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Introduction

The literature pertaining to the Hohenbuehelia species is limited and confined to the following reports. Vafina and Molodtsov described the synthesis of some p-nitrophenyl 2-acylamino-2-deoxy-D-glucosides and their hydrolysis using Hohenbuehelia serotina β-D-hexosaminidase (1). The culture filtrate of Hohenbuehelia geogenius inhibited the growth of two rapidly growing grafted tumors (Ehrlich ascites carcinoma and L1210 lymphoid leukemia) and a slow-growing spontaneous mammary tumor in mice. An active substance was isolated by solid-liquid extraction and column chromatography and its chemical structure was elucidated (2).

Anti-A agglutinins, the reaction of which has been shown to be strongly inhibited by N-acetyl-D-galactosamine, have been detected in Hohenbuehelia serotina extracts (3). Two strains of Hohenbuehelia atrocaerulea (Pleurotaceae) that produce pleurotin, a naphthoquinone antibiotic originally obtained from Pleurotus griseus, have been identified. Solid substrate fermentation for two months yielded 1–2 mg/l of pleurotin. Shipley et al (4) detailed the developmental process, which depended on inclusion in the medium of an aqueous extract of alder woodm leading to a yield of pleurotin exceeding 300 mg/l from liquid fermentation. Bala et al reported that water, ethanol and hexane extracts of the Hohenbuehelia species inhibited growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli (5). Bala et al (6) further demonstrated that a water extract of Hohenbuehelia species inhibited six pathogens each comprising of two Gram-positive and -negative bacteria together with two fungi.

Polysaccharides extracted from the fruiting bodies of Hohenbuehelia serotina have exhibited antitumor activity in sarcoma 180-bearing mice (7). The molecular weights of these polysaccharides ranged from 1.19×103 to 1.55×104 Da and composed of ribose, arabinose, mannose, glucose and galactose at a ratio of 0.65:0.69:9.35:14.24:5.47; they were isolated by Li et al (8). However, there is a dearth of information on the proteinaceous constituents of the Hohenbuehelia species.

Ribonucleases (RNases) have been isolated and characterized from a multitude of organisms, including parasites, bacteria, fungi, plants and a variety of tissues from mammals (9–17) RNases display different activities, such as antitumor (18–25), immunosuppressive (26), antifungal (27), and antiviral (28,29) activities. Due to the array of potentially exploitable activities, RNases have drawn the attention of many researchers.

RNases have been purified from the fruiting bodies or mycelia of a diversity of mushroom species. These mushrooms include some common edible and medicinal species as follows: Pleurotus ostreatus (30,31), Irpex lacteus (32), Volvariella volvacea (33), Pleurotus tuber-regium (34), Pleurotus pulmonarius (35), Agrocybe cylindracea (36), Russula virescens (37), Termitomyces globules (38), Cantharellus cibarius (39), Pleurotus sajor-caju (40), Ganoderma lucidum (41), Clitocybe maxima (42), Thelephora ganbajun (43), Boletus griseus (44), Hypsizigus marmoreus (45), Russula delica (16,46) and Lyophyllum shimeiji (47). The aim of this study was to isolate and characterize an RNase isolated from the dried fruiting bodies of the edible fungus, Hohenbuehelia serotina, and to compare its characteristics and N-terminal sequence with those of RNases isolated from the aforementioned species. The comparison would reveal any differences between RNases from different species and our findings may expand the knowledge and provide further information on this fungus.

Materials and methods

Isolation of RNase from Hohenbuehelia serotina

The dried fruiting bodies (200 g) of the edible mushroom, Hohenbuehelia serotina, obtained from North China were extracted with distilled water (2 ml/g) using a Waring blender (Waring Laboratory Supplies, Torrington, CT, USA). Tris-HCl buffer (pH 7.2, 1 M) was added to the supernatant, obtained by centrifugation of the homogenate, until the concentration of Tris reached 10 mM. The supernatant was subjected to ion exchange chromatography on a 5×15 cm column of DEAE-cellulose (Sigma, St. Louis, MO, USA) in 10 mM Tris-HCl buffer (pH 7.2). Following elution of the unadsorbed proteins (fraction D1) with the same buffer, the adsorbed proteins were desorbed sequentially with 0.2 M NaCl and 1 M NaCl in Tris-HCl buffer to form fractions D2 and D3, respectively. Fraction D1 was then directly chromatographed on a 5×15 cm Affi-gel blue gel column (Bio-Rad, Hercules, CA, USA) in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed proteins were eluted as fraction B1. The adsorbed proteins were eluted sequentially with 0.2 M NaCl and 1 M NaCl in Tris-HCl buffer and collected as fractions B2 and B3, respectively. Fraction B2 was dialyzed against 10 mM NH4OAc buffer (pH 5) subjected to ion exchange chromatography on a 2.5×20 cm CM-cellulose (Sigma) column in 10 mM NH4OAc buffer (pH 5). Following the removal of the unadsorbed proteins (fraction CM1), the adsorbed proteins were eluted with 10 mM NH4OAc buffer (pH 5) containing a linear gradient of 0–1 M NaCl, and collected as fractions CM2 and CM3. Fraction CM3 was dialyzed, lyophilized and further purified on a Superdex 75 HR 10/30 column in 0.2 M NH4HCO3 buffer (pH 8.5) using an AKTA Purifier (GE Healthcare, Piscataway, NJ, USA).

Molecular mass determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by fast protein liquid chromatography (FPLC)-gel filtration

SDS-PAGE was conducted following the protocol of Laemmli and Favre (48), using a 12% resolving gel and a 5% stacking gel. At the conclusion of electrophoresis, staining of the gel with Coomassie brilliant blue was carried out. FPLC-gel filtration was performed using a Superdex 75 HR 10/30 column that had been calibrated with molecular mass standards using an AKTA Purifier (GE Healthcare).

Analysis of N-terminal amino acid sequence

The amino-acid sequence of the purified protein was determined by means of automated Edman degradation. The amino acid sequence was determined using an HP G1000A Edman degradation unit and an HP 1000 HPLC system (Agilent Technologies, Santa Clara, CA, USA).

Assay for RNase activity

The activity of the purified RNase toward yeast tRNA (Sigma) was assayed by determining the generation of acid-soluble UV-absorbing species (reaction products) with the method of Wang and Ng (52). The RNase was incubated with 200 μg tRNA in 150 μg of 100 mM MES buffer (pH 6.0) at 37°C for 1 h. The reaction was terminated by the addition of 350 μl of ice-cold 3.4% perchloric acid. After remaining on ice for 15 min, the sample was centrifuged (15,000 × g) at 4°C for 15 min. The OD260 of the supernatant was read after appropriate dilution. One unit of enzymatic activity is defined as the amount of enzyme that induces an increase in OD260 of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified conditions.

Activity of RNase toward polyhomoribonucleotides

The ribonucleolytic activity of the purified RNase toward various polyhomoribonucleotides as substrates was determined with a modification of a previously described method (33). The incubation of RNase with 100 μg poly(A), poly(C), poly(G) or poly(U) was carried out at 37°C for 1 h in 250 μl of 100 mM sodium acetate buffer (pH 5.0), prior to the addition of 250 μl of ice-cold 1.2 N perchloric acid containing 20 mM lanthanum nitrate to terminate the reaction. The reaction mixture was left on ice for 15 min prior to centrifugation at 15,000 × g for 15 min at 4°C. After appropriate dilution, the absorbance of the supernatant was read at 260 nm [for substrates poly(A), poly(G) and poly(U)] or at 280 nm [for substrate poly(C)].

Assay for ability to inhibit human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT)

The assay to determine the HIV-1 RT inhibitory activity of Hohenbuehelia serotina RNase was executed using a non-radioactive reverse transcriptase ELISA kit (Sigma-Aldrich, Trading Co., Ltd., Shanghai, China) as previously described (49).

Assay for anti-proliferative activity of RNase isolated from Hohenbuehelia serotina

The anti-proliferative activity of the purified RNase was determined as follows: the L1210 and MBL2 cells were maintained in Dulbecco’s modified Eagle’s 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 atmosphere of 5% CO2. Cells (1×104) in their exponential growth phase were seeded into each well of a 96-well culture plate and incubated for 3 h prior to the addition of the purified RNase followed by incubation for a further 48 h. The radioactive precursor, 1 μCi [3H-methyl]-thymidine, was added to each well followed by incubation for 6 h before the cultures were harvested by means of a cell harvester. The incorporated radioactivity was determined by liquid scintillation counting, as previously described (40).

Results

Isolation of RNase

Ion exchange chromatography of the extract on DEAE-cellulose yielded three fractions D1, D2 and D3 containing similar amounts of proteins. RNase activity was found only in the unadsorbed fraction D1. D1 was separated on Affi-gel blue gel into one unadsorbed and also the largest fraction (B1), together with two adsorbed fractions (B2 and B3). Fraction B2 was the only fraction with strong RNase activity. This fraction was resolved on CM-cellulose into an unadsorbed fraction CM1 devoid of RNase activity, an adsorbed fraction CM2 with weak RNase activity, and the most strongly adsorbed fraction CM3 containing the bulk of RNase activity (Fig. 1). Fraction CM3 was resolved into three fractions, SU1, SU2 and SU3 upon gel filtration on Superdex 75. RNase activity resided in the second fraction SU2 (Fig. 2 and Table I).

Figure 1 Ion exchange chromatography on CM-Sepharose. Sample: fraction B2 which was previously adsorbed on Affi-gel blue gel. Column dimensions: 2.5×20 cm. Starting buffer: 10 mM NH4Ac (pH 5.2). Slanting dotted line across the right side of the chromatogram represents the linear 0–1 M NaCl gradient used to elute adsorbed proteins.

Figure 2 FPLC-gel filtration of fraction CM3 on a Superdex 75 HR10/30 column. Buffer, 0.2 M NH4HCO3 (pH 8.5); flow rate, 0.4 ml/min; fraction size, 0.8 ml.

Table I Yields (from 650 g fresh Hohenbuehelia serotina fruiting bodies) and RNase activity (determined in 0.1 M MES buffer, pH 6.0, 37°C) of various chromatographic fractions.

Table I

Yields (from 650 g fresh Hohenbuehelia serotina fruiting bodies) and RNase activity (determined in 0.1 M MES buffer, pH 6.0, 37°C) of various chromatographic fractions.

Fraction	Yield (mg)	RNase activity (U/mg)	Fraction	Yield (mg)	RNase activity (U/mg)
Extract	2018	35.7	CM1	19.0	<1
D1	459	98.6	CM2	13.4	51.8
D2	403	12.5	CM3	23.5	983.0
D3	529	<1	SU1	3.6	102.8
B1	165	<1	SU2	6.9	2705.3
B2	79.3	369.5	SU3	5.9	86.1
B3	81.2	15.7

[i] RNase, ribonuclease.

Determination of molecular mass

Fraction SU2 appeared as a single band with a molecular mass of 27 kDa, as shown by SDS-PAGE (Fig. 3).

Figure 3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified Hohenbuehelia serotina ribonuclease (RNase) (fraction SU2). The molecular mass of the marker proteins are, from top to bottom, 94, 67, 43, 30, 20 and 14.4 kDa.

Analysis of N-terminal amino acid sequence

The amino acid sequence was abtained by an HP 1000 HPLC system depending on Edman degradation. The N-terminal sequence was as follows: TVGGSLAEKGN, which showed homology to other fungal RNases to a certain degree (Table II).

Table II N-terminal sequence of Hohenbuehelia serotina (HS) ribonuclease (RNase) in comparison with RNases isolated from other mushrooms.

Table II

N-terminal sequence of Hohenbuehelia serotina (HS) ribonuclease (RNase) in comparison with RNases isolated from other mushrooms.

RNase	N-terminal sequence
HS:	TVGGSLAEKGN
TG:	DADIAVWAPPVNAQN
CM:	ETAHTHAGIQYSTVDVNNSIMKAVGGGAGN
PP:	AISANNERKGVNQQSVQNTYQENDV
VV:	APYVQLFRPLIQPQVLATFAIANNMAQY
LE:	ISSGCGTTGALSCSSNAKGTCCFEAPGGLI
IL:	VNSGCGTSGAESCSNSDDGTCCFEAPGGLL
DI:	GQPRQPQPQLLV
PE:	GEVVQYYP
PS:	DNGEAGRAAR
GL:	HLPBVPSFAYGSIKVYIN
RV:	TDHTLDTMMTHTLRD
PO:	ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGN
PT:	ALTAQDNRVRVGNRIVGNNFNFAAVQAAYY

[i] TG, Thelephora ganbajun; CM, Clitocybe maxima; PP, Pleurotus pulmonarius; VV, Volvariella volvacea; LE, Lentinus edodes; IL, Irpex lacteus; DI, Dictyophora indusiata; PE, Pleurotus eryngii; PS, Pleurotus sajor-caju; GL, Ganoderma lucidum; RV, Russulus virescens; PO, Pleurotus ostreatus; PT, Pleurotus tuber-regium.

Determination of optimum pH and optimum temperature

The optimum pH (Fig. 4) and temperature (Fig. 5) for the purified RNase were pH 5.0 and 80°C, respectively.

Figure 4 pH dependence of Hohenbuehelia serotina ribonuclease (RNase). Temperature used: 37°C. Duration of incubation: 15 min. Buffer concentration: 0.1 M.

Figure 5 Temperature dependence of Hohenbuehelia serotina ribonuclease (RNase). Buffer used: 0.1 M NH4Ac buffer (pH 4.5). Duration of incubation, 15 min.

Determination of polyhomoribonucleotide specificity

The RNase exerted a ribonucleolytic activity of 455.1, 311.2, 119.4 and 105 U/mg toward poly(C), poly(U), poly(A) and poly(G), respectively.

HIV-1 RT inhibitory activity

The RNase inhibited HIV-1 RT with an IC50 of 50 μM (Table III).

Table III Inhibition rates (%) of RNase on growth of MBL2 cells, L1210 cells, and HIV-1 RT.

Table III

Inhibition rates (%) of RNase on growth of MBL2 cells, L1210 cells, and HIV-1 RT.

	Inhibition rates (%)
Dose (μM)	HIV-1 RT	L1210	MBL2
10	13.5±0.9	24.6±1.8	18.7±1.1
20	21.4±1.6	40.9±2.7	31.4±2.3
40	42.1±2.7	72.1±5.2	49.7±3.8
80	73.4±4.3	94.3±5.5	73.6±5.9
IC50 (μM)	49.9	24.8	40.3

[i] RNase, ribonuclease; HIV-1 RT, human immunodeficiency virus type 1 reverse transcriptase.

Anti-proliferative activity toward tumor cells

The RNase inhibited [3H-methyl]-thymidine uptake by L1210 cells and MBL2 cells with an IC50 of 25 and 40 μM, respectively (Table III).

Discussion

RNase isolated from Hohenbuehelia serotina is characterized by an N-terminal sequence distinctly different from that of previously reported mushroom and non-mushroom RNases. The molecular weight of previously reported mushroom RNases ranges from 9 to 42.5 kDa. The molecular mass of Hohenbuehelia serotina RNase (27 kDa) lies within this range. It is larger than the mass of RNases isolated from Clitocybe maxima (42), Hypsizigus marmoreus (45), Lyophyllum shimeiji (47), Pleurotus djamor (50), Pleurotus eryngii (51), Pleurotus ostreatus (31), Pleurotus pulmonarius (35), Pleurotus sajor-caju (40), Russula delica (46) and Thelephora ganbajun (43), but smaller than the mass of RNases isolated from Boletus griseus (44), Dictyophora indusiata (52), Ganoderma lucidum (43), Pleurotus tuber-regium (34), Russulus virescens (37) and Volvariella volvacea (33). Its mass is close to that of Dictyophora indusiata RNase (28 kDa) and Russulus virescens RNase (28 kDa). All these RNases are monomeric, and all of them are acid RNases.

RNases isolated from different mushrooms may have different optimum pH values and temperatures for RNase activity. The optimum pH of Hohenbuehelia serotina RNase (pH 5.0) is similar to that of Hypsizigus marmoreus RNase (pH 5.0) [Guan et al (45)] and Russula delica RNase (pH 5.0) [Zhao et al (46)]; however, Hohenbuehelia serotina RNase is much more thermostable. The optimum temperature for the purified Hohenbuehelia serotina RNase was found to be 80°C, which is the highest optimum temperature for RNases purified from mushrooms.

Some RNases display antiviral activity. Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation [Zhu et al (53)]. Rana catesbeiana RNase inhibits Japanese encephalitis virus (JEV) replication and enhances apoptosis of JEV-infected BHK-21 cells.

Li et al (54) suggested that the targeted RNase is an alternative anti-hepatitis B virus (HBV) agent. The HIV-1 RT inhibitory activity of Hohenbuehelia serotina RNase has been shown to be similar to that of RNases isolated from other mushrooms, such as Lyophyllum shimeiji (47) and Thelephora ganbajun (43). The mechanisms responsible for the inhibitory effects on HIV-1 RT may involve protein-protein interaction, as in the case of the inhibition of HIV-1 RT by the homologous protease (55) and the inhibition of HIV-1 protease by cathelicidin [Wong et al (56)].

Some RNases exert inhibitory effects on tumor cells. The binding of AS RNase and CM-AS RNase to leukaemic cells from patients with chronic lymphatic leukaemia has been demonstrated by indirect immunofluorescence, while no binding to normal leucocytes and leucocytes from patients with other hemoblastoses was observed (24). Bovine seminal RNase (BS-RNase) manifests specific cytotoxic effects on tumor cells, and non-malignant cells are not affected. In view of the finding that success was met only when BS-RNase was applied intratumorally, the properties of BS-RNase were improved by attachment to polylactic acid nanoparticles. The nanoparticle preparation and pure BS-RNase showed no difference when tested against leukemia (MOLT-4) and lymphoma (H9) cell lines sensitive and resistant to cytarabine in vitro. The aspermatogenic and anti-embryonal activities were augmented in the nanoparticle preparation of BS-RNase. It remains to be seen how well BS-RNase attached to polylactic acid nanoparticles performs as an antitumoral agent in vivo [Michaelis et al (57)]. The anticancer effect of the amphibian RNase Onconase as demonstrated experimentally and in clinical trials has been reported (58). Plant RNases have also been shown to exhibit antitumor effects in a number of studies (18,20,22).

Mushroom RNases have been shown to be effective against hepatoma and breast cancer cells (46). Zhao et al (16) noted that Schizophyllum commune RNase had no effect on the proliferation of leukemia and lymphoma cells. Only a few mushroom RNases have been shown to inhibit the growth of leukemia cells (36,40,59,60). We found that the anti-proliferative activities of Hohenbuehelia serotina RNase toward L1210 cells are not as remarkable as those of Pleurotus sajor-caju RNase (40), but more effective than those of Hypsizigus marmoreus RNase (45). To our knowledge, we also demonstrated for the first time that mushroom RNases exhibit anti-proliferative activity toward MBL2 cells.

Some plant RNases have antifungal activity (27,34,61) and have been classified as one family of pathogenesis-related proteins. It is interesting to note that Hohenbuehelia serotina RNase, similar to all previously reported mushroom RNases, is devoid of antifungal activity. However, its antiproliferative activity against cancer cells and its inhibitory activity toward HIV-I RT signify that it is a defense protein. Its RNase activity can be deployed against invaders.

In conlcusion, the RNase isolated from Hohenbuehelia serotina in the present study is a novel RNase, as evidenced by a novel N-terminal sequence and a high optimum pH. It manifests potent anti-proliferative activity toward cancer cells and inhibitory activity toward HIV-1 RT. These biological activities are potentially exploitable. In this regard it is noteworthy that not all previously reported mushroom RNases were assayed for or demonstrate anti-proliferative and HIV-1 RT inhibitory activities.

Acknowledgements

This study was financially supported by the National Grants of China (no. 2010CB732202) and the Special Fund for Agro-scientific Research in the Public Interest (no. 201303080).

References