First biochemical characterization of a novel ribonuclease from wild mushroom Amanita hemibapha

Ribonucleases (RNases) exist in a wide range of life forms from prokaryotes to eukaryotes (Fang and Ng. [2010]). RNases from different mushrooms also differ in biochemical properties such as molecular weight, carbohydrate content, and N-terminal sequence among others (Wang and Ng. [2006]). RNases isolated from different tissues may have different structures (Hofsteenge et al. [1989]; Iwama et al. [1993]; Sasso et al. [1991]) and it has long been claimed that wild mushrooms are beneficial to health in manifestation of anti-tumor (Kobayashi et al. [2000]), antiviral and antifungal (Wang and Ng. [2000]), immunomodulatory (Matousek et al. [1995]; Fang and Ng. [2010]) and immunosuppressive activities (Wang and Ng. [2000]; Ngai et al. [2003]). 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. [2010]). Ribonucleases isolated from roots of Panax ginseng (Chinese ginseng), P. notoginseng (sanchi ginseng), and P. quinquefolius (American ginseng) have antifungal properties (Wang and Ng. [2000]). RNases of both Chinese and American ginseng are homodimeric and demonstrate HIV-1 reverse transcriptase inhibitory activity (Wang and Ng, [2000]).

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. [2003]). 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. [2000]).

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.

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. [1999]). RNases from Boletus griseus (Wang and Ng. [2006]), Clitocybe maxima (Wang and Ng. [2004a]), Dictyophora indusiata (Wang and Ng. [2003a]), Hypsizigus marmoreus (Guan et al. [2007]), Lyophyllum shimeiji (Zhang et al. [2010]), Pleurotus djamor (Wu et al. [2010]), Pleurotus eryngii (Ng and Wang, [2004]), Pleurotus ostreatus (Ye and Ng. [2003]), Pleurotus pulmonarius (Ye and Ng, [2002]), Pleurotus sajor-caju (Ngai and Ng. [2004]), Pleurotus tuber-regium (Wang and Ng. [2001]), Russula delica (Zhao et al. [2010]), 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. [2004]) and Volvariella volvacea (Wang and Ng. [1999]) 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. [2001]) and P. ostreatus (Nomura et al. [1994]) RNases are specific for poly G. L. edodes RNase exhibits preference for polyA (Kobayashi et al. [1992]). Others like an ubiquitin-like peptide from mushroom Cantharellus cibarius (Wang et al. [2003]) 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. [2004]). 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.