Hindawi Publishing Corporation

BioMed Research International

Volume 2014, Article ID 340467, 7 pages
http://dx.doi.org/10.1155/2014/340467

Research Article
A Novel Hemagglutinin with Antiproliferative
Activity against Tumor Cells from the Hallucinogenic
Mushroom Boletus speciosus

Jian Sun,1,2 Tzi-Bun Ng,3 Hexiang Wang,2 and Guoqing Zhang1

1
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Sciences and Engineering, Beijing
University of Agriculture, Beijing 102206, China
2
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China
3
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Correspondence should be addressed to Hexiang Wang; hxwang@cau.edu.cn and Guoqing Zhang; zhanggq1001@gmail.com

Received 28 January 2014; Revised 30 March 2014; Accepted 29 April 2014; Published 29 May 2014

Academic Editor: Marcelo A. Soares

Copyright © 2014 Jian Sun et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Little was known about bioactive compounds from the hallucinogenic mushroom Boletus speciosus. In the present study, a
hemagglutinin (BSH, B. speciosus hemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also
tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange
chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75.
The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to
60∘ C, while there was a precipitous drop in activity when the temperature was elevated to 70∘ C. BSH retained 25% hemagglutinating
activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg2+ and slightly inhibited
by Fe2+ , Ca2+ , and Pb2+ . None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human
erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards
hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210) in vitro, with IC50 of 4.7 𝜇M and 7.0 𝜇M, respectively. It also
exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 7.1 𝜇M.

1. Introduction on parasitic infections, and molecular recognition during the

early stages of mycorrhization [3].
Lectins and hemagglutinins are nonimmune proteins or Mushrooms of the Boletus genus have been known since
glycoproteins that exhibit specific binding to carbohydrates ancient times for their delicious taste as edible wild food.
on the cell surface. They can agglutinate cells through sugar- However, some of them are toxic. There are only several
specific binding sites for polysaccharides and glycoconjugates reports about bioactive substance such as lectins [6, 7],
[1]. Lectins were first identified in plants and are now known ribonucleases [8], and toxic proteins [9] from this genus.
to be distributed widely throughout the nature, including Boletus speciosus is a rare wild hallucinogenic mushroom
plants, animals, fungi, bacteria, and viruses [2]. In plants, and can cause “lilliputian hallucination” when cooked in a
they are mainly storage proteins and serve in defence against wrong way or eaten too much. The stipe of the mushroom
insects or fungi [3]. Lectins or hemagglutinins are impor- is yellow and becomes blue quickly after bruise. That is why
tant receptors in animals, such as human C-type lectin locals of Southwest China call the mushroom “Jian Shou
langerin [4] and rat liver asialoglycoprotein receptor [5]. Qing” which means the mushroom changes to blue when
In mushrooms, lectins play a pivotal role in dormancy, touched. The toxicity of the mushroom is neuropsychiatric,
growth, morphogenesis, morphological changes consequent and the patient can see little joyous creatures jumping and
2 BioMed Research International

dancing around. The toxin was defined as hallucinogens but 2.3. Assay for Hemagglutinating Activity. A serial twofold
not isolated up to now [10]. On the other hand, the mushroom dilution of the hemagglutinin solution in microtiter U-plates
is considered as a delicious mushroom by local people (25 𝜇L) was mixed with 25 𝜇L of a 2% suspension of rabbit red
since the poisoning symptoms vanished after being boiled erythrocytes in phosphate-buffered saline (pH 7.2) at room
up. temperature. The results were recorded after approximately
There is dearth information pertaining to bioactive sub- half an hour when full sedimentation was observed in the
stances of B. speciosus. The present investigation aimed to blank. The hemagglutination titer, defined as the reciprocal
isolate and characterize a hemagglutinin from the fruiting of the highest dilution exhibiting hemagglutination, was
body of B. speciosus and compare its characteristics with other regarded as one hemagglutination unit. Specific activity is
known lectins or hemagglutinins from Boletus genus such as the number of hemagglutination units/mg protein [2]. All
Boletus edulis lectin [11]. determinations were performed in triplicate.
The hemagglutinating inhibition tests to investigate the
inhibitory activities of various carbohydrates towards the
2. Materials and Methods hemagglutinin were performed in a manner analogous to
the hemagglutination test. Serial twofold dilutions of sugar
2.1. Isolation of Hemagglutinin. Ion exchange chromatogra-
samples (200 mM to 0.195 mM) were prepared in phosphate-
phy of dried B. speciosus fruiting bodies (20 g) was carried out
buffered saline. All of the dilutions were mixed with an
using a 10 × 100 cm column of Q-Sepharose (GE Healthcare).
equal volume (25 𝜇L) of a solution of the hemagglutinin
The extract was first homogenized in 0.15 M NaCl (10 mL per
with 32 hemagglutination units. The mixture was allowed
gram) and extracted overnight at 4∘ C. Then the homogenate
to stand for 30 minutes at room temperature and then
was centrifuged at 12,000 rpm for 30 minutes at 4∘ C. The
mixed with 50 𝜇L of a 2% rabbit erythrocyte suspension.
supernatant was collected and (NH4 )2 SO4 was added to the
The minimum concentration of the sugar in the final reac-
supernatant until 80% saturation is reached. The mixture was
tion mixture, which completely inhibited 32 hemaggluti-
left at 4∘ C for 8 hours before centrifugation at 10,000 rpm
nation units of the protein preparation, was calculated.
for 25 min. The precipitate was redissolved and dialyzed to
The sugars tested included inositol, D-melibiose, D(−)-
remove (NH4 )2 SO4 before applying to a Q-Sepharose column
fructose, L(+)-rhamnose, sorbose, D(+)-galactose, D(+)-
in 10 mM Tris-HCl buffer (pH 8.0). After removal of the
mannose, 𝛼-lactose, D(+)-xylose, L(+)-arabinose, D-glucose,
unadsorbed fraction Q1, adsorbed proteins were desorbed
maltose, raffinose, cellobiose, inulin, p-nitrophenyl-𝛼-D-
stepwise with 50 mM NaCl in the Tris-HCl buffer to yield
glucopyranoside, p-nitrophenyl-𝛽-D-glucopyranoside, and
fraction Q2 and then with 300 mM and 1 M NaCl in the Tris-
inositol.
HCl buffer to yield fractions Q3 and Q4. Fraction Q3 with
The effects of temperature, NaOH solution, HCl solution,
hemagglutinating activity was then subjected to ion exchange
and solutions of metallic chlorides (including those of Fe2+ ,
chromatography on a 2.5 × 30 cm column of CM-Cellulose
(Sigma) in 10 mM sodium acetate-acetic acid buffer (NaAc- K+ , Ca2+ , Cd2+ , Cu2+ , Hg2+ , Mg2+ , Mn2+ , Pb2+ , Zn2+ , Al3+ ,
HAc, pH 5.2). After removal of the unadsorbed fraction C1 and Fe3+ ) on hemagglutinating activity of the protein were
with the same buffer, adsorbed proteins were eluted with examined as previously described [2].
150 mM NaCl in the NaAc-HAc buffer to yield fraction
C2 and then with 300 mM and 1 M NaCl in the NaAc-
HAc buffer to yield fractions C3 and C4. Fraction C3 with 2.4. Assay of Antiproliferative Activity on Tumor Cell Lines.
hemagglutinating activity was then subjected to ion exchange The tumor cell lines Hep G2 (hepatoma) and L1210
chromatography on a 1.0 × 10 cm column of SP-Sepharose (leukemia) were purchased from American Type Culture
(GE Healthcare) in 10 mM NaAc-HAc buffer (pH 5.2). After Collection (ATCC). The cell lines were maintained in Dul-
removal of the unadsorbed fraction S1 with the same buffer, becco modified Eagle’s medium (DMEM) supplemented with
adsorbed proteins were eluted with a linear 0–0.5 M NaCl 10% fetal bovine serum (FBS), 100 mg/L streptomycin, and
gradient in the NaAc-HAc buffer. The adsorbed fraction 100 IU/mL penicillin at 37∘ C in a humidified atmosphere of
S3 was further purified by gel filtration on a fast protein 5% CO2 . Cells (1 × 104 ) in their exponential growth phase
liquid chromatography Superdex 75 10/300 GL column (GE were seeded into each well of a 96-well culture plate (Nunc,
Healthcare). Denmark) and incubated for 3 hours before addition of the
hemagglutinin. Incubation was carried out for another 48
hours. Radioactive precursor, 1 𝜇Ci, ([3 H-methyl] thymidine,
2.2. Determination of Molecular Weight and N-Terminal from GE Healthcare) was then added to each well and
Sequence. The molecular weight of the active fraction (SU1) incubated for 6 hours. The cultures were then harvested by
was subsequently determined by sodium dodecyl sulfate- using a cell harvester. The incorporated radioactivity was
polyacrylamide gel electrophoresis (SDS-PAGE) in accor- determined by liquid scintillation counting [14].
dance with the procedure of Laemmli and Favre [12]. Molec-
ular weight determination was also performed by using
FPLC-gel filtration as described above. N-terminal sequence 2.5. Assay for HIV-1 Reverse Transcriptase (HIV-1 RT)
determination of the protein was carried out using an HP Inhibitory Activity. The assay for HIV-1 RT inhibitory activity
G-1000A Edman degradation unit and an HP 1000 HPLC was assessed by using an enzyme-linked immunosorbent
system [13]. assay (ELISA) kit from Boehringer Mannheim (Germany).
BioMed Research International 3

Table 1: Yields and specific hemagglutinating activities of various chromatographic fractions at different stages of B. speciosus hemagglutinin
(BSH) purification.

Fraction Yield (mg) Specific activity (U/mg) Total activity (U) Recovery of activity (%) Folds of purification
Extract 1496.6 5.3 7904.0 100.0 1.0
Q3 176.4 30.4 5299.2 67.0 5.7
C3 35.9 88.7 3187.2 40.3 16.8
S3 10.5 193.0 2035.2 25.8 36.6
SU1 5.7 220.2 1255.1 15.9 41.7

Marker

94 k
67 k

0.3 43 k
SU1

30 k
0.2
A 280 nm

SU2

20 k
0.1

14.4 k
0
0 4 8 12 16 20 24
Elution volume (mL)
(a) (b)

Figure 1: (a) Gel filtration of fraction on Superdex 75 HR 10/30 column by FPLC. Fraction SU1 represents purified hemagglutinin BSH. (b)
SDS-PAGE of purified BSH. Right lane: molecular weight markers, from top downwards, phosphorylase b (94 kDa), bovine serum albumin
(67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and lactalbumin (14.4 kDa).

The assay takes advantage of the ability of reverse transcrip- 2.6. Assay for Antifungal Activity. The assay for antifungal
tase to synthesize DNA, starting from the template/primer activity towards the phytopathogenic fungi Fusarium oxyspo-
hybrid poly(A) oligo(dT)15 . The digoxigenin- and biotin- rum, Rhizoctonia cerealis, Rhizoctonia solani, and Sclerotinia
labeled nucleotides in an optimized ratio are incorporated sclerotiorum was carried out in 100 × 15 mm petri dishes
into one of the same DNA molecules, which is freshly containing 10 mL of potato dextrose agar (PDA). After the
synthesized by the RT. The detection and quantification mycelial colony had developed, sterile blank paper disks
of synthesized DNA as a parameter for RT activity follow (0.625 cm in diameter) were placed at a distance of 0.5 cm
sandwich ELISA protocol. Biotin-labeled DNA binds to the away from the rim of the mycelial colony. An aliquot (15 𝜇L)
surface of microtiter plate modules that have been precoated of the hemagglutinin was added to a disk. The dishes were
with streptavidin. An antibody to digoxigenin, conjugated incubated at 23∘ C for 72 hours until mycelial growth had
to peroxidase (anti-DIG-POD), subsequently binds to the enveloped the disks containing the control and had formed
digoxigenin-labeled DNA. Finally, the peroxidase substrate crescents of inhibition around disks containing samples with
is added. The peroxidase enzymes catalyze the cleavage of antifungal activity [13].
the substrate and produce a colored reaction product. The
adsorbance of the samples at 405 nm can be determined 3. Results
by using a microtiter plate (ELISA) reader and is directly
correlated to the level of RT activity. A fixed amount (4– 3.1. Purification of BSH. The precipitate from ammonium
6 ng) of recombinant HIV-1 RT was used. The inhibitory sulfate precipitation was redissolved and dialyzed as crude
activity of the hemagglutinin was calculated as percentage protein extract. Then it was applied to an ion exchange
inhibition as compared to a control without the protein chromatography Q-Sepharose column. One of the adsorbed
[14]. fractions, fraction Q3, showed hemagglutinating activity
4 BioMed Research International

Table 2: Hemagglutinating activity of BSH on rabbit and human erythrocytes.

Human erythrocytes
Rabbit erythrocytes
A B O AB
Hemagglutinating activity (%) 100 50 50 25 50
Note: initial hemagglutinating activity was 32 U.

Table 3: Effect of temperature and pH values on hemagglutinating activity of BSH.

Temperature (∘ C) 30 40 50 60 70 80
Residual hemagglutinating activity (%) 100 100 100 100 12.5 0
pH values 2.2 1.9 1.6 1.3 1.0 0.7
Residual hemagglutinating activity (%) 100 50 25 0 0 0
pH values 11.8 12.1 12.4 12.7 13.0 13.3
Residual hemagglutinating activity (%) 100 100 50 50 25 0
Note: initial hemagglutinating activity was 32 U.

(Table 1). Subsequently, the active fraction Q3 was applied Table 4: Effects of cations on hemagglutinating activity of BSH.
to CM-Cellulose and yielded an unadsorbed fraction C1 and
three adsorbed fractions C2, C3, and C4. Hemagglutinating Cation (mM) 10 5 2.5 1.25
activity resided in fraction C3 (Table 1). Upon ion exchange Hg2+ 0 0 0 2
chromatography on SP-Sepharose, fraction C3 was resolved Fe2+ 8 16 32 32
into a large unadsorbed fraction S1 and two smaller adsorbed Ca2+ 16 16 32 32
fractions (S2 and S3) of approximately the same size. Activity Pb2+ 16 16 16 32
resided in fraction S3. Upon gel filtration on Superdex 75, K+ 32 32 32 32
S3 was resolved into a larger peak SU1 (with a molecular Cd2+ 32 32 32 32
mass of 31 kDa) and a smaller peak SU2 (Figure 1(a)). Hemag- Cu2+ 32 32 32 32
glutinating activity was enriched in SU1 which appeared as Mg2+ 32 32 32 32
a single band with a molecular mass of 15.5 kDa in SDS-
Mn2+ 32 32 32 32
PAGE (Figure 1(b)). About 40-fold purification was achieved
(Table 1). Zn2+ 32 32 32 32
Al3+ 32 32 32 32
Fe3+ 32 32 32 32
3.2. Biological Activities of BSH. The interactions of lectins or Note: initial hemagglutinating activity was 32 U.
hemagglutinins with cells can, in many instances, be inhibited
specifically by simple sugars. However, BSH showed no speci-
ficity towards the various carbohydrates tested, including
inositol, O-nitrophenyl-b-D-galactopyranoside, L-sorbose, BSH showed antiproliferative activity towards human
raffinose, L-rhamnose, D-fructose, D-mannose, cellobiose, hepatoma Hep G2 cells and mouse lymphocytic leukemia
L-arabinose, D-xylose, D-melibiose, lactose, inulin, maltose, cells (L1210) with IC50 of 4.7 𝜇M and 7.0 𝜇M, respectively
D-galactose, and D-glucose. It is suggested that the protein is (Figure 2). It exhibited HIV-1 reverse transcriptase inhibitory
a hemagglutinin but not a lectin. Its lower activities on human activity with an IC50 of 7.1 𝜇M (Figure 3). No inhibitory
type O erythrocytes compared with type A, type B, and type activity against the test with four phytopathogenic fungi was
AB indicate the binding is related to some ligand which has found when the hemagglutinin amount was up to 2.5 mg
different distribution to blood types (Table 2). (data not shown).
The activity of BSH was stable up to 60∘ C followed by
a precipitous decline from 64 to 8 hemagglutination units
when the temperature was raised to 70∘ C. At and above
4. Discussion
80∘ C activity was undetectable (Table 3). The hemagglutinin Although mushroom species from genus Boletus are delicious
was stable in 6 mM HCl (pH 2.2) and 12.5 mM NaOH (pH (e.g., B. edulis), some of them are toxic or even deadly. B.
12.1), while it was reduced by 75% in 25 mM HCl (pH 1.3) magnificus and B. speciosus cause hallucinogenic toxins and
and 100 mM NaOH (pH 13.0). The activity disappeared in B. erythropus and B. luridus manifest insecticidal activities,
50 mM HCl (pH 1.9) and 200 mM NaOH (pH 13.3) (Table 3). while B. satanas causes deadly liver damage, gastrointestinal
The hemagglutinating activity was unaffected in the presence upset, and hemolysis [10, 15]. Very little is known about mush-
of K+ , Cd2+ , Cu2+ , Mg2+ , Mn2+ , Zn2+ , Al3+ , and Fe3+ ions room lectins or hemagglutinins from the genus Boletus, since
(1.25–10 mM) but was reduced by Fe2+ (5–10 mM), Ca2+ (5– they are rare in nature and cannot yet be artificially cultured.
10 mM), and Pb2+ (2.5–10 mM) and potently reduced by Hg2+ B. speciosus hemagglutinin (BSH) is compared herein with
(1.25–10 mM) ions (Table 4). a lectin from another Boletus species, B. edulis lectin [11]
BioMed Research International 5

Table 5: Comparison of characteristics of BSH with B. edulis lectin.

Characteristics Boletus speciosus hemagglutinin B. edulis lectin [11]

Chromatographic behavior
DEAE ion exchanger Unadsorbed Unadsorbed
Q ion exchanger Adsorbed NA
SP ion exchanger Adsorbed NA
CM ion exchanger Adsorbed Adsorbed
Molecular mass (kDa) 31 32.6
Subunit molecular mass (kDa) 15.5 16.3
N-terminal amino acid sequence ANVKIVK TYGIALRV
Thermostability 60∘ C 60∘ C
pH stability <12.5 mM NaOH; <6.25 mM HCl <25 mM NaOH; <25 mM HCl
Sugar specificity Unknown Xylose, melibiose
Inhibited by Hg2+ , Fe2+ , Ca2+ , inhibited by Mg2+ , Mn2+ , and
Effect of cations on
and Pb2+𝑖 ions; no augmentation Zn2+ ions; augmented by Fe3+
hemagglutinating activity
observed and Al3+ ions
Antifungal activity Undetectable Undetectable
Antiproliferative activity IC50 of 4.7–7.0 𝜇M NA
HIV-1 RT inhibitory activity IC50 of 7.1 𝜇M IC50 of 14.3 𝜇M
NA: not available.

100 100
h
e
g
Inhibition of [3 H-methyl ] thymidine

80 e 80
Inhibition rate (%)

d
60
incorporation (%)

60 f
d
c
40
40
c
c b
20
20 b
a
0
a 0 5 10 15 20
0
0 5 10 15 20 Lectin concentration (𝜇M)
Lectin concentration (𝜇M) HIV-RT
HepG2
Figure 3: Inhibitory activity of BSH towards HIV-1 reverse tran-
L1210
scriptase. It exhibited HIV-1 RT inhibitory activity with an IC50
Figure 2: Antiproliferative activities of BSH towards Hep G2 and of 7.1 𝜇M. Each value in both panels represents the means ± SD
L1210 cell lines in vitro. The IC50 values towards Hep G2 and L1210 (𝑛 = 3). Different letters (a, b, c,. . .) next to the data points indicate
cells were 4.7 𝜇M and 7.0 𝜇M, respectively. Each value in both panels statistically significant differences (𝑃 < 0.05) when the data were
represents the means ± SD (𝑛 = 3). Different letters (a, b, c,. . .) next analyzed by analysis of variance followed by Duncan’s multiple range
to the data points indicate statistically significant differences (𝑃 < tests.
0.05) when the data were analyzed by analysis of variance followed
by Duncan’s multiple range tests.

dimeric lectins from Agaricus edulis [18], Volvariella volvacea

[19], Xerocomus spadiceus [20], and Lactarius flavidulus [17].
(Table 5). The two blood cell hemagglutinating proteins Regarding thermostability, they can withstand temperatures
share similar chromatographic behavior on cationic and no higher than 60∘ C. Nevertheless, they are more ther-
anionic exchangers; both are adsorbed on CM-Cellulose and mostable than lectins from some other mushrooms, like
unadsorbed on DEAE-Cellulose. They are distinct from some Pleurotus ostreatus [21], Schizophyllum commune [22], and L.
other mushroom lectins which are adsorbed on both chro- flavidulus [17], which are unstable above 40∘ C. None of these
matographic media [16, 17]. Their molecular mass is around mushroom lectins showed antifungal activity. However, BSH
32 kDa. They are both dimeric, which is in line with reports on showed a lower pH stability and a higher HIV-1 RT inhibitory
6 BioMed Research International

activity (IC50 was 7.1 𝜇M compared with 14.3 𝜇M for B. edulis Conflict of Interests
lectin). The two Boletus hemagglutinating proteins demon-
strate different sugar specificities. B. edulis lectin exhibited a The authors declare that they have no conflict of interests
unique sugar specificity towards xylose and melibiose, while regarding the publication of this paper.
BSH showed no specificity towards the variety of sugars
tested. There are reports about lectins inhibited by fetuin and Acknowledgments
glycoprotein rather than simple saccharides [23]. The effects
of various cations on the two proteins are also different. Fe3+ This work was financially supported by National Grants
and Al3+ ions augmented the hemagglutinating activity of of China (31200070, 2010CB732202, and 2012BAD14B09),
B. edulis lectin but none of the ions tested enhanced the Beijing Higher Education Young Elite Teacher Project
hemagglutinating activity; Hg2+ ions potently inhibited the (YETP1714), and Beijing Innovative Grant of Modern Agri-
hemagglutinating activity of BSH but did not affect B. edulis cultural Technology System (PXM2013-014207-000096).
lectin. Besides, the N-terminal sequence of BSH (ANVKIVK)
exhibited no homology to B. edulis lectin (TYGIALRV) or any References
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lepida (1.6 𝜇M towards Hep G2 cells and 0.9 𝜇M towards Langerhans cells that recognizes opportunistic and pathogenic
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cells and 6.81 𝜇M towards L1210 cells) [17], and Hericium [5] S. R. Dalton, R. L. Wiegert, and C. A. Casey, “An in vivo
erinaceus (56.1 𝜇M towards Hep G2 cells and 76.5 𝜇M towards method for determination of endosomal distribution of both
MCF-7 cells) [27], also manifested this activity. The potent ligand and asialoglycoprotein receptor in rat liver,” Comparative
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1 reverse transcriptase activity with an IC50 of 7.1 𝜇M. Other and F. Stirpe, “Mitogenic activity and immunological properties
lectins, such as L. flavidulus lectin (IC50 of 5.68 𝜇M) [17], P. of bolesatine, a lectin isolated from the mushroom Boletus
adipose lectin (IC50 of 1.9 𝜇M) [26], and H. erinaceus (IC50 satanas Lenz,” International Journal of Biochemistry, vol. 25, no.
of 31.7 𝜇M) [27], also exhibited anti-HIV-1 RT activity, while 5, pp. 789–792, 1993.
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RT can be classified into two groups: nucleoside RT inhibitors Boletus griseus,” Applied Microbiology and Biotechnology, vol. 72,
(NRTIs) and nonnucleoside RT inhibitors (NNRTIs) [29]. no. 5, pp. 912–916, 2006.
BSH, as a protein, belongs to NNRTIs group. The mechanism [9] M. Horibe, Y. Kobayashi, H. Dohra et al., “Toxic isolectins from
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BioMed Research International 7

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