- Open Access
First biochemical characterization of a novel ribonuclease from wild mushroom Amanita hemibapha
© Sekete et al.; licensee Springer. 2012
- Received: 22 September 2012
- Accepted: 4 December 2012
- Published: 27 December 2012
A 45-kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild mushroom Amanita hemibapha. It was adsorbed on DEAE-cellulose, S-sepharose, and finally purified on Superdex 75. The RNase exhibited maximal RNase activity at pH 5 and in a temperature range between 60-70°C. It demonstrated no ribonucleolytic activity toward four polyhomoribonucleotides. The amino acid sequence analysis (GDDETFWEHEWAK) showed this RNase was a ribonuclease T2-like RNase. It exhibited strong inhibitory activity against HIV-1 reverse transcriptase (HIV-1 RT) with an IC50 of 17 μM.
Ribonucleases (RNases) exist in a wide range of life forms from prokaryotes to eukaryotes (Fang and Ng. ). RNases from different mushrooms also differ in biochemical properties such as molecular weight, carbohydrate content, and N-terminal sequence among others (Wang and Ng. ). RNases isolated from different tissues may have different structures (Hofsteenge et al. ; Iwama et al. ; Sasso et al. ) and it has long been claimed that wild mushrooms are beneficial to health in manifestation of anti-tumor (Kobayashi et al. ), antiviral and antifungal (Wang and Ng. ), immunomodulatory (Matousek et al. ; Fang and Ng. ) and immunosuppressive activities (Wang and Ng. ; Ngai et al. ). In this manner, their potential clinical importance may also one day find application in the treatment of chronic diseases such as cancer and HIV- 1 infection.
Ribonucleases are capable of offering protective measures to various organisms due to their host defense mechanisms (Wong et al. ). Ribonucleases isolated from roots of Panax ginseng (Chinese ginseng), P. notoginseng (sanchi ginseng), and P. quinquefolius (American ginseng) have antifungal properties (Wang and Ng. ). RNases of both Chinese and American ginseng are homodimeric and demonstrate HIV-1 reverse transcriptase inhibitory activity (Wang and Ng, ).
RNases play a key role in RNA metabolism. They are involved in host defense and physiological cell death pathways. RNases possess therapeutic potentials for cancer treatment, as RNA damage caused by RNases could be an important alternative to standard DNA-damaging chemotherapeutics. (Makarov and Ilinskaya. ). Four members of the RNase A superfamily : Onconase from oocytes of Rana pipiens, BS-RNase from bull semen, and two closely related sialic acid-binding lectins from oocytes of Rana catesbeiana and Rana japonica are endowed with antitumor activity and show cytotoxicity toward several tumor cell lines (Notomista et al. ).
In the present study, a ribonuclease was isolated from the fryiting bodies of Amanita hemibapha for determination of biochemical characteristics and comparison with previously reported ribonucleases.
Dried fruiting bodies of the mushroom Amanita hemibapha from Sichuan China were homogenized in 0.15 M NaCl solution using a Waring blender, and then stored at 4°C overnight before centrifugation (10000 g, 15 min). Ammonium sulfate precipitation was carried out by adding (NH4)2SO4 to the supernatant to 80% saturation to precipitate proteins. After centrifugation (10000 g, 15 min), the precipitated proteins were dissolved in distilled water and dialyzed to remove (NH4)2SO4. NaAc-HAc buffer (pH 5.6, 1 M) was added to the solution, until the concentration of NaAc reached 10 mM. The supernatant was subjected to ion exchange chromatography on a column of DEAE-cellulose (Sigma) in 10 mM NaAc-HAc buffer (pH 5.6). After elution of unadsorbed proteins (fraction D1) with the same buffer, adsorbed proteins were desorbed sequentially with 50 mM NaCl, 150 mM NaCl, and 1 M NaCl to yield fractions D2, D3, and D4, respectively. Fraction D3 with RNase activity was dialyzed and subsequently chromatographed on a 2.5×10 cm of S-Sepharose (Sigma) in 10 mM NaAc-HAc buffer (pH 3.6). After removal of unadsorbed proteins (fraction S1), adsorbed proteins were eluted with a linear concentration gradient (0–500 mM) of NaCl and 1 M NaCl in 10 mM NaAc-HAc buffer (pH 3.6) to yield fraction S2 and S3. The peak (S3) with RNase activity was then further purified on a Superdex 75 HR 10/30 column (GE health) in 0.15 M NH4HCO3 buffer (pH 8.5). The first peak (SU1) obtained represented purified RNase.
Assay for activity of ribonuclease
Activity of A. hemibapha RNase toward yeast tRNA (Sigma) was assayed by measuring the production of acid-soluble, UV-absorbing species with a modification of the method of (Wang and Ng [2003a]). The RNase was incubated with 100 μg of tRNA in 150 μl 100 mM MES buffer (pH 4.6) at 37°C for 15 min. The reaction was terminated by addition of 350 μl of 3.7% perchloric acid. The sample was centrifuged at 15,000 g for 5 min. The absorbance of the resulting supernatant, after suitable dilution, was measured at 260 nm. One unit of enzymatic activity is defined as the amount of enzyme that produces an absorbance increase at 260 nm of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified conditions. The optimal pH and temperature were determined following the same method as described using buffer with different pH values as the reaction buffer and different temperatures instead of 37°C.
Molecular mass determination by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by FPLC-gel filtration
SDS-PAGE was conducted in accordance with the procedure of ([Laemmli and Favre 1973]) using a 12% resolving gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained with Coomassie brilliant blue. FPLC-gel filtration was carried out using a Superdex 75 HR 10/30 column that had previously been calibrated with molecular-mass standards using an AKTA Purifier (GE Healthcare).
Analysis of partial amino acid sequence
The single band of the purified RNase from SDS-PAGE was cut out and sent to National Center of Biomedical Analysis (Beijing) for analysis of amino acid sequence by Q-TOF.
Activity of A. hemibapha RNase toward polyhomoribonucleotides
The ribonucleolytic activity of A. hemibapha RNase toward polyhomoribonucleotides was determined with a modification of the method of ([Wang and Ng 2001]). Incubation of A. hemibapha RNase with 100 μg of poly A, poly C, poly G or poly U in 250 μl of 100 mM sodium acetate (pH 5.0) was carried out at 37°C for 1 h, prior to addition of 250 μl of ice-cold 1.2 N perchloric acid containing 20 mM lanthanum nitrate to terminate the reaction. After 15 min on ice, the sample was centrifuged at 15,000 ×g for 15 min at 4°C. The absorbance of the supernatant, after appropriate dilution, was read at 260 nm (for poly A, poly G and poly U) or at 280 nm (for poly C).
Assay for ability to inhibit human immunodeficiency virus reverse transcriptase (HIV-1 RT)
The assay for ability of A. hemibapha RNase to inhibit HIV-1 RT activity was carried out as detailed by (Collins et al. ) using a non-radioactive reverse transcriptase ELISA kit. The assay was executed following instructions supplied with the assay kit from Boehringer– Mannheim (Germany). The assay uses the ability to synthesize DNA with reverse transcriptase, starting 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 (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follow an ELISA protocol. Biotin labeled DNA binds to the surface of microtiter plate modules that have been pre pre-coated with streptavidin. In the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigenin-labeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzymes catalyze the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be determined using 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 isolated ribonuclease was calculated as percent inhibition as compared to a control without the protein (Collins et al. ).
Yields and RNase activities of various chromatographic fractions
Specific activity (U/mg)
Recovery of RNase activity (%)
Ammonium sulfate precipitate
Partial sequence of A. hemibapha RNase in comparison with other reported RNases
A. hemibapha RNase of this study
ribonuclease Trv [Metarhizium anisopliae]
125 GDDETFWEHEWAK 137
ribonuclease T2 [Puccinia graminis]
144 GN DETFWEHEWAK 156
ribonuclease T2 family, putative [Trichophyton verrucosum]
125 GDDETFWEHEWN K 137
ribonuclease T2 [Morchella esculenta]
124 GDDES FWEHEWS K 136
ribonuclease M [Cordyceps militaris]
117 GDDES FWEHEWG K 129
ribonuclease T2 precursor [Aspergillus terreus]
123 GDDES FWEHEWN K 135
ribonuclease T2 [Aspergillus fumigates]
128 GDDES FWEHEWN K 140
S-like RNase [Volvox carteri]
120 DETFWEHEWS K 130
Comparison of characteristics of various mushroom ribonucleases
Mol mass (kDa)
HIV-1 RT inhibitory activity (IC50 in μM)
Antiproliferative activity (IC50 in μM)
Hep G2 cells
Pleurotus ostreatus (Nomura et al.)
Pleurotus ostreatus (Ye et al.)
A 45-kDa ribonuclease (RNase) purified from dried fruiting bodies of the wild mushroom Amanita hemibapha is reported herein. Its molecular size of 45 kDa falls outside the range exhibited by all mushroom ribonucleases (9-45 kDa) reported so far, being greater than that of straw mushroom (42 kDa) (Wang and Ng. ). RNases from Boletus griseus (Wang and Ng. ), Clitocybe maxima (Wang and Ng. [2004a]), Dictyophora indusiata (Wang and Ng. [2003a]), Hypsizigus marmoreus (Guan et al. ), Lyophyllum shimeiji (Zhang et al. ), Pleurotus djamor (Wu et al. ), Pleurotus eryngii (Ng and Wang, ), Pleurotus ostreatus (Ye and Ng. ), Pleurotus pulmonarius (Ye and Ng, ), Pleurotus sajor-caju (Ngai and Ng. ), Pleurotus tuber-regium (Wang and Ng. ), Russula delica (Zhao et al. ), Russulus virescens (Wang and Ng [2003b]) and Thelephora ganbajun (Wang and Ng [2004b]), are all smaller than 32 kDa while those of Ganoderma lucidum (Wang et al. ) and Volvariella volvacea (Wang and Ng. ) are higher than 30 kDa but less than 45 kDa. A. hemibapha RNase demonstrated no ribonucleolytic activity toward four polyhomoribonucleotides. Most reported RNases such as P. tuber-regium (Wang and Ng. ) and P. ostreatus (Nomura et al. ) RNases are specific for poly G. L. edodes RNase exhibits preference for polyA (Kobayashi et al. ). Others like an ubiquitin-like peptide from mushroom Cantharellus cibarius (Wang et al. ) showed ribonuclease activity against various polyhomoribonucleotides.
The optimum pH was 5 and the optimal temperature was 60~70°C. Its optimum pH is very different from that of Russulus virescens ribonuclease (optimum pH of 4.5) (Wang and Ng [2003b]) and of Ganoderma lucidum ribonuclease (optimum pH of 4.0) (Wang et al. ). The temperature dependence curve for the activity of A. hemibapha ribonuclease indicates that it is a fairly thermostable enzyme. It retains more than half of its maximal activity at 80°C and is totally inactivated only at 100°C. The partial sequence of A. hemibapha RNase reveals 100% similarity to ribonuclease Trv Metarhizium anisopliae and slight difference from ribonuclease T2 Puccinia graminis, ribonuclease T2 family, putative Trichophyton verrucosum, ribonuclease T2 Morchella esculenta, ribonuclease M Cordyceps militaris, ribonuclease T2 precursor Aspergillus terreus, ribonuclease T2 Aspergillus fumigatus, S-like RNase Volvox carteri as depicted in Table 2. But this RNase inhibited HIV-1 reverse transcriptase with an IC50 of 17 μM. This anti-HIV-1 reverse transcriptase activity has not been demonstrated for the majority of the previously isolated mushroom RNases.
This work was financially supported by National Grants of China (2010CB732202).
- Collins RA, Ng TB, Fong WP, Wan CC, Yeung HW: A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs. Life Sci 1997, 60: L345-L351.Google Scholar
- Fang EF, Ng TB: Ribonucleases of different origins with a wide spectrum of medicinal applications. Biochimica et Biophysica Acta 2010, 1815: 65-74.Google Scholar
- Guan GP, Wang HX, Ng TB: A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochim Biophys Acta 2007, 1770: 1593-1597. 10.1016/j.bbagen.2007.07.014View ArticleGoogle Scholar
- Hofsteenge J, Matthies R, Stone SR: Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily. Biochemistry 1989, 28: 9806-9813. 10.1021/bi00451a040View ArticleGoogle Scholar
- Iwama M, Sanda A, Ohgi K, Hofsteenge J, Irie M: Purification and primary structure of a porcine kidney non-secretory ribonuclease. Biosci Biotechnol Biochem 1993, 57: 2133-2138. 10.1271/bbb.57.2133View ArticleGoogle Scholar
- Kobayashi H, Inokuchi N, Koyama T, Watanabe H, Iwami M, Ohgi K: Primary structure of a base nonspecific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinula edodes. Biosci Biotechnol Biochem 1992, 55: 2003-2010.View ArticleGoogle Scholar
- Kobayashi H, Hara J, Itagaki T, Inokuchi N, Koyama T, Sanda A, Iwama M, Ohgi K, Irie M: Relationship of two ribonucleases with molecular masses of 45 kDa and 37 kDa from the culture medium of Lentinula edodes. Biol Pharm Bull 2000, 23: 800-804. 10.1248/bpb.23.800View ArticleGoogle Scholar
- Laemmli U, Favre M: Gel electrophoresis of proteins. J Mol Biol 1973, 80: 575-599. 10.1016/0022-2836(73)90198-8View ArticleGoogle Scholar
- Makarov AA, Ilinskaya ON: Cytotoxic ribonucleases: molecular weapons and their targets. FEBS Lett 2003,540(1–3):15-20.View ArticleGoogle Scholar
- Matousek J, Soucek J, Riha J, Zankel TR, Benner SA: Immunosuppressive activity of angiogenin in comparison with bovine seminal ribonuclease and pancreatic ribonuclease. Comp Biochem Physiol B Biochem Mol Biol 1995, 112: 235-241. 10.1016/0305-0491(95)00075-5View ArticleGoogle Scholar
- Ng TB, Wang HX: A novel ribonuclease from fruiting bodies of the common edible mushroom Pleurotus eryngii. Peptides 2004, 25: 1365-1368. 10.1016/j.peptides.2004.01.027View ArticleGoogle Scholar
- Ngai PHK, Ng TB: A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju. Peptides 2004, 25: 11-17. 10.1016/j.peptides.2003.11.012View ArticleGoogle Scholar
- Ngai PH, Wang HX, Ng TB: Purification and characterization of a ubiquitin-like peptide with macrophage stimulating, antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea. Peptides 2003, 24: 639-645. 10.1016/S0196-9781(03)00136-0View ArticleGoogle Scholar
- Nomura H, Inokuchi N, Kobayashi H, Koyama T, Iwama M, Ohgi K: Purification and primary structure of a new guanylic acid specific ribonuclease from Pleurotus ostreatus. J Biochem 1994, 116: 26-33.Google Scholar
- Notomista E, Catanzano F, Graziano G, Dal Piaz F, Barone G, D'Alessio G, Di Donato A: Onconase: An unusually stable protein. Biochemistry 2000, 39: 8711-8718. 10.1021/bi000415xView ArticleGoogle Scholar
- Sasso MP, Carsana A, Confalone E, Cosi C, Sorrentino S, Viola M, Palmieri M, Russo E, Furia A: Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain. Nucleic Acids Res 1991, 19: 6469-6474. 10.1093/nar/19.23.6469View ArticleGoogle Scholar
- Wang H, Ng TB: Isolation of a new ribonuclease from fresh fruiting bodies of the straw mushroom. Biochem Biophys Res Commun 1999, 264: 714-718. 10.1006/bbrc.1999.1571View ArticleGoogle Scholar
- Wang HX, Ng TB: Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from American ginseng roots. Biochem Biophys Res Commun 2000, 269: 203-208. 10.1006/bbrc.2000.2114View ArticleGoogle Scholar
- Wang HX, Ng TB: Purification and characterization of a potent homodimeric guanine-specific ribonuclease from fresh mushroom (Pleurotus tuber-regium) sclerotia. Int J Biochem Cell Biol 2001, 33: 483-490. 10.1016/S1357-2725(01)00038-3View ArticleGoogle Scholar
- Wang H, Ng TB: A novel ribonuclease from the veiled lady mushroom Dictyophora indusiata. Biochem Cell Biol 2003, 81: 373-377. 10.1139/o03-067View ArticleGoogle Scholar
- Wang H, Ng TB: A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens. Biochem Biophys Res Commun 2003, 312: 965-968. 10.1016/j.bbrc.2003.10.201View ArticleGoogle Scholar
- Wang H, Ng TB: Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima. Peptides 2004, 25: 935-939. 10.1016/j.peptides.2004.03.008View ArticleGoogle Scholar
- Wang HX, Ng TB: Purification of a novel ribonuclease from dried fruiting bodies of the edible wild mushroom Thelephora ganbajun. Biochem Biophys Res Commun 2004, 324: 855-859. 10.1016/j.bbrc.2004.09.132View ArticleGoogle Scholar
- Wang H, Ng TB: A ribonuclease from the wild mushroom Boletus griseus. Appl Microbiol Biotechnol 2006, 72: 912-916. 10.1007/s00253-006-0385-7View ArticleGoogle Scholar
- Wang HX, Ngai HK, Ng TB: A ubiquitin-like peptide with ribonuclease activity against various polyhomoribonucleotides from the mushroom Cantharellus cibarius. Peptides 2003, 24: 509-513. 10.1016/S0196-9781(03)00116-5View ArticleGoogle Scholar
- Wang HX, Ng TB, Chiu SW: A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. Biochem Biophys Res Commun 2004, 314: 519-522. 10.1016/j.bbrc.2003.12.139View ArticleGoogle Scholar
- Wong JH, Ng TB, Randy CF, Cheung Ye XJ, Wang HX, Lam SK, Lin P, Chan YS, Evandro FF, Patrick Ngai HK, Xia LX, Ye XY, Jiang Y, Liu F: Proteins with antifungal properties and other medicinal applications from plants and mushrooms. Appl Microbiol Biotechnol 2010, 87: 1221-1235. a minireview 10.1007/s00253-010-2690-4View ArticleGoogle Scholar
- Wu X, Zheng S, Cui L, Wang H, Ng TB: Isolation and characterization of a novel ribonuclease from the pink oyster mushroom Pleurotus djamor. The Journal of general and applied microbiology 2010,56(3):231-239. 10.2323/jgam.56.231View ArticleGoogle Scholar
- Ye XY, Ng TB: A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius. Biochem Biophys Res Commun 2002, 293: 857-861. 10.1016/S0006-291X(02)00301-7View ArticleGoogle Scholar
- Ye XY, Ng TB: Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus. J Pept Sci 2003, 9: 120-124. 10.1002/psc.437View ArticleGoogle Scholar
- Zhang RY, Zhang GQ, Hu DD, Wang HX, Ng TB: A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. Biochem Genet 2010, 48: 658-668. 10.1007/s10528-010-9347-yView ArticleGoogle Scholar
- Zhao S, Zhao Y, Li S, Zhang G, Wang H, Ng TB: An antiproliferative ribonuclease from fruiting bodies of the wild mushroom Russula delica. J Microbiol Biotechnol 2010, 20: 693-699. 10.4014/jmb.0911.11022View ArticleGoogle Scholar
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