Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 342-348
doi:10.1111/j.1745-7270.2006.00163.x
Analysis of the
Resveratrol-binding Protein using Phage-displayed Random Peptide Library
Lei FENG, Jian JIN, Lian-Feng
ZHANG, Ting YAN, and Wen-Yi TAO*
The
Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern
Yangtze University, Wuxi 214036, China
Received: January
18, 2006
Accepted: February
28, 2006
This work was
supported in part by the Key Laboratory of Bioreactor Engineering, Institute of
New World Biotechnology, East China University of Science and Technology, Shanghai
200237, China
*Corresponding
author: Tel, 86-510-85860236; 86-510-85860721; E-mail, [email protected]
Abstract Resveratrol, a plant polyphenol, is found
in significant amounts in the skin of grapes and in some traditional herbs. It is
reported to exert different biological activities, such as inhibiting lipid
peroxidation, scavenging free radicals, inhibiting platelet aggregation, and
anticancer activity. In order to screen the resveratrol-binding proteins, we
synthesized biotinylated resveratrol, purified by liquid chromatography and
immobilized it into streptavidin-coated microplate wells.
3-(4,5-Demethylthiazol-)-2,5-diphenyl tetrazolium bromide assay showed little
change in the anticancer activity of biotinylated resveratrol in vitro.
A random library of phage-displayed peptides was screened for binding to
immobilized resveratrol to isolate resveratrol-binding proteins. Several
peptides were found to bind to resveratrol specifically, which was proven by
enzyme-linked immunosorbent assay. Through amino acid sequence analysis of the
selected peptides and human proteins using the BLAST program, the results
showed that resveratrol has an affinity for various proteins such as breast
cancer-associated antigen, breast cancer resistance protein, death-associated
transcription factor, and human cyclin-dependent kinase. These results
demonstrate that our study provides a feasible method for the study of binding
proteins of natural compounds using a phage-displayed random peptide library.
Key words resveratrol; binding protein; phage
display; anticancer activity
Resveratrol (3,5,4‘-trihydroxystilbene)
is a phytoalexin, a natural polyphenol that has been found in a variety of
dietary and medicinal plants including grapes and the root of Polygonum
cuspidatum. The latter has traditionally been used in China for the
treatment of inflammation, hepatitis and osteomyelitis [1,2]. Resveratrol was
first detected from grapevines in 1976 by Langcake and Pryce [3], who found
that this compound was synthesized by leaf tissues in response to fungal
infection (mainly Botrytis cinerea) or exposure to ultraviolet light.
Because of its high concentration in grape skin, a significant amount of
resveratrol is present in wine, especially red wine. It has been suggested that
resveratrol might be partially responsible for the beneficial effect of red
wine in protecting against coronary heart disease (e.g. the French paradox)
[4,5]. However, the possible application of resveratrol in cancer treatment has
recently been proposed. Resveratrol was demonstrated to have chemopreventive
effects in different systems based on its remarkable inhibition of diverse
cellular events associated with three major cancer stages, tumor initiation,
promotion and progression [6]. Resveratrol has also been shown to inhibit the
growth of a number of human cancer cell lines in vitro, including human
breast cancer cell lines MCF-7 and MDA-MB-231 [7], human liver cancer cell line
Hep G2 [8], and human prostate cancer cell lines DU-145, PC-3 and JCA-1 [9].
Resveratrol could inhibit the growth of breast cancer cells in spite of
estrogen receptor (ER)-positive (MCF-7) or ER-negative (MCF-10, MDA-MB-231). It
has been suggested that resveratrol has several important biological functions,
such as inhibition of protein kinase C, D and protein kinase (CKII) activity
[10–12], and modulation of human mammary
epithelial cell O-acetyltransferase, sulfotransferase, and kinase
activation of the heterocyclic amine carcinogen N-hydroxy-PhIP [13], and
others. However, the molecular mechanism of anticarcinogenesis of resveratrol
is still unknown.
Methods to identify proteins that interact with a specific ligand
are very limited. Among them, the phage-displayed random peptide library is a
relatively successful molecular tool for investigating novel peptides that bind
to a target. In this library the dodecapeptides are displayed on the surface of
bacteriophage M13 [14,15]. Such phages thereby become vehicles for expression
that not only carry the nucleotide sequence encoding expressed proteins, but
also have the capacity to replicate [16]. Peptides with high affinity and
specificity for a target molecule can be identified and isolated through
multiple rounds of phage-based selection for binding to a target molecule. Such
phage libraries have been used successfully to identify proteins that
specifically bind to immobilized doxorubicin [17].Because of the extraordinarily high affinity of the binding interaction
of biotin to streptavidin, the key step is to make a biotinylated compound
(bifunctional molecules), which contains both a biotin and a substrate unit for
phage display screening [18], without changing its original bioactivity.The objective of this work was to identify potential resveratrol
targets using phage display technology. We synthesized a novel compound,
biotinylated resveratrol, and compared its anti-tumor effects on human breast
cancer cell line MCF-7 with natural resveratrol. Phage clones were then
selectively amplified during the biopanning procedure using a phage display
library. Finally we determined the amino acid sequences for the peptides
selected, and compared these with the protein sequences available from the
BLAST databases. We hope that these findings have valuable implications of the
anti-tumor activities of resveratrol.
Materials and Methods
Reagents
Resveratrol and 3-(4,5-demethylthiazol-)-2,5-diphenyl tetrazolium bromide
(MTT) were purchased from Sigma Chemical (St. Louis, USA). The Ph.D.-12 Phage
Display Peptide Library Kit (#E8110SC; New England Biolabs, Beverly,
USA) contained 1.5?1013
plaque-forming units (pfu) per milliliter with a complexity of 2?109 independent peptide sequences. All other
chemicals and solvents were of analytical grade.
Preparation of biotinylated
resveratrol
Biotinylated resveratrol was synthesized in the presence of
1,3-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine as catalyst.
Resveratrol (744 mg, 3.26 M) was mixed with DCC (168 mg, 0.815 M) and
4-dimethylaminopyridine (10 mg, 0.08 M) in dry N,N-dimethylformamide (DMF; 10
ml). Biotin in DMF (199 mg, 0.815 M) was slowly added to this mixture in a
dropwise manner. This solution was stirred for 24 h at room temperature. In
order to obtain and purify the target compound, the reaction mixture was
separated by a chromatographic column (Resource RPC 100 ml; Amersham Pharmacia
Biotech, Uppsala, Sweden) connected with a fully automated liquid
chromatography system (AKTA Explorer 100; Amersham Pharmacia Biotech), which
was designed for method development and research applications. We selected
acetonitrile and water for the mobile phase at a flow rate of 10 ml/min.
Finally, the freeze-dried biotinylated resveratrol was prepared and identified
by reversed phase-high performance liquid chromatograph-mass spectrometry
(RP-HPLC-MS) (Platform ZMD 4000; Waters, Milford, USA) [19].
Cell culture
The human breast epithelial cell line MCF-7, an estrogen
receptor-positive cell line derived from an in situ carcinoma, and human
liver cancer cell line Hep G2 were obtained from ATCC (Drive Rockville, USA).
RPMI 1640 medium (Gibco, Grand Island, USA) supplemented with 10%
heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine, 100 U/ml
penicillin and streptomycin (Gibco), 0.4% trypsin was used for cell culture as
the basal medium. MCF-7 was routinely cultured with the above culture medium at
37 ?C in a humidified atmosphere of 5% CO2.The human breast epithelial cell line MCF-7, an estrogen
receptor-positive cell line derived from an in situ carcinoma, and human
liver cancer cell line Hep G2 were obtained from ATCC (Drive Rockville, USA).
RPMI 1640 medium (Gibco, Grand Island, USA) supplemented with 10%
heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine, 100 U/ml
penicillin and streptomycin (Gibco), 0.4% trypsin was used for cell culture as
the basal medium. MCF-7 was routinely cultured with the above culture medium at
37 ?C in a humidified atmosphere of 5% CO2.
MTT assay for cell viability
For treatment with drugs, resveratrol and biotinylated resveratrol
were dissolved in culture medium containing 0.1% dimethylsulfoxide
(DMSO) to obtain a 400 mM stock solution, and then diluted with culture medium to obtain the
working solution. Untreated control cells were incubated under identical
conditions with the same volume of culture medium containing 0.1% DMSO. Cell
viability was assessed by MTT assay [20]. Briefly, cells were counted and plated
at the same initial density into a 96-well plate with 8000 cells per well in
100 ml medium. After 24 h incubation, the old medium was removed and the
fresh medium containing drugs of different concentrations was added to the
wells, which was further incubated for 48 h. Then 20 ml MTT solution (5 mg/ml)
was added to each well and, after 4 h incubation, the medium containing MTT was
replaced with 150 ml DMSO. The plate was further incubated for 15 min at 37 ?C in the
dark. Finally we measured the absorbance (A570) of
each well on a microplate reader (Multiskan MK3; Thermo Labsystems, Marietta,
USA). All experiments were performed three times.
Immobilization of biotinylated
resveratrol and biopanning experiments
The biotinylated resveratrol was dissolved in NaHCO3 buffer (0.1 M, pH 8.6) and transferred to a well of the
streptavidin-coated plate. After incubation for 12 h at 4 ?C, the well was
washed six times with TBST [0.1% (V/V) Tween-20 solution in TBS
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl)]. The Ph.D.-12 phage-displayed
random peptide library (1?1011 pfu) in 100 ml of TBS buffer was added to the resveratrol-immobilized plate, and the
plate was shaken gently for 1 h at room temperature. To remove the unbound
phages the plate was washed 10 times with TBST, and the phages, which had
adsorbed resveratrol, were eluted by 100 ml of 0.2 M glycine-HCl (pH
2.2). The elution was neutralized to pH 7.5 with 15 ml Tris-HCl (pH 9.1)
immediately. The bound phages were amplified using Escherichia coli
ER2738 to make enough copies for the next round of biopanning. After five
rounds of biopanning (the concentration of Tween-20 in the washing solution used
in the second, third, fourth and fifth eluting rounds was increased to 0.3%,
0.5%, 0.5% and 0.5%, respectively), the bound phages were eluted and plated on
Luria Broth agar plates containing isopropyl b–D-thiogalactopyranosid
and X-gal to prevent contamination. The blue monoclone was picked and amplified
to sequence its DNA. The sequencing primers were M13 U: 5‘-GTTCCTTTCTATTCTCACTC-3‘
and M13 L: 5‘-TCGTCACCAGTACAAACTAC-3‘. Amino acid sequence
comparisons with all available human protein sequences were performed with the
BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
ELISA binding assay
To test whether or not the four selected monophages could bind to
biotinylated resveratrol specifically, ELISA assays were carried out. The
plates were coated with streptavidin for 24 h at 4 ?C, blocked with bovine
serum albumin, and washed six times with 0.5% PBST [0.5% (V/V)
Tween-20 solution in phosphate-buffered saline buffer]. Biotinylated
resveratrol was added and allowed to bind for 24 h at 4 ?C. After washing with
0.5% PBST, 1010 amplified monophages were added, incubated
for 2 h at 37 ?C. After washing, horseradish peroxidase-conjugated anti-M13
antibody (Amersham Pharmacia Biotech) was added, incubated for 2 h at 37 ?C.
ABTS (Amresco, Cleveland, USA) was used in color-development. Absorbance at 405
nm (A405) of each well was read on a microplate reader (Multiskan MK3,
Thermo Labsystems). Wells coated with streptavidin or streptavidin-biotin
conjugate were used as negative controls.
Results
Characterization of
biotinylated resveratrol
In this study, for the analysis and identification of biotinylated
resveratrol, we developed a method by validated RP-HPLC system with diode array
and ion-trap mass spectrometric detection. RP-HPLC-MS, using electrospray
ionization with 25% ammonia solution as the sheath liquid, was performed in the
selected ion monitoring mode at m/z 453 [M–H]–. It is well known that MS detection is highly linear for all
investigated analytes including all kinds of reagent, catalyst and production,
and the limits of detection were in the low nanogram range.The RP-HPLC conditions were as follows: chromatographic column
[SunFire C18, 5 mm, 2.1 mm?150 mm
(Part No. 186002541, Lot No. 0108143521, Waters, Milford, USA)]; Eluent A was
15% methanol+1% acetic acid, Eluent B was 85% methanol+1% acetic acid and
Eluent C was pure methanol; the elution gradient was that 90% Eluent A+10%
Eluent B to 0% Eluent A+100% Eluent B in 20 min, to 100% Eluent C at 5 min;
flow-rate 0.3 ml/min; temperature 30 ?C; injection volume 10 ml.Fig. 1 shows a typical MS spectrum of
biotinylated resveratrol in the negative ion mode. A molecular ion
corresponding to the most intense peak was observed at m/z 227.8
and corresponded to resveratrol. The [M–H]– peak at m/z 453.9 was assigned to biotinylated
resveratrol. In positive mode, the RP-HPLC-MS experiments also gave a similar
spectrum (not shown) with an m/z value of 455.6 for the
biotinylated resveratrol. Altogether, the results indicated that our designed
chemical synthesis with catalyst led to the formation of biotinylated
resveratrol in vitro with a molecular weight of 454 Da, as expected.
Anti-tumor effects of
biotinylated resveratrol on cancer cell growth in vitro
Resveratrol has also been shown to possess strong anti-tumor
activity against several cancer cell lines in vitro, including MCF-7 [7]
and Hep G2 [8], at micromolar concentrations. To evaluate the bioactivity of
biotinylated resveratrol compared to natural resveratrol, MCF-7 and Hep G2
cells were cultured in the presence of biotinylated resveratrol or natural
resveratrol. The bioactivity was determined with a growth inhibition ratio of
cells after 48 h culture.The results, shown in Fig. 2, illustrate that more than 30%
cell growth was inhibited in the two cell lines exposed to 300 mM biotinylated
resveratrol, and 40% growth inhibition ratio on resveratrol in the same
conditions. We also found the inhibition of the two reagents was dose-dependent
in both MCF-7 and Hep G2 cells. As shown in Fig. 2(A), when Hep G2 cells were treated with
biotinylated resveratrol at a low concentration (50 mM), the cells propagated without influence. However, as the
concentration of biotinylated resveratrol increased, the number of cells
decreased sharply. The same phenomenon is shown in MCF-7 cells in Fig. 2(B),
suggesting the same sensitivity to biotinylated resveratrol in both cell types.
Because of the low concentration of biotinylated resveratrol in medium (400 mM), it has an inhibition
ratio of 34.68% and 32.95% in MCF-7
and Hep G2 cells, respectively. The MTT results suggested that resveratrol and
biotinylated resveratrol could inhibit the cell viability of MCF-7 and Hep G2
[21].Based on the results above, we consider that although the anti-tumor
activity and solubility of biotinylated resveratrol decreased to some degree,
this effect was so limited that we predict that resveratrol linked with biotin
changed its anti-tumor activity in vitro only a little.
Resveratrol-specific peptide
biopanning by phage-displayed random peptide library
The phage-displayed random peptide library that we use was pooled to
contain a diversity of 2?109 independent and different peptide sequences using a previously
reported design. The M13 phage particles whose
recombinant pIII proteins might bind to resveratrol were isolated by biopanning
as detailed in “Materials and Methods” using a derivatized
resveratrol with a biotin group and immobilized on a streptavidin-coated plate.
Five pIII structural proteins present at the tip of the virion each possessed a
random 12 amino acids extension at their amino terminus, coded by a random
synthetic oligonucleotide inserted into the corresponding position in the gene
for pIII. The sequences of inserted oligonucleotides were determined and
translated to obtain the sequence of the displayed peptide [16,22]. The M13 phage infects E. coli ER2738 and replicates without
lysis of the host during the procedure of the selected phage’s amplification.
The titer of eluted phages and their apparent affinity increased with each
round despite the use of increasingly stringent wash conditions (Fig. 3).
The phage titer of the eluted solutions increased from 6.1?102 pfu after the first round to 1.2?104 pfu at the fifth round of binding and elution.
In Fig. 3, we found that the titer of eluted phages fell off sharply
when the concentration of Tween-20 in the washing solution was increased from
0.3% to 0.5%. Note that 0.5% Tween-20 can wash away the majority of inferior
affinity phages binding to resveratrol more efficiently than 0.1% and 0.2% Tween-20
while superior affinity phages remain. So we performed the fourth and fifth
biopanning rounds until we obtained a constant yield of eluted phages. These
results indicated that phages binding to resveratrol were selectively isolated
and amplified by the biopanning procedure.After five rounds of biopanning, 20 eluted phages plated on Luria
Broth agar plates containing isopropyl b–D-thiogalactopyranosid
and X-gal were randomly chosen to be amplified for extracting single-stranded
DNA of the M13 phage, which was used as the polymerase chain reaction template.
Then we finally obtained the polymerase chain reaction products of
approximately 200–300 bp. The sequencing primers were M13 U: 5‘-GTTCCTTTCTATTCTCACTC-3‘
and M13 L: 5‘-TCGTCACCAGTACAAACTAC-3‘. Amino acid sequence
comparisons were carried out using the BLAST program and all available human
protein sequences. The results (Table 1) indicated that the primary
structure analysis did not reveal any homology for four peptides exposed on the
surface of the selected phages. As shown in Fig. 4, absorbance at 405 nm
(A405) of the well coated with streptavidin-biotinylated resveratrol
conjugate was much higher than the other two, which meant that all four
selected phage monoclones could bind to biotinylated resveratrol specifically.
Discussion
Resveratrol, also known as 3,5,4‘-trihydroxystilbene, is a
phytoalexin, a polyphenol used by plants to defend themselves from fungal and
other forms of aggression. It is found in grape skin and red wine in substantial
amounts [23]. As a plant polyphenol, resveratrol is an antioxidant and a free
radical scavenger, and it has therefore been suspected to be responsible for
the cardioprotective effects of red wine, usually described as the “French
paradox” [24,25]. As the molecular mechanisms of the anticancer and
chemopreventive effect of resveratrol are unknown, it is worth examining the
resveratrol-binding protein.It is well known that the interreaction of the properties of
proteins or polypeptide is an important ramification in many areas of biology,
ranging from medicine, to chemistry, to food. The use of the phage-displayed
random peptide library is a popular approach to achieving this goal, because of
its convenient amplification and evaluation. This technology is based on
diverse answers to the same question: What is the binding target? This
phage-displayed system requires molecules, which can be captured by a solid
phase after reaction or which contain chiral substrates tethered to a solid
phase. The immobilization of the drug is the first key step in the experiment.
In this study we chose the biotin-streptavidin system because of the
extraordinarily high affinity of the binding interaction of biotin to
streptavidin, which is commonly used in phage display biopanning experiments.
We had already synthesized a novel compound, biotinylated resveratrol. The
reactants (biotin and excess resveratrol) brought about the single substitution
reaction of esterification at room temperature, using DCC and DAMP as catalysts.
By RP-HPLC coupled with electrospray ionization MS, biotinylated resveratrol
(molecular weight 454 Da) was obtained and identified (Fig. 1). As a
result, resveratrol could be immobilized on a solid support
(streptavidin-coated plate) by non-covalent binding of the biotin-streptavidin
system for phage display panning experiments. Fig. 2 shows that
biotinylated resveratrol could inhibit the proliferation of MCF-7 and Hep G2
cells in a dose-dependent manner, the same as the results for resveratrol using
MTT assay. It is considered that the virgin spatial configuration of
resveratrol binding a biotin group underwent little change, and its anticancer
activity remained the same, although its solubility was lower. These results
also mean that biotinylated resveratrol could provide the same binding site or
binding spatial structure as natural resveratrol to bind the target protein
after immobilization, which would only marginally affect the reliability of the
biopanning experiment results.Using a phage library displaying random peptides of 12 amino acids
on its surface, four peptides were found that bind to resveratrol. No obvious
homology was found for these peptides after five rounds of biopanning (Fig.
3; Table 1). Results from ELISA assay (Fig. 4) confirmed that
the affinity between the four peptides and resveratrol was specific. In order
to find out the potential target proteins of resveratrol, the above four
selected resveratrol-binding peptides were compared with all human protein
sequences available from the BLAST databases. Sequences with at least five
residues coinciding with those of a peptide were selected. The results from
sequence comparison revealed high homology with diverse proteins, including
death-associated transcription factor, immunoglobulin E, F-box and leucine-rich
repeat protein, protein kinase C and CKII, retinoblastoma-binding protein,
breast cancer-associated antigen BRCAA1, oncostatin M receptor, polymerase (DNA
directed), GTPase activating protein, UDP (uridine diphosphate)-glucuronosyltransferase,
G-protein coupled receptor, breast cancer resistance protein (BCRP), voltage
dependent t-type calcium channel alpha-1H subunit, transcription factor-like
protein, purine nucleoside phosphorylase, human cyclin-dependent kinase 2,
activating transcription factor, and zinc finger protein. According to
published reports, some of these proteins have been identified as relevant to
the anticancer activity of resveratrol. For example, protein kinase CKII is
involved in cell proliferation and oncogenesis [25], and resveratrol was shown
to inhibit the phosphotransferase activity of CKII. Studies revealed that
resveratrol acted as a competitive inhibitor with respect to the substrate ATP
and inhibited the catalytic reaction of CKII with guanosine triphosphate as
substrate. These results suggested that resveratrol was likely to function by
inhibiting oncogenic disease, at least in part, through the inhibition of CKII
activity [12].We also found that resveratrol can inhibit the viability of both
MCF-7 and MCF-7/ADM cells induced to resistance with ADM by the gradually
increasing concentrate method (resistant index 200 multiple) in a dose- and
time-dependent manner in vitro. Flow cytometry showed that resveratrol
induces the G1 phase accumulation in MCF-7/ADM cells (data
not shown). In this study, we tested the high affinity of the binding
interaction of resveratrol to BCRP. BCRP is a more recently discovered
multidrug ATP-binding cassette transporter, a member of the ATP-binding
cassette gene ?alf-transporter subfamily [27]. It has a drug resistance profile
similar though not identical to P-glycoprotein and, like P-glycoprotein, is
located at barrier sites [28] where it might influence entry of xenobiotic
material. In recent years some published work has reported that many plant
polyphenols interact directly with BCRP, including modulating its transport
function and ATPase activity [29]. So it could be implied that resveratrol is
directly transported by BCRP, resulting in possible conformational alterations
to BCRP or effects on the cellular function of BCRP. This might explain why
resveratrol inhibits the viability of MCF-7/ADM cells. However, this rational
conjecture demands more powerful proof, such as ELISA assay and iRNA
technology. The BLAST results also provided valuable information about the
bioactivity of resveratrol, which includes not only antitumor, but also
anti-inflammatory, immunomodulating, and antiviral activities.Our study provides an example of a feasible method of investigating
the binding proteins of natural products, compounds or drugs. We made use of
biotinylation, which did not significantly alter the activities of the original
molecules, and a phage-displayed random peptide library.
Acknowledgements
The authors wish to thank Dr. Jun-Hua XIAO (the Key Laboratory of
Bioreactor Engineering, Institute of New World Biotechnology, East China
University of Science and Technology, Shanghai 200237, China) for helpful
suggestions.
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