Nebrodeolysin, a novel hemolytic protein from mushroom Pleurotus nebrodensis with apoptosis-inducing and anti-HIV-1 effects.
A novel hemolysin was isolated from the edible mushroom Pleurotus nebrodensis by ion exchange and gel filtration chromatography on DEAE-Sepharose and Sephacryl S-100. The hemolysin from Pleurotus nebrodensis hemolysin (nebrodeolysin) is a monomeric protein with a molecular weight of approximately 27 kDa as determined by gel filtration and SDS-PAGE. Nebrodeolysin exhibited remarkable hemolytic activity towards rabbit erythrocytes and caused efflux of potassium ions from erythrocytes. Subsequently, this hemolysin showed strong cytotoxicity against Lu-04, Bre-04, HepG2, L929, and HeLa cells. It was also found that this hemolysin induced apoptosis in L929 and HeLa cells as evidenced by microscopic observations and DNA ladder, respectively. Moreover, this hemolysin was shown to possess anti-HIV-1 activity in CEM cell culture. [C] 2008 Elsevier GmbH. All rights reserved.
Keywords: Pleurotus nebrodensis hemolysin (nebrodeolysin); Cytotoxicity; Apoptosis; Anti-HIV-1 activity
Extensive studies have revealed that a number of mushroom species are of value in the prevention and/or treatment of cancers, viral diseases, hypercholesterolemia, and hypertension (Breene, 1990; Tong et al., 1991). A variety of proteins like ubiquitin-like proteases, proteoglycans, ribosome-inactivating proteins, antifungal proteins and lectins with immunomodulatory, antitumor, antifungal and hypotensive activities have been isolated from mushrooms (Wang et al., 1996; Lam and Ng, 2001a, b; Lam et al., 2001; Liu et al., 2008).
However, few hemolytic proteins have been isolated from the edible mushrooms (Seeger, 1975; Suzuki et al., 1990; Luo et al., 2007; Berne et al., 2002; Ngai and Ng, 2006). The genus Pleurotus is a cosmopolitan group of mushrooms with high nutritional value, healthy and potential therapeutic properties in food and pharmaceutical applications (Cohen et al., 2002; Wasser and Weis, 1999). So far, there were only two kinds of mushroom hemolysins, Pleurotus ostreatus and Pleurotus eryngii isolated from the edible mushrooms.
In the present study, this hemolysin was reported for the first time that it was isolated and characterized from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. Also, this hemolysin was found to possess dual biological functions, apoptosis-inducing and anti-HIV-1 activities.
Materials and methods
Basidiocarps of Pleurotus nebrodensis was purchased from the institution of domestic fungus in Chengdu. The various human cancer cell lines employed, i.e., Lu-04 (lung), Bre-04 (breast), and HepG2 (liver), were procured from Di'ao Group, Chengdu, China. The host cell lines and the tested viruses were obtained from Rega Institute for Medical Research, Katholieke Universiteit Leuven.
Purification of hemolysin from Pleurotus nebrodensis (nebrodeolysin)
Fresh basidiocarps of Pleurotus nebrodensis (300 g, wet weight) were homogenized in 300 ml of 140 mM NaCl buffer, pH 7.2, and after soaking at 4[degrees]C for 24 h, the supernatants were obtained by filtration. The supernatants were dialyzed against the 20 mM HAc-NaHAC buffer pH 5.0 at 4[degrees]C for 24 h. The crude extracts were applied onto a CM-sepharose column (Pharmacia, Sweden) and eluted with a linear gradient of NaCl of 0-0.5 M in the same buffer. The fraction of the C3 peak, which was hemolytic, had been dialyzed against the 50 mM Tris-HCl buffer, pH 8.8 at 4 [degrees]C for 24 h prior to separation with a DEAE-sepharose column (Pharmacia, Sweden). Proteins were eluted with a linear NaCl gradient (0-0.5 M) in the same buffer. The hemolytic peak was collected, concentrated by ultrafiltration, and applied onto a Sephacryl S-200 column (Pharmacia, Sweden). The column was eluted with 20 mM sodium phosphate buffer, pH 7.2 at a flow rate of 0.3 ml/min and hemolytic fractions were collected and stored at -20 [degrees]C until use.
Protein concentrations were determined as described by Lowry et al. (1951), using crystalline bovine serum albumin (BSA) as a standard.
Protein analysis and molecular mass determination were carried out through SDS-PAGE by using 15% separating gel according to Laemmli (1970). The samples were dissolved in electrophoresis buffer containing SDS with or without 5% B-mercaptoethanol. After electrophoretic separation the proteins were stained with 0.1% Coomassie blue R-250.
Native molecular mass determination
Gel filtration for measuring the molecular weights of native Hemolysin was carried out on a Sephacryl S-200 column (1.5 x 100 cm Pharmacia), which had been calibrated with the following standard proteins: bovine serum albumin (67 kDa), Galanthus nivalis agglutinin (48 kDa), trypsin (23.3 kDa), equine myoglobin (17 kDa), and cytochrome c (12.4 kDa). The eluting buffer is 20 mM sodium phosphate buffer (pH 7.2), and the flow rate is 0.3 ml/min.
The hemolytic assay was performed as described previously (Tomita et al., 2004). Erythrocytes (3 x [10.sup.7] cells/ml) were incubated with nebrodeolysin at 25 [degrees]C for 30 min. After centrifugation at 600g for 5 min, the supernatants obtained were assayed for absorbance at 541 nm. 100% lysis was defined as the absorbance of the supernatants obtained from the osmotically lysed cells.
Efflux of potassium ions in nebrodeolysin-treated erythrocytes
Rabbit erythrocytes were washed five times by centrifugation for 5 min at 600g to remove extracellular potassium ions. The washed erythrocytes (approx. 6 x [10.sup.8] cells/ml) were incubated with nebrodeolysin (0.1 and 0.3 [micro]g/ml) at 25 [degrees]C, and were centrifuged for 1 min at 4 [degrees]C and 5000g. A small portion of the supernatants was withdrawn for the haemolytic assay. The rest of the supernatants were centrifuged at 18,000g for 20 min to remove erythrocyte membranes, if any. The supernatants were dried on a hot plate at 70-80 [degrees]C, decomposed at 550 [degrees]C, and dissolved in a portion of 0.02 M HC1. The atomic absorption spectrophotometric assay for potassium was performed at the wavelength of 766.5 nm with a SpectrAA-220FS (Varian, American).
MTT colormetric assay
Tests were prepared as the method referred by Mosmann (1983). Lu-04 (lung), Bre-04 (breast), and HepG2 (liver) cells as well as L929 and HeLa cells with logarithmic growth phase (1 x [10.sup.5] cells/ml) were seeded independently in a 96-well plate with the final volume 100 [micro]l containing 1 x [10.sup.4] cells per well. These plates were incubated at 37 [degrees]C for 24 h. And then various concentrations of this nebrodeolysin were added. After another 24 h, 0.05 mg (10 [micro]l of 5 mg/ml) MTT was added to each well and incubated at 37 [degrees]C for 4h. The absorbance of the samples was measured at 570 nm with a spectrophotometer [Model 3550 Microplate Reader (BIO-RAD)]. The percentage of cell growth inhibition was calculated as follows:
Cell viability(%) = OD570(drug)/OD570(control) x 100.
Observations of cell morphologic changes
Lu-04 (lung), Bre-04 (breast), and HepG2 (liver) cells as well as L929 and HeLa cells in RPMI-1640 containing 10% FBS were seeded into 96-well culture plates and cultured for 24 h. These three kinds of cells were treated with the 0.05% dimethyl sulfoxide (DMSO). This nebrodeolysin (1 x [10.sup.-3] mg/ml) was added to the cells and the cellular morphology was observed using phase contrast microscopy (Leica, Wetzlar, Germany). The changes in nuclear morphology of two kinds of apoptotic cells, L929 and HeLa cells out of the five, were investigated by fluorescent microscopy (Cheng et al., 2008). After being treated with or without this nebrodeolysin (4 pM) for 24 h, L929 cells were stained with 20 mg/ml AO for 15 min and then the nuclear morphology was observed under fluorescence microscopy (Olympus, Tokyo), whereas the nuclear morphology of HeLa cells were also observed under the same microscopy.
DNA fragmentation assay
L929 cells cultured with or without nebrodeolysin at 37 [degrees]C for 12, 24, and 48 h were harvested and suspended with 1 ml medium. The total DNA was isolated by using a DNA extraction kit (Shanghai Wason Co. Ltd) and analyzed by electrophoresis on 1.5% agarose gel containing 0.1 [micro]g/ml ethidium bromide and visualized under UV light.
Using previously established procedures (Balzarini et al., 2004), the in vitro antiviral activities of compounds were determined against a variety of DNA and RNA viruses, and their cytotoxicities for the host cell lines were assayed in parallel with those of standard drugs with known antiviral activities. The viruses and cells used were herpes simplex virus type 1 (HSV-1) (strain KOS), herpes simplex virus type 2 (HSV-2) (strain G), vaccinia virus, vesicular stomatitis virus, and thymidine-kinase-deficient HSV-1 ([TK.sup.-]) (strain KOS) in HEL cells; vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus in HeLa cells; parainfluenza virus type 3, reovirus type 1, Sindbis virus, Coxsackie B4 virus and Punta Toro virus in Vero cells; and HIV-1 ([III.sub.B]) and HIV-2 (strain ROD) in human T-lymphocyte (CEM) cells (at compound concentrations up to 100 [micro]g/ml). The antiviral activities were determined as percent inhibition of microscopically visible virus-induced cytopathicity in the cell cultures.
Statistical analysis of the data
All the data were confirmed in at least three independent experiments. These data were expressed as mean [+ or -] S.D. Statistical comparisons were made by Student's t-test. p < 0.05 was significant.
Purification of nebrodolysin
A single haemolytic protein from the basidiocarps of Pleurotus nebrodensis was purified by successive column chromatography using a CM-Sepharose column, a DEAE-Sepharose column, and a Sephacryl S-100 column as shown in Fig. 1. The molecular size of the purified protein was determined to be 27 kDa on SDS-PAGE under non-reducing and reducing conditions (Fig. 2A; lanes 1 and 2, respectively). The molecular mass of the purified protein was also estimated to be 27 kDa by gel filtration (data not shown). These data suggested that the haemolytic protein is a 27 kDa monomer.
[FIGURE 1 OMITTED]
Hemolytic activity of nebrodeolysin
We tested the haemolytic activity of nebrodeolysin towards rabbit erythrocytes at 25 [degrees]C for 30 min, and the concentration of nebrodeolysin for inducing 50% hemolysis was estimated to be 0.1 [micro]g/ml (Fig. 2B).
[FIGURE 2 OMITTED]
Since swelling of cells is generally caused by an increased permeability of cell membranes, we assayed nebrodeolysin-induced leakage of potassium ions (a representative of small molecules and ions) from rabbit erythrocytes and lysis of the cells. Leakage of potassium ions ([black square]) and haemoglobins ([white square]) from rabbit erythrocytes was monitored after exposure of the cells to nebrodeolysin at the concentration of 0.1 and 0.3 [micro]g/ml, respectively, at 25 [degrees]C, approx. 50% and 90%, respectively, of the intracellular potassium ions leaked within 3 min after the addition of hemolysin, whereas onset of haemolysis occurred after a 5 min- or longer incubation time under the same conditions.
Assay of the growth inhibitions in five cancer cells
Inhibition of cell proliferation of this hemolysin was measured by the MTT assay. The five cell groups treated with this hemolysin from 0.1 to 60 [micro]g/ml showed that the inhibition ratio reached above 50% at 40 [micro]g/ml (1.5 pM), respectively (Fig. 3A).
[FIGURE 3 OMITTED]
Morphological observation of apoptotic cells
The five cells were observed under the fluorescent microscopy, respectively. As shown in Fig. 3B, RNA in cytoplasm and nucleolus appeared red, while DNA in nucleus was green or chartreuse observed. While among this hemolysin (1.5 pM) treated cells, dense chartreuse nucleolus, condensed chromatin, and fragmented nuclei were observed in L929 cells. Also, there was a reduction in nuclear volume. Furthermore, the apoptotic phenomenon was also observed in HeLa cells. Some of the nucleus degenerated into discretely spherical fragments of highly condensed chromatin. These demonstrated that this hemolysin induced L929 and HeLa cell apoptosis. Yet, this apoptotic phenomenon was not observed in the other three cells (data not shown).
DNA ladder assay of apoptosis in L929 cells
The most distinctly biochemical hallmark of apoptosis is the activation of the endogenous [Ca.sup.2+]/[Mg.sup.2+] -dependent endonuclease and endonuclease-mediated cleavage of internucleosomes to generate oligonucleotide fragments with about 180-200 bp length or their polymers. Characteristic ladder bands can be obtained by agarose gel electrophoresis of DNA extracted from apoptotic cells. In this study, DNA ladder bands could be observed in nebrodeolysin-treated group after 24h, whereas in the presence of nebrodeolysin for 12h or in the control group, smear-like DNA degradation was observed (Fig. 3C). These phenomena demonstrated that nebrodeolysin induced L929 apoptosis in a time-dependent manner.
Antiviral activity of nebrodeolysin in vitro
To measure the inhibitory effect on the viral cytopathogenic effects (CPE) in cell culture, nebrodeolysin was exposed to different cell cultures that were infected by a variety of DNA and RNA viruses. However, none of the viruses were inhibited at subtoxic concentrations (see the Supplementary material). Only the replication of HIV-1 ([III.sub.B]) but not HIV-2 (ROD) was inhibited by nebrodolysin in CEM cell cultures at an [EC.sub.50] of 2.4 pM (Table 1) (An et al., 2006; Chu and Ng, 2006; Wong and Ng, 2003). The cytotoxic concentration of nebrodolysin in HEL and Vero cell cultures is 3.7 x [10.sup.-3][micro]M and in HeLa cell cultures is 7.4 x [10.sup.-3] [micro]M. These data are in the same range as found for its cytostatic activity against the above described tumor cell lines (see the Supplementary material).
Table 1. Anti-HIV activity of nebrodeolysin in CEM cell culture Compounds [CC.sub.50] (a) [EC.sub.50] (b) ([micro]g) ([micro]g) HIV-1 (IIIB) HIV-2 (ROD) Pleurotus 100 65 100 nebrodolysin Ground bean > 100 4,280,000 (73 [micro]M) - lectin Smilaxin > 100 168,000 (5.6 [micro]M) - Polygonatum 74 0.92 0.78 cyrtonema Hua lectin Lycoris 7.10 0.79 0.59 radiate agglutinin UDA (Urtica 50 1.0 - dioica agglutinin) Galanthus 50 0.37 - nivalis agglutinin (a) 50% Cytostatic concentration or compound concentration required to inhibit CEM cell proliferation by 50%. (b) 50% Effective concentration or compound concentration required to inhibit HIV-induced syncytia formation in CEM cell cultures by 50%.
Hemolysin, which is produced by a large number and variety of bacteria and mushroom, has been implicated as a virulence factor (Hildebrand et al., 1991; Bhakdi et al., 1984; Chu et al., 1991). It has been suggested that they may have a role in processes such as modulation of bacterial sporulation, virulence of A. fumigatus and mushroom fruiting (Chu and Holt, 1994; Yanagihara et al., 1982; Geooy et al., 1987).
In this study, we isolated and purified a novel mushroom hemolysin (designated nebrodolysin) from Pleurotus nebrodensis for the first time. The procedure involves gel filtration on CM-Sepharose column, DEAE-Sepharose column, and Sephacryl S-100 column. [([NH.sub.4]).sub.2][SO.sub.4] precipitation was not used here because it can cause severe protein loss in the purification process (Tomita et al., 1998). The multi-step ion exchange chromatography and gel filtration chromatography were also useful in the purification of other mushroom hemolysins. The results of SDS-PAGE and gel filtration analysis indicated that nebrodolysin is a monomeric protein. This finding is well in agreement with the properties of eryngeolysin, ostreolysin, and aegerolysin isolated from mushrooms (Sepcica et al., 2004). The results of hemolysis and efflux of potassium ions from nebrodeolysin-treated erythrocytes closely resemble flammutoxin and pleurotolysin (Tadjibaeva et al., 2000; Sakurai et al., 2004). The immediate efflux of potassium ions and the delayed onset of haemolysis suggested pore formation by hemolysin, leading to osmotic burst of human erythrocytes (Amoakoa et al., 1997).
The nebrodolysin exhibited only hemolytic activity. Though we have not observed agglutinin properties of nebrodedolysin, there are some other proteins containing both agglutinin properties and hemolytic activity, such as Laetiporus sulphureus lectin (LSL). LSL is a novel pore-forming lectin homologous to bacterial toxins, its agglutinin properties is ascribed to lectin-carbohydrate interactions in organisms (Tateno and Goldstein, 2003). The fact that LSL is hemolytic, and shows homology to bacterial toxins, could indicate that it might have evolved from the ancestral protein to acquire an oligomerization domain in forming pores at the membrane (Tateno and Goldstein, 2003). In term of the correlation between nebrodedolysin and LSL, both of them have the hemolytic activity, but the outstanding point is that LSL also has agglutinin properties. The existence of the proteins such as LSL may give us some insights into the relations between hemolysins and lectins. Thus, further investigations need to be carried, especially in the evolutionary field.
As to nebrodedolysin, its cytotoxicities against some typical tumor cells were shown in vitro by several mushrooms or mushroom components (Li et al., 2003) e.g. water-soluble polysaccharides (Tao et al., 2006) proteoglycans (Zhao et al., 2003), lectins (Liu et al., 2008), ubiquitin-like proteases, and ribosome-inactivat-ing proteins. Some of these mushroom hemolysins also exhibit potential anti-proliferative activity on several cancer cell lines in vitro, e.g. Phallolysin from Amanita phalloides is cytotoxic to HeLa cells and ascites tumor cells (sarcoma P43 of the mouse), Pleurotus ostreatus hemolysin possesses antiproliferation activities against human fibrosarcoma cancer cells HT-1080 and against human breast adenocarcinoma cancer MCF-7 cell lines, and Pleurotus eryngii hemolysin reduces the viability of leukemia L1210 cells (Berne et al., 2002).
Here, we found that this hemolysin induced L929 and HeLa cell apoptosis. However, this apoptotic phenomenon was not observed in the other three human cancer cells. Therefore, like other anti-tumor agents, this hemolysin executed a strong growth-inhibitory effect on human cancer cell lines (Wong and Ng, 2003). Based on the observation of morphologic changes in cellular nuclei, we concluded that this hemolysin induced L929 and HeLa cell apoptosis. Thus, this hemolysin would act as a very potent inducer of apoptosis and this offers an interesting potential therapeutic strategy in the treatment of cancer therapy.
The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and therapeutic effects. In the past decade, some compounds have been isolated from the fruiting bodies of basidio-mycetes among Pleurotus, and found to have antiviral effects. However, there was no report available on hemolysin from the edible mushrooms (Zhang et al., 1994).
Herein, nebrodeolysin lacked significant antiviral activity toward a variety of tested viruses. It was noteworthy that nebrodeolysin exhibits a suppressive action on HIV-1 and reproducible antiviral effect was observed. Although the anti-HIV-1 activity of this nebrodeolysin was not very remarkable when compared with other known lectins in Table 1, it was reported for the first time that a hemolysin from the mushrooms possesses anti-HIV-1 activity. Although the mechanism of the antiviral effect of the nebrodeolysin remains to be elucidated, we suggested that nebrodeolysin might act in a different way by interacting with the cell membrane and subsequently suppressing the infection of the virus.
In summary, a novel hemolysin has been isolated and characterized from the edible mushroom Pleurotus nebrodensis for the first time. This hemolysin was strong cytotoxic against human cancer cells; moreover, it was found to induce apoptosis in L929 and HeLa cells. And, this nebrodeolysin was not shown an anti-virus activity against a broad range of viruses, but against HIV-1. Thus, further investigations determining the mechanisms of antineoplastic and anti-HIV-1 activities of nebrodeolysin might spark off the development in cancer and HIV therapies in the near future.
We are grateful to Dr. Qin Tian for her skillful assistance with this manuscript. Furthermore, this work was supported in part by grants from the National Natural Science Foundation of China (General Programs: Nos. 30270331 and 30670469). Also, this work was also supported by Director Fund of State Key Laboratory of Oral Diseases (Sichuan University).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.phymed. 2008.07.004.
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Hui Lv (a), (1), Yang Kong (a), (1) Qing Yao (a), Bo Zhang (a), Fang-wei Leng (a), He-jiao Bian (a), Jan Balzarini (b), Els Van Damme (c), Jin-ku Bao (a), *
(a) College of Life Sciences, State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610064, China
(b) Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
(c) Department of Molecular Biotechnology, Ghent University, 9000 Gent, Belgium
* Corresponding author. Tel./fax: + 86 28 85410672.
E-mail address: email@example.com (J.-K. Bao).
(1) These authors contributed equally to this work.