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Doi:10.1016/j.peptides.2006.10.004

A peptide with HIV-1 reverse transcriptase inhibitoryactivity from the medicinal mushroom Russula paludosa Jianbin Wang H.X. Wang , T.B. Ng a Department of Chemistry, Peking University, Beijing 100080, Chinab State Key Laboratory of Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100094, Chinac Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Hot water extracts of 16 species of mushrooms, including both edible and medicinal Received 11 September 2006 mushrooms, were screened for human immunodeficiency virus (HIV)-1 reverse transcrip- Received in revised form tase (RT) inhibitory activity. Extracts of Lactarius camphoratus, Trametes suaveolens, Sparassis crispa, Pleurotus sajor-caju, Pleurotus pulmonarius, and Russula paludosa elicited over 50% Accepted 9 October 2006 inhibition when tested at the concentration of 1 mg/ml. The extract of R. paludosa demon- Published on line 17 November 2006 strated the highest inhibitory activity on HIV-1 RT (97.6%). Fraction SU2, purified fromR. paludosa extract by anion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75, exhibited potent inhibitory activity on HIV-1 RT. At the concentrations of 1 mg/ml, 0.2 mg/ml, and 0.04 mg/ml, the inhibition ratios were 99.2%, 89.3%, and 41.8%, respectively, giving an IC50 of 11 mM. The molecular mass of SU2 was 4.5 kDa and its Reverse transcriptase (RT) N-terminal amino acid sequence was determined to be KREHGQHCEF. The peptide was devoid of hemagglutinating, ribonuclease, antifungal, protease, protease inhibitory, and Edible and medicinal mushrooms laccase activities.
# 2006 Published by Elsevier Inc.
receptor inhibitors used to block and prevent HIV entry intocells, such as rsCD4, and sulfated glucans, (b) RT inhibitors, The causative agent of acquired immunodeficiency syndrome comprising nucleotide analogs, such as AZT, ddC, ddT, and (AIDS) is human immunodeficiency virus (HIV) The ddT, and non-nucleotide analogs such as ramifications of symptoms of people infected with HIV encompass immuno- thiazole, (c) integrase inhibitors , (d) viral accessorial deficiency, decline in ability to combat infections, opportu- protein inhibitors, (e) viral nucleoside inhibitors , (f) nistic infections, malignant tumors, and nerve handicaps protease inhibitors, (g) glycosidase inhibitors and (h) virus The distribution of AIDS is worldwide, and it assembly inhibitors .
has become one of the most difficult viral diseases to treat.
Currently the prevailing strategy for the therapy of AIDS is In the past 20 years, rapid progress has been made in combinative application of several therapeutic agents, that is, developing natural products and chemically synthesized reverse transcriptase inhibitors, such as nucleotides or non- compounds as anti-HIV drugs. Through HIV kinetics studies, nucleotides used in conjunction with protease inhibitors medicines have been developed that are targeted at many . The treatment can reduce viral number to such a stages, for example, the infection and replication of HIV. These low level that the virus cannot be detected in blood and drugs include the following: (a) soluble molecules of CD4, and lymphoid tissues. But the drugs have toxicity and are extremely * Corresponding author.
E-mail address: (T.B. Ng).
0196-9781/$ – see front matter # 2006 Published by Elsevier Inc.
expensive. Resistance of HIV against the drugs can develop (fraction D1), adsorbed materials (fractions D2 and D3) were easily. About 90% of the 40 million HIV-infected people in the eluted with a linear concentration gradient of NaCl (0–1 mol/l) in world cannot afford the substantial sum incurred in treatment.
10 mmol/l phosphate buffer (pH 7.5). The second adsorbed Thus, research and development of drugs with higher efficacy, fraction, fraction D3, was dialyzed and further fractionated lower toxicity, and reduced cost are in urgent need.
by FPLC on a Superdex 75 HR 10/30 column (Amersham Chinese medicine is a treasure of China and the world. It is Biosciences) in 200 mmol/l NH4HCO3 (pH 8.6). The Superdex one of the hottest pharmacological issues to screen anti-AIDS 75 column had previously been calibrated with molecular mass drugs from Chinese medicinal materials. Abundant resources markers including bovine serum albumin (67 kDa), ovalbumin exist in edible medicinal mushrooms which possess a multi- tude of biological activities The advan- (13.7 kDa), and cytidine (0.246 kDa). The eluate was monitored tages of screening HIV-1 RT inhibitors from edible medicinal for HIV-1 RT inhibitory activity .
fungi are as follows: (i) abundant resources. There are about10,000 species of fungi which can form large fruiting bodies.
Assay of HIV-1 RT inhibitory activity of mushroom Among them, more than 2000 species are edible medicinal extract and peptide mushrooms. The wild fungi can be collected, some of them canbe cultivated, and the majority of the species can be cultured by The assay was performed as described in the protocol included large-scale fermentation to yield mycelia. (ii) Lower toxicity and with the kit (HIV-1 RT assay kit, Boehringer Mannheim, side effects. The use of edible medicinal fungi has a long Germany). The extract from a given mushroom species was history. Generally speaking, natural products extracted from dissolved in distilled water at the concentration of 4 mg/ml. RT edible medicinal fungi exhibit lower toxicity and fewer side solution (20 ml) was added to 20 ml test sample, and 20 ml lysis effects than chemical drugs. The natural products can be taken buffer. Then 20 ml solution of the reaction mixture was added over a prolonged period as medicine, or consumed as tonics by after 10 min to each well of a 96-well plate of the assay kit. The healthy people. (iii) More affordable prices. Because of the plate was incubated at 37 8C for 1 h. Washing buffer (250 ml) abundant resources and ready availability of most of the edible was added to each well and the wells were washed five times.
medicinal fungi, natural products extracted from these Antibody (anti-DIG-POD) (200 ml) was added to each well, materials could be produced at a lower cost than those in followed by incubation at 37 8C for 1 h. The wells were then clinical use. It means that more patients could be treated. RT is washed again. In the final step, the peroxidase substrate, one of the key enzymes in HIV replication. HIV replication 200 ml ABTS solution, was added. The plate was left at room would be interfered with if the enzyme is inhibited. So RT temperature for about 30 min. The absorbance of the sample inhibitors can be used to treat AIDS at 405 nm was determined using a microtiter plate reader andis directly correlated to the level of RT activity. The inhibitoryactivity of the purified peptide was calculated as percent Materials and methods inhibition as compared to a control without the purifiedpeptide Edible medicinal mushrooms Molecular mass determination by sodium dodecyl Tricholoma robostum, Morchella esculenta, Pholiota adiposa, Schi- sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by zophyllum commune, Sparassis crispa, Lentinus tigrinus, Trametes FPLC–gel filtration suaveloens, Grifola umbellatum, Pleurotus nebrodensis, Pleurotuspulmonarius, Pleurotus sajor-caju, Russula paludosa, and Lactarius SDS-PAGE was carried out in accordance with the procedure of camphorates were gifts from the Laboratory of Edible and Nielsen and Reynolds using a 12% resolving gel and a 5% Medicinal Fungi at China Agricultural University.
stacking gel. At the end of electrophoresis, the gel was stainedwith Coomassie brilliant blue. FPLC–gel filtration was carried Extraction of mushroom fruiting bodies out using a Superdex 75 column and also on a Superdexpeptide column which had been calibrated with molecular Dried fruiting bodies of edible or medicinal mushrooms (5 g) mass standards (Amersham Biosciences).
were homogenized in distilled water and then extracted withdistilled water at 95 8C for 6 h. The mixture was centrifuged at N-terminal amino acid sequence analysis 4000  g for 10 min, and the supernatant was collected. Threevolumes of 95% ethanol were added, and the mixture was The N-terminal amino acid sequence of R. paludosa peptide centrifuged again. The precipitate was stored at 60 8C. The was analysed by means of automated Edman degradation.
dried samples were powdered for the next step.
Microsequencing was carried out using a Hewlett Packard1000A protein sequencer equipped with an HPLC system Purification of R. paludosa peptide Assay of HIV-1 integrase inhibitory activity A DEAE-cellulose (Sigma) column (1.5 cm  20 cm) which hadpreviously been equilibrated with 10 mmol/l phosphate buffer Expression and purification of recombinant HIV-1 (pH 7.5) was used. The extract, prepared from 50 g dried fruiting bodies and dissolved in the same buffer, was chromatographed The plasmid used, pT7-7-His(YjTX)-HIV-1-IN, expressed His- on the column. After elution of inactive unadsorbed materials tagged wild-type HIV-1 integrase. To express the protein, a 1 l culture of E. coli BL21(DE3) cells containing the expression Assay of laccase activity plasmid was grown at 37 8C until OD600 reached 0.7–0.8. Cellswere induced by addition of 0.8 mM IPTG and harvested after a The isolated peptide was assayed for this activity in view of a 4 h incubation by centrifugation at 6000  g for 10 min at 4 8C.
report that some laccases possessed HIV-1 reverse transcrip- Cells were suspended at a concentration of 10 ml/g wet cell tase inhibitory activity . Laccase activity was assayed by paste in 20 mM Tris–HCl (pH 8.0), containing 0.1 mM EDTA, measuring the oxidation of 2,70-azinobis[3-ethylbenzothiazo- 2 mM b-mercaptoethanol, 0.5 M NaCl, and 5 mM imidazole.
lone-6-sulfonic acid]diammonium salt (ABTS). A modification Lysozyme was added to a concentration of 0.2 mg/ml. After 1 h of the method of Ng and Wang was used. An aliquot of a incubation at 4 8C, the lysate was sonicated and centrifuged at solution of the peptide was incubated in 1.3 ml of 67 mM 40,000  g at 4 8C for 20 min. The pellet was homogenized in sodium acetate buffer (pH 4.5) containing 1.54 mm ABTS at 50 ml buffer A (20 mM Tris–HCl, pH 8.0, 2 M NaCl, 2 mM b- 30 8C. One unit of enzyme activity was defined as the amount mercaptoethanol) containing 5 mM imidazole. The suspen- of enzyme required to produce an absorbance increase at sion was rotated at 4 8C for 1 h and cleared by centrifugation at 405 nM of 1 min1 ml1 of reaction mixture under the 40,000  g at 4 8C for 20 min. The supernatant was loaded onto a 1 ml chelating Sepharose column charged with 50 mMimidazole. The column was washed with five column volumes Assay of ribonuclease activity of buffer A containing 5 mM imidazole and the protein waseluted with three column volumes of buffer A containing Some ribonucleases exhibited HIV-1 reverse transcriptase 200 mM and 400 mM imidazole, respectively. Protein-contain- inhibitory activity Hence the activity of the purified ing fractions were pooled and EDTA was added to a final peptide toward yeast tRNA (Sigma) was assayed by determin- concentration of 5 mM. The protein was dialyzed against ing the generation of acid-soluble, UV-absorbing species with buffer B (20 mM HEPES, pH 7.5, 1 mM EDTA, 1 M NaCl, 20% the method of Wang and Ng The peptide was glycerol) containing 2 mM b-mercaptoethanol and then incubated with 200 mg tRNA in 150 ml of 100 mM MES buffer against buffer B containing 1 mM dithiothreitol. Aliquots of (pH 5) at 37 8C for 15 min. The reaction was terminated by the protein were stored at 70 8C introduction of 350 ml of ice-cold 3.4% perchloric acid. Afterleaving on ice for 15 min, the mixture was centrifuged HIV-1 integrase assays (15,000  g, 15 min) at 4 8C. The OD260 of the supernatant A non-radioactive ELISA-based HIV-1 integrase assay was was read after appropriate dilution. One unit of enzymatic performed according to the DNA-coated plates method as activity is defined as the amount of enzyme that brings about detailed by Ng et al. In this study, 1 mg of Smal-linearized p an increase in OD260 of 1 min1 in the acid-soluble fraction per Bluescript SK was coated onto each well in the presence of 2 M milliliter of reaction mixture under the specified condition.
NaCl as target DNA. The donor DNA was prepared by annealingVU5BR (50-biotin-GTGTGGAAAATCTCTAGCAGT-30) and VU5 Assay of antifungal activity (50-ACTGCTAGAGATTTTCCACAC-30) in 10 mM Tris–HCl, pH8.0, 1 mM EDTA, and 0.1 M NaCl at 80 8C followed by 30 min at Some antifungal peptides inhibited HIV-1 reverse transcrip- room temperature. Integrase reaction was performed in 20 mM tase . The assay of the purified peptide for antifungal HEPES (pH 7.5), containing 10 mM MnCl2, 30 mM NaCl, 10 mM activity toward Mycosphaerella arachidicola and Physalospora dithiothreitol, and 0.05% Nonidet-P40 (Sigma). After the piricola was carried out in 100 mm  15 mm petri plates integrase reaction, the biotinylated DNA immobilized on the containing 10 ml of potato dextrose agar. After the mycelial wells was detected by incubation with streptavidin-conjugated colony had developed, sterile blank paper disks (0.625 cm in alkaline phosphatase (Boehringer Mannheim) followed by diameter) were placed at a distance of 0.5 cm away from the colorimetric detection with 1 mg/ml p-nitrophenyl phosphate rim of the mycelial colony. An aliquot (15 ml) of a solution of in 10% diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl2.
the purified peptide was added to a disk. The plates were The absorbance due to the alkaline phosphatase reaction was incubated at 23 8C for 72 h until mycelial growth had measured at 415 nm enveloped the disks containing the control and had formedcrescents of inhibition around disks containing samples with Assay of hemagglutinating activity antifungal activity .
The isolated peptide was assayed for lectin (hemagglutinating) Assay of protease activity activity in view of the report that some lectins exhibited HIV-1reverse transcriptase inhibitory activity A serial two-fold A solution of casein, which was used as substrate in the dilution of a solution of R. paludosa peptide in microtiter U-plates protease assay, was freshly prepared as follows. To 0.1 g (50 ml) was mixed with 50 ml of a 2% suspension of rabbit red casein 10 ml 200 mM phosphate buffer (pH 7.5) were added.
blood cells in phosphate buffered saline (pH 7.2) at 20 8C. The Subsequently, the solution was heated at 60 8C for 30 min. The results were read after about 1 h when erythrocytes in the blank precipitate was removed and the resulting solution could be had fully sedimented and formed a dot at the bottom of the well.
used. The test sample or trypsin solution (25 ml) was mixed The hemagglutination titer, defined as the reciprocal of the with 140 ml of the above casein solution and the reaction highest dilution exhibiting hemagglutination, was reckoned as mixture was incubated at 37 8C for 15 min. Subsequently, one hemagglutination unit. Specific activity is the number of 600 ml 5% trichloroacetic acid (TCA) was added. The reaction hemagglutination units per milligram of protein .
mixture was allowed to stand at room temperature for 30 min

before centrifugation at 8000  g for 15 min. The absorbance ofthe supernatant was read at 280 nm against water as blank.
Protease activity was calculated based on the activity oftrypsin (7900 BAEE units/mg according to Sigma) in theprotease assay using casein as substrate .
Assay of protease inhibitory activity Some protease inhibitors inhibit HIV-1 reverse transcriptaseThus, the isolated peptide was assays for proteaseinhibitory activity. The assay was similar to the assay ofprotease activity except for the use of trypsin as the protease.
The activity of trypsin toward casein was determined in the Fig. 1 – Ion exchange chromatography of R. paludosa extract presence and also in the absence of the isolated peptide.
on a DEAE-cellulose column (1.5 cm  20 cm) in 10 mmol/lphosphate buffer (pH 7.5). The slanting broken line acrossthe chromatogram represents the linear concentration (0–1 mol/l) gradient of NaCl used to elute the adsorbedpeaks. Buffer flow rate: 2 ml/min; fraction size: 6 ml/tube.
Extraction of mushrooms fruiting bodies Extracts were prepared from 16 mushrooms according to thefollowing steps: hot water extraction, centrifugation, ethanol Purification and characterization of R. paludosa precipitation, centrifugation, precipitate drying, and homo- The extract of R. paludosa exhibited strong absorption at HIV-1 RT inhibition activity of mushroom extracts 280 nm and was selected for fractionation because it showedthe most potent HIV-1 RT inhibitory activity. DEAE-cellulose The HIV-1 RT inhibition ratio of samples from 16 edible was employed to purify a peptide from the extract. Three medicinal mushrooms ranged from 9.2% to 97.6% when the absorbance peaks, designated as D1, D2, and D3, respectively, extracts were tested at the concentration of 1 mg/ml ().
were obtained (). Only D3 distinctly showed HIV-1 RT The inhibition ratio of the following five extracts was above inhibitory activity, At the concentrations of 1 mg/ml, 0.2 mg/ 50%: Lactarius camphorates, Rametes suaveloens, S. crispa, P. sajor- ml, and 0.04 mg/ml, it inhibited 98.4%, 75.2%, and 28.4% of HIV- caju, P. pulmonarius, and R. paludosa. Peptide from R. paludosa 1 RT activity, respectively. The IC50 value of D3 was 0.086 mg/ demonstrated the highest inhibition ratio of 97.6% at 1 mg/ml.
When the concentration was reduced to 0.2 mg/ml and Fraction D3 was subjected for further purification by gel 0.04 mg/ml, the inhibition ratios dropped to 42.6% and filtration on an FPLC-Superdex 75 column. Two peaks, SU1 and 14.3%, respectively. The IC50 value was 0.25 mg/ml.
SU2, resulted (SU2, with a molecular mass of 4.5 kDa,demonstrated potent HIV-1 RT inhibitory activity. At theconcentrations of 1 mg/ml, 0.2 mg/ml, and 0.04 mg/ml, itinhibited 99.2%, 89.3%, and 41.8% of HIV-1 RT activity, Table 1 – HIV-1 RT inhibitory activity of extracts from 16edible medicinal mushrooms when tested at 1 mg/ml Grifola umbellate Lactarius camphorates Pleurotus eryngii Pleurotus nebrodensis Polyporus umbellatus Rubella esculenta Fig. 2 – FPLC–gel filtration of fraction D3 on an FPLC Tricholoma gambosay Superdex 75 HR 10/30 column. Buffer: 200 mmol/lNH Bold value represents highest inhibitory activity.
4HCO3 (pH 8.6); flow rate: 0.4 ml/min; fraction size:

and laccases of mushroom origin are known to inhibitHIV-1 reverse transcriptase. It is noteworthy that the peptideisolated in the present study does not belong to any of theseclasses of these proteins because it does not possess any of thebioactivity specific to these proteins. In fact its N-terminalsequence does not bear any resemblance to publishedsequences. Its HIV-1 RT inhibitory potency is within the rangeof potencies exhibited by other anti-HIV natural products It exerts its inhibitory action probably by protein-proteininteraction, just like the inhibition of HIV-1 RT by HIV-1protease [ ]. It is noteworthy that it does not inhibit HIV-1integrase. The lack of other biological activities in the isolatedpeptide indicates that it is a new type of peptide.
We thank the Medicine Panel of CUHK Research Committeefor award of a direct grant and Ms. Fion Yung for secretarialassistance.
Fig. 3 – SDS-PAGE of R. paludosa peptide. Left lane:R. paludosa peptide; right lane: molecular mass markers.
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