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
# 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
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
Fig. 2 – FPLC–gel filtration of fraction D3 on an FPLC
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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Googling Trends in Conservation Biology EL PROULX, PHILIPPE MASSICOTTE, AND MARC P´ Centre de recherche sur les interactions bassins versants—´ emes aquatiques (RIVE) and Groupe de recherche interuni- versitaire en limnologie (GRIL), Universit´ eres, C.P. 500, Trois-Rivi ebec, G9A 5H7, Canada, Abstract: Web-crawling approaches, that is, automated programs data mining the internet to obtain in-formation about a particular process, have recently been proposed for monitoring early signs of ecosystemdegradation or for establishing crop calendars. However, lack of a clear conceptual and methodologicalframework has prevented the development of such approaches within the field of conservation biology.Our objective was to illustrate how Google Trends, a freely accessible web-crawling engine, can be used totrack changes in timing of biological processes, spatial distribution of invasive species, and level of publicawareness about key conservation issues. Google Trends returns the number of internet searches that weremade for a keyword in a given region of the world over a defined period. Using data retrieved online for13 countries, we exemplify how Google Trends can be used to study the timing of biological processes, such asthe seasonal recurrence of pollen release or mosquito outbreaks across a latitudinal gradient. We mapped thespatial extent of results from Google Trends for 5 invasive species in the United States and found geographicpatterns in invasions that are consistent with their coarse-grained distribution at state levels. From 2004through 2012, Google Trends showed that the level of public interest and awareness about conservationissues related to ecosystem services, biodiversity, and climate change increased, decreased, and followed bothtrends, respectively. Finally, to further the development of research approaches at the interface of conservationbiology, collective knowledge, and environmental management, we developed an algorithm that allows therapid retrieval of Google Trends data.