A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal Fungus Agaricus placomyces

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, Km values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50 of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein.


Introduction
Laccase (E.C. 1.10.3.2), a polyphenol oxidase containing copper atoms in its catalytic center that plays a key role in lignin degradation in nature, was first demonstrated in plants at the end of 19th century [1,2]. It is widely distributed among plants, insects, bacteria, and especially fungi [3]. With a research history of more than a century, laccase has gained importance as a versatile industrial enzyme with a repertoire of applications in lignin degradation, environmental detoxification, and a variety of industries including paper, textile, bioremediation, biocatalysis, diagnostic, and food industries [4,5]. Studies on laccases have mainly focused on characterization in terms of molecular mass, protein sequence, pH and temperature optima, enzyme kinetics, substrate specificity, purification protocol, cloning, structure, applications, and chemical synthesis [1,3,[6][7][8].
Agaricus placomyces, belonging to Family Agaricaceae, is a species of mycorrhizal fungus widely distributed in Asia, Europe, and North America. It is reportedly edible, but for some people it could cause gastrointestinal problems. Few studies on A. placomyces except those on its toxic components have been published [9]. On the other hand, articles about laccases from the same genus Agaricus, for example, laccases from A. bisporus [10] and A. blazei [11], are available. The objective of the present study was to isolate a laccase from A. placomyces and investigate its characteristics.

Materials and Reagents. Fruiting bodies of A. placomyces
were collected in the forest of Baiwangshan National Forest Park (Beijing, China) and authenticated by experts in the Department of Microbiology and Immunology, College of Biosciences, China Agricultural University. DEAEcellulose, CM-cellulose, Trizma-base, 2,7 -azinobis[3ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS), catechol, hydroquinone, 2-methylcatechol, N,N-Dimethyl-1,4-phenylenediamine, pyrogallol, and tyrosine were purchased from Sigma, USA. Q-Sepharose and molecular mass standards were obtained from GE Healthcare, USA. MCF 7 tumor cell lines and Hep G2 tumor cell lines were purchased from the American Type Culture Collection, USA. All other reagents used were of reagent grade and from China unless otherwise mentioned.

Molecular Mass Determination by SDS-PAGE and FPLC
Gel Filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a 12% resolving gel and a 5% stacking gel [13]. At the end of electrophoresis, the gel was stained with Coomassie brilliant blue R-250. FPLC-gel filtration was carried out using a Superdex 75 column that had been calibrated with molecular mass standards [14].

Determination of N-Terminal
Amino Acid Sequence of Isolated Laccase. Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system [12].
2.5. Assay of Laccase Activity. Laccase activity was assayed by measuring the oxidation of 2,7 -azinobis[3ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS). A modification of the method of Shin and Lee was used [15]. In brief, 5 μL laccase solution was incubated with 140 μL 1 mM ABTS (in 50 mM sodium acetate buffer, pH 5.2) at 30 • C for 5 min. Subsequently, the reaction was ended by adding 255 μL 10% (w/v) trichloroacetic acid. One unit of enzyme activity was defined as the amount of enzyme required to produce an absorbance increase at 405 nm of 1/min/mL of reaction mixture under the aforementioned conditions. All determinations were performed in triplicate.

Determination of Temperature Optimum and Thermostability of Isolated Laccase.
To determine the temperature optimum of A. placomyces laccase, the standard laccase assay mentioned above was conducted over a temperature range of 10-80 • C. In the thermostability assay, enzyme solutions were incubated at various temperatures (20,40,60,70, and 80 • C) and for various durations (0, 10, 20, 30, 40, 50, and 60 min), respectively. The residual activity was measured using the standard assay after the enzyme solutions had been cooled down to room temperature.

Determination of pH Optimum of Isolated Laccase.
In the assay for determining optimal pH, two substrates, ABTS and hydroquinone, and a series of substrate solutions in buffers with different pH values were used. The assay buffers were prepared in 50 mM Na 2 HPO 4 -citric acid buffers (pH 2.4-8.0). The assay temperature was 30 • C.

Assay of Substrate Specificity of Isolated Laccase and Enzyme Kinetics.
To determine the substrate specificity of the purified laccase, several aromatic substrates (at 5.0 mM concentration) other than ABTS were used in the enzyme assay. The substrates tested comprised ABTS, catechol, hydroquinone, 2-methyl-catechol, N,N-dimethyl-1,4-phenylenediamine, pyrogallol, and tyrosine. Equal volumes of substrate solutions and buffers were mixed as the assay substrate solutions. The substrate oxidation rate was followed by measuring the change in absorbance using the molar extinction coefficient (e) obtained from the literature [5]. In the enzyme kinetics assay, the Michaelis-Menten constants (K m ) were determined at 30 • C using ABTS and hydroquinone dissolved in sodium acetate buffer (50 mM, pH 5.2) as substrates. The K m value was determined from a Lineweaver-Burk plot [2].

Effects of Metal Ions and Chemical Reagents on Enzyme
Activity of Isolated Laccase. The effects of different metal chlorides and chemical reagents were tested at the final concentrations of 1.25, 2.5, and 5.0 mM, respectively. The purified enzyme solution was preincubated with the metal ions or chemical reagents at 4 • C for 60 min before the standard laccase assay was performed. The control did not contain metal ions or chemical reagents.

Assay of Antiproliferative Activity and HIV-1 Reverse
Transcriptase Inhibitory Activity of Isolated Laccase. Antiproliferative activity of the purified laccase towards MCF 7 human breast cancer and Hep G2 hepatoma cell lines was determined using the MTT assay [14]. Inhibitory activity toward human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was determined by using the assay kit from the Boehringer-Mannheim (Germany) and following the standard protocol of Zhang et al. [2].

Isolation of Laccase.
A. placomyces laccase was purified with a protocol that entailed three consecutive steps of ion exchange chromatography and a single step of gel filtration, resulting in a 14.42% recovery of activity and a purification factor of 43.7-fold ( Table 1). The crude extract was resolved into three fractions: D1, D2, and D3 after elution with 0, 300, and 1000 mM NaCl, respectively, in NH 4 HCO 3 buffer  (10 mM, pH 9.4). Fraction D2 with laccase activity was further purified by adsorption on CM-cellulose and eluted as fraction C2 with 50 mM NaCl in the elution buffer (pH 5.2). Subsequently, fraction C2 was applied to Q-Sepharose and eluted with a linear gradient of 0-1 M NaCl. Laccase activity was concentrated in fraction Q2 (Table 1 and Figure 1(a)). Fraction Q2 was separated, upon FPLC on Superdex 75, into a large peak (SU1) with laccase activity and a small peak (SU2) devoid of laccase activity (Table 1 and Figure 1(b)).

Determination of Molecular Mass and N-Terminal
Sequence. Based on a comparison of its volume of elution from the Superdex 75 column with those of molecular mass standards, SU1 manifested a molecular mass of 68 kDa (data not shown). SDS-PAGE showed a single band with the same molecular mass (Figure 2). It indicated that the laccase is a monomeric protein with a molecular mass of 68 kDa. Its Nterminal amino acid sequence was DVIGPQAQVTLANQD (UniProt accession number: B3EWI3). A comparison of N-terminal amino acid sequence of A. placomyces laccase with other mushroom laccases is presented in Table 2.
The purified laccase showed little N-terminal amino acid sequence homology to other mushroom laccases.

Characteristics of A. placomyces
Laccase. An optimal temperature of 30 • C was required for the purified A. placomyces  laccase to express maximal activity. The activity underwent a sharp decline as the temperature was increased from 40 • C to 80 • C. Laccase activity vanished when the temperature reached 80 • C (Figure 3(a)). The purified enzyme was stable below 40 • C and a decline of laccase activity was noted, when the enzyme was incubated at 60 • C for 10-60 min. After incubation for one hour at 70 • C, about 20% of the maximal activity remained. When the laccase was preincubated at   80 • C for more than 20 min, it lost almost all the enzyme activity (Figure 3(b)).
The purified A. placomyces laccase exhibited optimal pH values of 5.2 and 6.8 towards ABTS and hydroquinone, respectively ( Figure 4). When the ambient pH was raised to 5.5 (towards ABTS) or 7.0 (towards hydroquinone), an abrupt decrement in enzyme activity took place. The degradative activity towards ABTS dropped further at pH 6.0 Journal of Biomedicine and Biotechnology and decreased to an undetectable level at pH 7.6. About 40% of total activity towards hydroquinone remained when the pH value increased to 8.0 (Figure 4). A. placomyces laccase oxidized a variety of substrates, including polyphenols (hydroquinone, pyrogallol, catechol), methoxy-substituted phenols (2-methylcatechol), aromatic diamines (N,N-dimethyl-1,4-phenylenediamine), and the nonphenolic heterocyclic compound ABTS. The highest degradative activity was demonstrated towards hydroquinone, 77% as much activity was observed towards ABTS, and 54% activity towards N,N-dimethyl-1,4phenylenediamine. Activity towards other substrates was considerably attenuated or indiscernible: 9% activity towards pyrogallol, 3% activity towards catechol, 1% activity towards 2-methyl-catechol, and undetectable activity towards tyrosine. The K m values at 30 • C and pH 5.2 towards hydroquinone and ABTS were 0.392 mM and 0.775 mM, respectively (using the Lineweaver-Burk plots, Figure 5).
The laccase activity was not significantly affected by the presence of cations such as K + , Ca 2+ , Mg 2+ , Mn 2+ , and Zn 2+ ions at the concentrations of 1.25-5.0 mM. However, the activity was reduced in the presence of Cu 2+ , Hg 2+ , Pb 2+ , and Fe 3+ ions and also EDTA. Al 3+ ions could enhance the enzyme activity by 14-57% at the concentrations of 1.25-2.5 mM (Table 3) A comparison of characteristics of laccases from A. bisporus [10], A. blazei [11], and A. placomyces (this study) is shown in Table 4.

A. bisporus [10]
A. blazei [11] A A. placomyces laccase demonstrates a low temperature optimum at 30 • C and a sharp decline in enzyme activity at temperatures higher than than 30 • C, thus the purified laccase is quite thermo sensitive. Similar research has been reported on the laccase from A. blazei, which is stable at 20 • C, but loses activity rapidly at 40 • C [11]. A. placomyces laccase has nearly 80% of total activity remaining after incubation for one hour at 60 • C, while A. blazei laccase loses 90% of total activity after incubation at 60 • C for half an hour. The laccase from A. bisporus remains completely stable at 40 • C and below for at least 24 h and undergoes a 50% loss of total activity after 10 min at 70 • C, 40 min at 60 • C, and 3 h at 50 • C [10]. On the contrary, many other laccases possess higher temperature optima, including A. biennis (60 • C) [5], Albatrellus dispansus (70 • C) [23], C. maxima (60 • C) [2], and Ganoderma lucidum (70 • C) [24].
In line with laccases reported in the literature, the isolated enzyme manifests degradation activities towards a variety of substrates including ABTS, aromatic diamines, and phenols. However, in contradistinction to many other laccases which prefer ABTS to hydroquinone, A. placomyces laccase expresses the highest activity towards hydroquinone (K m value = 0.392 mM), followed by ABTS with a K m value of 0.775 mM. A. biennis laccase manifests an activity towards hydroquinone which is only about 10% of that towards ABTS (K m = 33.5 μM) [5]. The degradative activity of C. maxima laccase towards various substrates differs as follows: activity towards ABTS (K m = 61.7 μM) > hydroquinone > N,Ndimethyl-1,4-phenylenediamine > pyrogallol > catechol > 2methylcatechol [2]. The laccase from A. blazei demonstrates K m values of 63 μM and 1.03 mM towards ABTS and 2,6dimethoxyphenol (DMP), respectively [11]. Based on the kinetics data, A. blazei laccase appears to be more sensitive towards ABTS than the laccase from A. placomyces.
Many previous studies indicate that laccases can be enhanced by low levels of Cu 2+ ions (0.5-3.5 mM). Addition of 1 mM Cu 2+ ions increases the activity of P. ostreatus laccase by eightfold [25]. Cu 2+ ions at 6.25 mM concentration bring about 11.7% augmentation in activity of A. biennis laccase [5]. In the present study, Cu 2+ ions in the concentration range of 1.25-5.0 mM reduce the enzyme activity of A. placomyces laccase by about 30-40%, reminiscent of the observation that the laccase from Fusarium solani with approximately 30% inhibition by Cu 2+ ions at 20 mM Journal of Biomedicine and Biotechnology 7 concentration [26]. On the other hand, the activity of C. maxima laccase is not significantly affected by Cu 2+ ions at the concentrations ranging from 6.25 mM to 50 mM [2].
The purified enzyme possesses potential application with inhibitory activities towards MCF 7 and Hep G2 tumor cells and HIV-1 RT. Many fungal lectins manifest antiproliferative activities towards tumor cells, but few laccases are reported to inhibit tumor cell proliferation [27]. Laccases from Hericium coralloides and shiitake mushroom are devoid of antiproliferative activities against Hep G2 or MCF 7 tumor cells at a concentration of 60 μM [12,28]. Among the laccases with antiproliferative activities towards tumor cells, A. placomyces lacase possesses lower IC 50 values. A. biennis laccase demonstrates antiproliferative activities against Hep G2 and MCF-7 cells with IC 50 values of 12.5 μM and 6.7 μM, respectively [5]. C. maxima laccase shows antiproliferative activity against Hep G2 and MCF-7 tumor cells with IC 50 values of 12.3 μM and 3.0 μM, respectively [2]. It indicates that the present lacccase shows potential applications in cancer treatments.
It is noteworthy that A. placomyces laccase exhibits antiproliferotive activity towards tumor cells and inhibitory activity toward HIV-1 RT, and manifests a slightly high pH optimum. It indicates that the enzyme has great potential for medical and industrial applications.