mushroom Pholiota adiposa

Little was known about biological activities of compounds from the medicinal mushroom of the genus Pholiota. A lectin from the Pholiota adiposa has now been isolated and its properties tested. The isolation procedure included ion exchange chromatography on DEAE-cellulose and CM-cellulose, and fast protein liquid chromatography-gel filtration (FPLC) on Superdex 75. The lectin was composed of two identical subunits, each with a molecular mass of 16 kDa. Its N-terminal amino-acid sequence showed little similarity to sequences of other Agaricales lectins. The hemagglutinating activity of the lectin was stable at temperatures up to 50 degrees C, and in NaOH and HCl solutions with concentrations less than 25 mM. It was inhibited by inulin (12.5-200 mM), but enhanced by Cu(2+) (6.25-25 mM), Fe(2+) (12.5-25 mM), and Al(3+) (6.25-25 mM) ions. The lectin showed antiproliferative activity toward hepatoma Hep G2 cells and breast cancer MCF7 cells with an IC(50) of 2.1 microM and approximately 3.2 microM, respectively. It exhibited HIV-1 reverse transcriptase inhibitory activity with an IC(50) of 1.9 microM. When compared with P. aurivella lectin, the only Pholiota lectin published to date, P. adiposa lectin differs in chromatographic behavior, molecular mass, N-terminal sequence, and effect of cations on hemagglutinating activity. In the case of the lectin from P. aurivella, its antifungal, antiproliferative, and HIV-1 reverse transcriptase inhibitory activities have not been determined.


G. Q. Zhang and others
Pholiota adiposa is an edible as well as a medicinal mushroom cultured in China and Japan.It belongs to Agaricales (Strophariaceae), and has antitumor, antimicrobial, antihypertensive, and antihyperlipidemic activities.An extract of P. adiposa inhibited the growth of sarcoma 180 and Ehrlich solid cancers in mice (Yu et al., 2007).Further research suggested that the antitumor activity of P. adiposa polysaccharide was due to immunostimulation (Zhao et al., 2007).A novel pentapeptide (Gly-Glu-Gly-Gly-Pro) with angiotensin converting enzyme inhibitory activity was isolated from the fruiting body of P. adiposa (Izawa & Aoyagi, 2006;Koo et al., 2006).Stigmasterol purified from methanol extracts of P. adiposa fruiting bodies inhibited β-hydroxy-β-methyl-glutaryl coenzyme A reductase, a rate-limiting enzyme in cholesterol biosynthesis, with an IC 50 of 6.8 μg/ml (Yu et al., 2007).A P. adiposa extract did not produce any significant change in total triglyceride contents or epididymal fat mass, but caused a decrease in retroperitoneal fat in mice on a high-fat diet (Cho et al., 2006).The present investigation aimed to isolate and characterize a lectin from the fruiting bodies of P. adiposa and to find out if it has some distinctive characteristics.To date only one lectin has been isolated from a representative of the Pholiota genus, Pholiota aurivella (Kawagishi et al., 1991).

Isolation of lectin.
Dried fruiting bodies of the mushroom P. adiposa (100 g) cultured in the Department of Microbiology of China Agricultural University, were homogenized in 0.15 M NaCl (10 ml/g) at 4°C and extracted overnight at 4°C.Then the homogenate was centrifuged at 8 000 × g for 15 min.To the supernatant (NH 4 ) 2 SO 4 was added to 80% saturation.The mixture was left at 4°C for 4 h before centrifugation at 8 000 × g for 15 min.The precipitate was redissolved and dialyzed to remove (NH 4 ) 2 SO 4 before applying to a DEAE-cellulose (Sigma) column (2.5 × 20 cm) in 10 mM NH 4 HCO 3 buffer (pH 9.4).After removal of the unadsorbed peak (D1), three adsorbed peaks, D2, D3 and D4, were eluted stepwise with 50 mM NaCl, 150 mM NaCl and 1 M NaCl in the buffer, respectively.Fraction D3 was then passed through a CM-cellulose (Sigma) column (1.5 × 10 cm) which had been equilibrated and then eluted with 10 mM NH 4 OAc (pH 4.6).Unbound material ((fractions C1) was eluted with the starting buffer, while bound proteins (fractions C2, C3) were desorbed sequentially with 50 mM NaCl and 1 M NaCl in the starting buffer.The active peak (C2) was subsequently purified by fast protein liquid chromatography (FPLC) on a gel filtration Superdex 75 HR 10/30 column (GE Healthcare) in 0.15 M NH 4 HCO 3 buffer (pH 8.5).The second peak (SU2) obtained constituted a purified lectin.
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 (1973).Gel filtration on an FPLC-Superdex 75 column, which had been calibrated with molecular mass markers (GE Healthcare), was conducted to determine the native 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 (Wang et al., 2002).
Assay for lectin (hemagglutinating) activity.In the assay, a serial twofold dilution of the lectin solution in microtiter U-plates (25 μl) was mixed with 25 μl of a 2% suspension of rabbit red blood cells in phosphate-buffered saline (pH 7.2) at 20 o C. The results were recorded after about 1 h when erythrocytes in 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 (Wang et al., 2000).
Hemagglutinating inhibition tests to investigate the inhibition of lectin-induced hemagglutination by various carbohydrates were performed in a manner analogous to the hemagglutination assay.The carbohydrates tested included inositol, lsorbose, raffinose, l-rhamnose, d-fructose, d-mannose, cellobiose, l-arabinose, d-xylose, d-melibiose, lactose, inulin, maltose, d-galactose, d-glucose and O-nitrophenyl-β-d-galactopyranoside.Serial twofold dilutions of sugar samples were prepared in phosphate-buffered saline (pH 7.2).All of the dilutions were mixed with an equal volume (25 μl) of a solution of the lectin with 16 hemagglutination units.The mixture was allowed to stand for 30 min at room temperature and then mixed with 50 μl of a 2% rabbit erythrocyte suspension.The minimum concentration of the sugar in the final reaction mixture which completely inhibited 16 hemagglutination units of the lectin preparation was calculated (Han et al., 2005).
The effects of temperature, NaOH, HCl, and metallic chlorides on the hemagglutinating activity of the lectin were determined as previously described (Wang et al., 1996).
Assay of antiproliferative activity on tumor cell lines.The tumor cell lines, human breast cancer (MCF 7) and hepatoma (Hep G2), were purchased from American Type Culture Collection (ATCC).They were maintained in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/l streptomycin and 100 IU/ml penicillin at 37 o C in a humidified atmosphere of 5% CO 2 .Cells (1 × 10 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 carried out for another 48 h.Radioactive precursor, 1 μCi, ([ 3 H-methyl]thymidine, from GE Healthcare) 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 (Li et al., 2009).
Assay for HIV-1 reverse transcriptase inhibitory activity.The inhibitory activity towards human immunodeficiency virus type 1 (HIV)-1 reverse transcriptase (RT) was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit from Boehringer Mannheim (Germany).The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(A)-oligo(dT) 15 .The digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into the DNA molecule synthesized by the RT.The detection and quantification of the synthesized DNA as a measure of RT activity follows sandwich ELISA protocol.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 & Ng, 2004).
Assays for antifungal and ribonuclease activities.The assays were conducted as described in (Han et al., 2005;Wang & Ng, 2006), respectively.

Isolation of lectin
One of the fractions adsorbed on DEAEcellulose, fraction D3, showed hemagglutinating activity (Table 1).Ion exchange chromatography on CM-cellulose resulted in an unadsorbed fraction C1 and two adsorbed fractions C2 and C3 (Fig. 1).The lectin activity resided in fraction C2 (Table 1).Subsequently, fraction C2 was resolved into a smaller peak SU1 and a larger peak SU2 upon gel filtration on a Superdex 75 HR 10/30 column by FPLC on an AKTA Purifier (GE Healthcare; Fig. 2).The hemagglutinating activity was   1).

Molecular mass determination and N-terminal sequence analysis
The purified lectin appeared as a single band with a molecular mass of 16 kDa in SDS/PAGE (Fig. 3) and a single peak with a molecular mass of 32 kDa in FPLC gel filtration (Fig. 2).Thus it appears that the purified lectin is composed of two identical subunits, each with a molecular mass of 16 kDa.The N-terminal sequence of the lectin was DILMGTYGML, different from the other mushroom lectins shown in Table 2.

Other biological activities
The lectin inhibited the proliferation of Hep G2 and MCF tumor cells with an IC 50 value of 2.1 μM and about 3.2 μM, respectively (Fig. 4).HIV-1 reverse transcriptase was inhibited with an IC 50 of 1.9 μM (Fig. 5).The lectin lacked antifungal and ribonuclease activities (not shown).

DISCuSSION
Pholiota adiposa lectin (PAL) isolated in the present investigation differs from lectins purified from other Agaricales mushrooms.PAL was purified using three kinds of columns: DEAE-cellulose, CM-cellulose and Superdex 75.PAL is adsorbed on DEAE-cellulose and CM-cellulose columns and eluted with 150 mM NaCl and 50 mM NaCl, respectively.It is strongly adsorbed on SP-Sepharose column and no activity can be eluted with 3 M NaCl.Compared with PAL, a lectin named PAA isolated from Pholiota aurivella, which belongs to the same genus as P. adiposa, is adsorbed during affinity chromatography on a fetuin, asialofetuin, BSM, or asialo-BSM column, and during ion exchange chromatography on DEAE-Toyopearl and CM-Toyopearl.No lectin activity is found when a variety of eluents is used.Only 0.1% SDS can elute the lectin from the adsorbents or matrices (Kawagishi et al., 1991).
Carbohydrate specificity is an important characteristic of lectins.It is interesting in that only inulin, a plant polysaccharide, is able to inhibit the hemagglutinating activity of PAL.To date only a few inulin-specific lectins have been reported (Liu et al., 2004;Feng et al., 2006).Hence PAL may be used in the production of immobilized lectin for affinity chromatography.
In the present study, Cu 2+ , Fe 2+ , and Al 3+ ions increased the hemagglutinating activity of PAL when present at 12.5 mM, 25 mM, and 12.5 mM, respectively, while Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Hg 2+ , and Fe 3+ ions were devoid of any effect.Different mushroom lectins may be affected differently by the ions.Al 3+ ions strongly increase the hemagglutinating ac-   tivity of Pleurotus ostreatus lectin (Wang et al., 2000), but do not affect the lectin from Schizophyllum commune (Han et al., 2005).Ca 2+ , Mg 2+ , Mn 2+ , and Zn 2+ ions do not affect the hemagglutinating activity of lectins isolated from Amanita pantherina (Zhuang et al., 1996), G. frondosa (Kawagishi et al., 1990), and Hericium erinaceum (Kawagishi et al., 1994), but inhibit a Schizophyllum commune lectin (Han et al., 2005).PAL is stable in the presence of 25 mM HCl and 50 mM NaOH.It exhibits some similarity to the lectin from Armillaria luteo-virens (Feng et al., 2006).Mushroom lectins differ from one another in thermostability.The hemagglutinating activity of PAL decreases when the lectin is exposed to temperatures above 50°C.At and above 70°C the lectin activity is completely abolished.The lectin from Ganoderma capense is not affected after exposure to 100°C for 60 min (Ngai & Ng, 2004), while the hemagglutinating activity of lectins from P. ostreatus is reduced at or above 40°C (Wang et al., 2000).
PAL is characterized by an ability to inhibit proliferation of two tumor cell lines, Hep G2 and MCF-7.The IC 50 toward Hep G2 cells and MCF7 cells is 2.1 μM and near 3.2 μM, respectively.Lectins from A. bisporus, P. ostreatus, Tricholoma mongolicum, and Volvariella volvacea have antitumor activity in vivo or antiproliferative activity in vitro (Wang et al., 1996;1998;2000).The potent antiproliferative activity of PAL is remarkable and hopefully it can be developed into an agent for cancer therapy.HIV RT is a key enzyme of the HIV life cycle.Screening of HIV RT inhibitors is currently a strategy to search for anti-HIV drugs.It is worth mentioning that PAL manifests a potent inhibitory activity toward HIV-1 RT with an IC 50 of 1.9 μM.
Compared with many other lectins, P. adiposa lectin has significant inhibitory activity toward HIV-1 reverse transcriptase.It is possible that the mechanism of inhibition is analogous to the protein-protein interaction involved in the inhibition of HIV-1 reverse transcriptase by the homologous protease (Bottcher & Grosse, 1997).
When compared with P. aurivella lectin, the P. adiposa lectin displays many differences in chromatographic behavior, molecular mass, N-terminal sequence, and effect of cations on hemagglutinating activity.For P. aurivella lectin, many biological activities, including antifungal, antiproliferative, and HIV-1 RT inhibitory activities have not been determined (Table 6) (Kawagishi et al., 1991).
In summary, a lectin with a distinctive Nterminal sequence, carbohydrate specificity, and potent antiproliferative activity was isolated from P. adiposa fruiting bodies.It represents an addition to the existing list of mushroom lectins since it is the second Strophariaceae lectin and the second lectin isolated from a mushroom belonging to the Pholiota genus.

Figure 1 .
Figure 1.Ion exchange chromatography of fraction D3 (adsorbed on DEAE-cellulose) on CM-cellulose.A CM-cellulose column (1.5 × 10 cm) was equilibrated and eluted with 10 mM NH 4 OAC buffer (pH 4.6) and then stepwise with 50 mM and 1 M NaCl in the same buffer.Arrows indicate the points at which buffer was changed.

Table 1 . Summary of purification of P. adiposa lectin.
Activity refers to hemagglutinating activity.