a novel lectin. The dimeric nature of RLL is in accord with reports on dimeric lectins from Agaricus edulis II (Eifler and Ziska, 1980), Ischnoderma resinosum (Kawagishi and Mizuno, 1988), and Pleurotus cornucopiae (Yoshida et al., 1994). RLL is similar to these mushroom lectins in both subunit and total molecular weight.
Biological activities of RLL
The interactions of lectins with cells can, in many instances, be inhibited specifically by simple sugars. This is refered to as sugar specificity. This finding has led to the conclusion that lectins bind specifically to saccharides on the surface of cells, and has provided an important tool for the investigation of the architecture of the cell surfaces (Dong et al., 1993; Ng et al., 1989). The sugar specificity of lectins can also be used for the isolation and purification of carbohydrate-containing polymers, and for the study of their chemical structure (Iobst and Drickamer, 1994). Among the variety of sugars tested, no hemagglutinating activity at 200 and 100 mM O-nitrophenyl-[beta]-D-galacto-pyranoside, and at 200-25 mM inulin (data not shown). Comparison of sugar specificity of R. lepida lectin and other mushroom lectins was listed in Table 3. To date, only a few inulin-specific lectins have been isolated (Feng et al., 2006; Liu et al., 2004; Zhang et al., 2009). Hence RLL may be used in carbohydrate banding research and the production of immobilized lectins for affinity chromatography.
Table 3 Comparison of characteristics of R. lepida lectin and other mushroom lectins. Species Molecular No. of Sugar specificity mass subunits (kDa) R. lepida 32 2 Inu, Oni-D-Gal[beta]-Pyr Agaricus bisporus 64 4 Gal[beta]1-3, GalNAc Agrocybe aegerita 31.6 2 Lac Armillaria 29.4 2 Inu luteo-virens Boletus edulis 32.5 2 -- Canoderma capense 18 1 Gal Ischnoderma 32 2 Lac, Gal resinosum Lactarius 37 2 [beta]-D-Gal (l [right arrow] deliciosus 3) GalNAc Lactarius 37 2 [beta]-D-Gal (l [right arrow] deterrimus 3) GalNAc Lactarius -- -- Not inhibited by simple lignyotus sugars Lactarius rufus 98 6 4-Nitr-(3-D-Glu; [alpha] -Phe-NAc-D-Glu Pholiota adiposa 32 2 Inu Pleurotus 32.4 2 Inu, Mal, Oni-D-Gai[beta]-Pyr citrinopileatus Pleurotus 31 2 No inhibition cornucopiae Pleurotus 41 2 Inu, Mel, Lac, Gal, Raf, ostreatus N-ace, [alpha]Met-D-Gal-Pyr Schizophyllum 64 2 Lac commune Tricholoma 37 2 Lac mongolicum Xerocomus 32.2 2 Inu spadiceus Xylaria hypoxylon 28.8 2 Xyl Species Acid or alkali Temperature References stability stability ([degrees]C) R. lepida 12.5 mM NaOH or 70 This study HC1 Agaricus bisporus -- -- Wang et al. (1998) Agrocybe aegerita 240 mM NaOH or 50 Sun et al. HC1 (2003) Armillaria 25 mM NaOH, 12.5 70 Feng et al. luteo-virens mM HCI (2006) Boletus edulis 25 mM NaOH or 40 Zheng et al. HC1 (2007) Canoderma capense pH4-ll 100 Ngai and Ng (2004) Ischnoderma resinosum -- -- Kawagishi and Mizuno (1988) Lactarius deliciosus -- -- Guillot et al. (1991) Lactarius deterrimus -- -- Giollant etal. (1993) Lactarius lignyotus -- -- Sychrova et al. (1985) Lactarius rufus pH 4-10 Less than 60 Panchak and Antoniuk (2007) Pholiota adiposa 25 mM NaOH or 50 Zhang et al. HC1 (2009) Pleurotus 100 mM NaOH, 6 60 Li et al. (2008) citrinopileatus mM HCI Pleurotus cornucopiae pH 3-12 70 Yoshida et al. (1994) Pleurotus ostreatus Lessr than 25 mM 30 Wang et al. NaOH or HCI (2000) Schizophyllum commune 125 mM NaOH, 25 40 Han et al. mM HCI (2005) Tricholoma mongolicum 50 mM NaOH, Less 80 Wang et al. than 25 mM HCI (1995) Xerocomus spadiceus Less than 30 mM 60 (Liu et al. NaOH or HCI (2004)) Xylaria hypoxylon 25 mMNaOH or 35 Liu et al. 12.5 mM HCI (2006) (a) Abbreviations: [alpha]Met-D-Gal-Pyr, [alpha]-Methyl-D-galactopyranoside; [alpha]-Phe-NAc-D-Glu, [alpha]-phenyl-N-acetyl-D-glucosaminopyranoside; [beta]-D-Gal (1 [right arrow] 3) GalNAc, [beta]-D-galactosyl (1[right arrow] 3)-D-N-acetyl galactosamine; Gal, Galactose; GalNAc, N-acetyl-glucosamine; Inu, Inulin; Lac, Lactose; Mal, Maltose; Mel, Melibiose; N-ace, N-Acetylneuraminic acid; Oni-D-Gai[beta]-Pyr, O-nitrophenyl-[beta]-D-galacto-pyranoside; Raf, Raffinose; Xyl, xylose; 4-Nitr-[beta]-D-Glu, 4-nitrophenyl-[beta]-D-glucosamine. b "--": No data available.
The lectin was stable up to 70 [degrees]C. The hemagglutinating activity was reduced to 50% when the temperature was elevated to 80 [degrees]C. No hemagglutinating activity was detected when the temperature was or above 90 [degrees]C (Table 4). The hemagglutinating activity was retained in the presence of 6 mM and 12.5 mM solutions of NaOH or HCl, but was reduced to 25% when the molarity of the acid or alkali was increased to 25 mM. The hemagglutinating activity disappeared altogether in 100 mM NaOH and HC1 solutions (Table 5). It is more thermostable than some mushroom hemagglutinatins, such as those from Schizophyllum commune (Han et al., 2005) and Pleurotus ostreatus (Wang et al., 2000), which are stable at and below 40 [degrees]C, respectively. The thermostability of RLL is very similar to that of Armillaria luteo-virens lectin which is stable up to 70 [degrees]C but inactivated at 80 [degrees]C and 90 [degrees]C (Feng et al., 2006). Comparisons of pH and temperature stability of R. lepida lectin and other mushroom lectins was showed in Table 3.
Table 4 Effect of temperature on hemagglutinating activity of R. lepida lectin (initial hemagglutinating activity: 64 U). Temperature ([degree]C) 20 30 40 50 60 70 80 90 100 Hemagglutinating activity (U) 64 64 64 64 64 64 32 0 0 The experiment was repeated twice and results were reproducible. Table 5 Effects of NaOH and HC1 on hemagglutinating activity of R. lepida lectin (initial hemagglutinating activity: 32 U). HC1 (mM) 6 12.5 25 50 100 200 Hemagglutinating activity (U) 32 32 8 4 0 0 NaOH (mM) 6 12.5 25 50 100 200 Hemagglutinating activity (U) 32 32 8 4 0 0 The experiment was repeated twice and results were reproducible.
The hemagglutinating activity of the lectin was unaffected by the presence of [Ca.sup.2+], [Mg.sup.2+], [Zn.sup.2+], [Fe.sup.2+], [Al.sup.3+], and [Fe.sup.3+] ions. It was increased by [Cu.sup.2+], but decreased by [Co.sup.2+] [Mn.sup.2+], and [Hg.sup.2+] ions (Table 6). It is noteworthy that [Cu.sup.2+] ions augment the hemagglutinating activity of R. lepida lectin. Dolichos biflorus lectin possesses bound [Cu.sup.2+] ions which, however, are not related to its sugar binding activity (Borrebaeck et al., 1981). The activity of R. lepida lectin is not affectied by [Fe.sup.2+], [Fe.sup.3+] and [Al.sup.3+] ions. In contrast, [Fe.sup.3+] and [Al.sup.3+] ions are important to the hemagglutinating activity of Boletus edulis lectin (Zheng et al., 2007). The hemagglutinating activity of RLL is inhibited by [Mn.sup.2+], [Co.sup.2+] and [Hg.sup.2+] ions. By comparison, the hemagglutinating activity of Boletus edulis (Zheng et al., 2007) and Schizophyllum commune (Han et al., 2005) lectins are also depressed by [Mn.sup.2+] ions.
Table 6 Effects of cations on hemagglutinating activity of R. lepida lectin (initial hemagglutinating activity: 64 U). Cation 50 mM 25 mM 12.5 mM 6.25 mM [Ca.sup.2+] 64 64 64 64 [Mg.sup.2+] 64 64 64 64 [Mn.sup.2+] 32 32 64 64 [Zn.sup.2+] 64 64 64 64 [Cu.sup.2+] 128 128 128 64 [Co.sup.2+] 32 32 64 64 [Fe.sup.2+] 64 64 64 64 [Hg.sup.2+] 4 32 64 64 [Al.sup.3+] 64 64 64 64 [Fe.sup.3+] 64 64 64 64 The experiment was repeated twice and results were reproducible.
RLL inhibited the proliferation of Hep G2 and MCF-7 tumor cells with an [IC.sub.50] value of 1.6 [micro]M and 0.9 [micro]M, respectively (Fig. 4), with a higher potency than French bean lectin (Lam and Ng, 2009). Daily intraperitoneal injections of the lectin (5.0 mg/kg body weight/day for 20 days) brought about 67.6 % reduction in weight of S-180 tumor (Table 7). RLL exhibited a weaker antitumor potency than Pleurotus citrinopileatus lectin (Li et al., 2008) which had the same molecular weight (Table 6) The lectin lacked anti HIV-1 RT, antifungal, and ribonuclease activities when tested up to 100 [micro]M (data not shown).
[FIGURE 4 OMITTED]
Table 7 Inhibition of Sarcoma 180 by R. lepida lectin (5.0 mg/kg/day for 20 days) in vivo. Control group (n = 6) R. lepida Lectin-treated group (n = 6) Tumor weight (g) 3.73 [+ or -] 0.55 1.21 [+ or -] 0.20 (P <0.05) % Inhibition of - 67.6 tumor growth P. citrinopileatus Lectin-treated group (n = 6) Tumor weight (g) 0.72 [+ or -] 0.15 (P <0.05) % Inhibition of tumor growth 80.8
RLL manifested potent antitumor activity in vivo and vitro. Previously, the antiproliferative effect of lectins on tumor cell lines and the antitumor action of lectins in tumor-bearing mice have been demonstrated using Agaricus bisporus lectin (Wang et al., 1998; Yu et al., 1993), Pleurotus ostreatus lectin (Wang et al., 2000), Tricholoma mongolicum lectin (Wang et al., 2000), and Volvariella volvacea lectin (Lin and Chou, 1984). A bisporus lectin (ABL) was reported to elicit a remarkable dose-dependent decline of [.sup.3]H-thymidine incorporation into human colon cancer cell lines HT29 and Caco-2, breast cancer cell line MCF-7, and rat mammary fibroblasts Rama-27 (Yu et al., 1993). P. ostreatus lectin was capable of potently inhibiting the growth of sarcoma S-180 and hepatoma H-22 in mice, and the lifespan of those tumor-bearing mice was also significantly prolonged (Wang et al., 2000). The effects of lectins from European mistletoe (Viscum album L.) on malignant melanoma have been investigated (Stauder and Kreuser, 2002). 212 (30.9%) patients relapsed or progressed, and 107 (15.6%) died. Long-term treatment with the European mistletoe fermentation extract (FEM) was shown to be safe and without any further tumour enhancement (Augustin et al., 2005).
An antifungal protein from Bacillus subtilis, designated as bacisubin, exhibits hemagglutinating and ribonuclease activities (Liu et al., 2007). However, RLL lacks ribonuclease and antifungal activities. To date, only several lectins have been reported to have antifungal activity, including wheat germ agglutinin (Ciopraga et al., 1999), stinging rettle lectin (Does et al., 1999), red kidney bean lectin (Ye et al., 2001), potato tuber lectin (Gozia et al., 1995) and flageolet bean lectin (Xia et al., 2005). In contrast to lectins that display HIV-1 reverse transcriptase inhibitory activity (Han et al., 2005; Li et al., 2008; Wong et al., 2006), RLL is devoid of this activity.
The mushroom family Russulaceae is composed of two genera Russula and Lactarius, the former being the greater majority. Four lectins have been isolated from Lactarius species whereas none has been reported from Russula species (Table 3). Lectins from L. delicosus and L. deterrimus are 37-kDa dimeric proteins with specificity toward [beta]-D-galactosyl (1 [right arrow] 3)-D-N-acetyl galactosa-mine (Giollant et al., 1993; Guillot et al., 1991). L. lignyotus lectin is inhibited by desialylated mucin and fetuin and edible bird's nest glycoprotein but not by simple sugars (Sychrova et al., 1985). L. rufus lectin is a 98-kDa hexamer with affinity toward 4-nitrophenyl-[beta]-D-glucosamine and [alpha]- phenyl-N-acetyl-D-gluco-saminopyranoside. It is stable in the pH range 4-10 and loses over 85% of its activity at 60 [degrees]C. It is not inhibited by divalent metal ions (Panchak and Antoniuk, 2007). Hence RLL differs from the aforementioned Lactarius mushrooms in molecular weight, sugar specificity, thermostability, and susceptibility to metal ions. RLL has been tested for various biological activities whereas Lactarius lectins have not been similarly assayed.
In summary, a lectin with a distinctive N-terminal sequence, sugar specificity, hemagglutinating activity with relatively high thermostability and [Cu.sup.2+] -induced enhancement, and potent antiproliferative and antitumor activities was isolated from R. lepida fruiting bodies. The potent antitumor activity of RLL is remarkable and hopefully it can be developed into an agent for cancer therapy. This report represents an addition to the existing list of mushroom lectins.
This work was financially supported by National Grants of China (nyhyzx07-008, 2007BAD89B00 and 2010CB732202).
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* Corresponding authors. Tel.: +852 2609 8031, +8610 62732578.
E-mail addresses: email@example.com (H. Wang), firstname.lastname@example.org (T.B. Ng).
G. Zhang (a), J. Sun (a), H. Wang (a), *, T.B. Ng (b), *
(a) State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China
(b) School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
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|Author:||Zhang, G.; Sun, J.; Wang, H.; Ng, T.B.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Aug 1, 2010|
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