Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 443-451
doi:10.1111/j.1745-7270.2008.00412.x
Screening and identification
of a novel target specific for hepatoma cell line HepG2 from the FliTrx
bacterial peptide library
Wenhan Li1,2#, Ping Lei1#, Bing Yu1,
Sha Wu1, Jilin Peng1,
Xiaoping Zhao1, Huifen Zhu1,
Michael Kirschfink2, and Guanxin Shen1*
1
Department of
Immunology, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
2
Institute of Immunology,
University of Heidelberg, 69120 Heidelberg, Germany
Received: December
15, 2007
Accepted: March 5,
2008
This work was
supported by the grants from the Hi-tech research
and development program of China (No. 2006AA02Z158) and
the Science Foundation of the Ministry of Education of China (No. 20060487024)
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-27-83692611; E-mail, guanxin_shen [email protected]
To explore
new targets for hepatoma research, we used a surface display library to screen
novel tumor cell-specific peptides. The bacterial FliTrx system was screened
with living normal liver cell line L02 and hepatoma cell line HepG2
successively to search for hepatoma-specific peptides. Three clones (Hep1,
Hep2, and Hep3) were identified to be specific to HepG2 compared with L02 and
other cancer cell lines. Three-dimensional structural prediction proved that
peptides inserted into the active site of Escherichia coli thioredoxin
(TrxA) formed certain loop structures protruding out of the surface. Western blot
analysis showed that FliC/TrxA-peptide fusion proteins could be directly used
to detect HepG2 cells. Three different FliC/TrxA-peptide fusion proteins
targeted the same molecule, at approximately 140 kDa, on HepG2 cells. This work
presented for the first time the application of the FliTrx library in screening
living cells. Three peptides were obtained that could be potential candidates
for targeted liver cancer therapy.
Keywords tumor target; hepatoma; bacterial
display; FliTrx system; counter-screening; living-cell panning; 3-D modeling
Estimates from the year 2000 indicate that liver cancer remains the
fifth most common malignancy in men and the eighth most common in women worldwide,
burdened by a constantly increasing frequency. This tumor accounted for 5.6% of
all human cancers (7.5% among men and 3.5% among women) [1,2]. The main obstacles to improving control and treatment of hepatoma
are the lack of biomarkers for early diagnosis [3] and selective delivery of
chemotherapeutic drugs into the tumor cells in vivo [4].Phage display technology is a very powerful tool for the
identification of critical amino acids responsible for protein-protein
interaction and the discovery of new therapeutic targets [5,6]. In the current
study, we chose a bacterial peptide library, the FliTrx Escherichia coli
thioredoxin (TrxA) scaffold random peptide library, which has been widely used
to identify conformationally constrained dodecamer peptide sequences
specifically recognized by soluble proteins, such as monoclonal antibodies
[7,8]. The FliTrx peptide library has an estimated diversity of 1.77108 different random dodecamer loop sequences and does not contain any
predefined structural motif. Thus, novel peptide sequences could be identified
in this library that mimic the functions of protein domains found in nature
[9]. In this flagella display library, peptides are directly displayed on the
surface of E. coli fused with two proteins, the major bacterial
flagellar protein (FliC) and TrxA. Various dodecapeptides are inserted within
the active loop of TrxA, and the fusion protein forms a stable protrusion from
the bacterial cell surface with the help of bacterial flagella [10,11]. We
wanted to identify FliTrx peptides specifically targeting tumor cells, not only
to investigate target molecules, but also to deliver drugs into malignant
tissues. We identified three FliTrx peptides from the bacterial random
library that are specific for hepatoma cell line HepG2 and do not bind to other
cancer cell lines or normal liver cells. Results of structure prediction
suggested that TrxA-peptide fusion proteins based on the TrxA active site might
be more valuable than linear peptides themselves. Immune blotting results
further proved that FliC/TrxA-peptide fusion proteins could be used to detect
HepG2 cells and indicate a novel target molecule on these cells.
Materials and Methods
Cell culture
Human hepatoma cell line HepG2 and human liver cell line L02 (both
cell lines were maintained in our laboratory) were cultured in RPMI 1640
(Gibco, Carlsbad, USA) supplemented with 10% (V/V) fetal bovine
serum (Gibco) at 37 ?C in a 5% CO2 incubator.
Selection of HepG2
cell-binding peptides
The FliTrx random peptide display library (Invitrogen, Carlsbad,
USA) containing 1.77108 primary clones was used to
identify specific HepG2 cell-binding peptides incorporating negative selection
using the normal liver cell line L02.Bacteria were treated according to the manufacturer’s instructions.
The bacterial cells were diluted 10-fold in IMC medium (1 M9 salts, 0.2%
casamino acids, 0.5% glucose, 1 mM MgCl2)
(Invitrogen) with 100 mg/ml ampicillin and cultured with shaking (225–250 rpm) at 25 ?C
for 15 h.The expression of the bacteria was induced by adding 3 ml of the
overnight culture (approximately 11010 cells) to 50 ml IMC
medium containing 100 mg/ml ampicillin and 100 mg/ml tryptophan (Invitrogen) and the mixture
was cultured with shaking for 6 h. Biopanning was carried out after induction
of the bacterial library with tryptophan, which activated the transcription of
the FliC/TrxA peptide fusion proteins.A confluent dish with L02 cells was first blocked with IMC medium
containing 1% bovine serum albumin (BSA) and 1% a-methyl mannoside
(Sigma-Aldrich, St. Louis, usa)
at 37 ?C for 1 h. Blocked cells were incubated with 10 ml induced bacterial
suspension in IMC medium containing 1% BSA and 1% a-methyl mannoside at room
temperature for 1 h. Unbound bacteria were transferred and incubated in another
dish with blocked L02 cells at room temperature for 1 h. Then the unbound
bacteria were transferred to the blocked HepG2 cells and incubated for another
1 h. Dishes with HepG2 cells were washed with IMC medium containing 1% a-methyl
mannoside for 5 min and the wash was repeated four more times. Binding bacteria
were obtained by stirring in a vortex for 30 s and subsequently amplified in
IMC medium containing ampicillin (100 mg/ml) overnight for the next selection round.
Recovered bacteria were counted by plating on RMG plates (1 M9 salts, 2%
casamino acids, 0.5% glucose, 1 mM MgCl2, and 1.5% agar) (Invitrogen) supplemented with 100 mg/ml ampicillin.
After five rounds of selection, individual clones were isolated and analyzed.
Clone polymerase chain
reaction (PCR)
Clone polymerase chain
reaction (PCR)
Individual clones were picked and lysed in 20 ml of 0.1% Triton
X-100 and the lysate was used as a template. Reactions were carried out with
FliTrx forward sequencing primer (5‘-ATTCACCTGACTGACGAC-3‘),
FliTrx reverse sequencing primer (5‘-CCCTGATATTCGTCAGCG-3‘)
(synthesized by BioAsia, Shanghai, China), and Taq DNA polymerase (MBI
Fermentas, St. Leon-Roth, Germany) in a volume of 20 ml for 2 min predenaturing
at 94 ?C and 30 cycles of 1 min denaturing at 94 ?C, 1 min annealing at 55 ?C,
and 1 min extension at 72 ?C. PCR products were analyzed by electrophoresis on
1% agarose gel. Clones containing inserts of 170 bp were selected for further
analysis.
Flow cytometry
Individual clones were further screened for binding with HepG2 by
flow cytometry, using L02 as negative control. Cells (1105 cells/well) were removed from the dish with trypsin, washed once
with phosphate-buffered saline (PBS)/1% BSA, and incubated with 50 ml induced
bacteria (1012
c.f.u./ml in PBS/1% BSA and 1% a-methyl mannoside) for 1 h
on ice. Cells were washed twice with PBS/1% BSA and incubated with mouse anti-thio
antibody (1:500 diluted) (TrxA; Invitrogen) for 30 min on ice. Cells were
washed twice with PBS/1% BSA and incubated with
fluorescein-isothiocyanate-conjugated anti-mouse immunoglobulin G (1:500
diluted) (Invitrogen) for 30 min on ice. Then the cells were washed twice with
PBS/1% BSA and analyzed using FACSCalibur (Becton Dickinson, Oxford, UK).
Clones that only bound HepG2, not L02, were selected for sequencing.
DNA sequencing and homology
Individual clones were isolated and sequenced by
BioAsia (Shanghai) with forward sequencing primer 5‘-ATTCACCTGACTGACGAC-3‘ and
reverse sequencing primer 5‘-CCCTGATATTCGTCAGCG-3‘. Sequences
of dodecapeptides were submitted to the PubMed (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi),
SwissProt (http://expasy.org/tools/blast/),
and European Molecular Biology Laboratory (EMBL) (http://www.ebi.ac.uk/blast2/index.html)
databases for homology analysis.
Characterization of
specificity with flow cytometry
To characterize specificity, individual clones from the last
screening round were analyzed using flow cytometry for binding with the
following cancer cell lines: SLC (squamous lung carcinoma), MKN28 (gastric
adenocarcinoma), K562 (chronic myelogenous leukemia), HeLa (cervix
adenocarcinoma), MCF7 (mammary gland adenocarcinoma), HL60 (peripheral blood
acute promyelocytic leukemia), and SMMC7721 (hepatocellular carcinoma). Similar
techniques were used as above described.
Structure prediction
The sequences of selected peptides were analyzed with
ExPASy proteomics and sequence analysis tools (http://expasy.org/tools/). Secondary structure prediction was carried out by agadir (http://www.embl-heidelberg.de/Services/serrano/agadir/agadir-start.html).
Information on TrxA-peptide fusion proteins was sent to the 3D-jigsaw comparative modeling server (http://bmm.cancerresearchuk.org/~3djigsaw/).
VMD 1.8.5 (http://www.ks.uiuc.edu/Research/vmd/vmd-1.8.5/)
was used to display and analyze the results of the modeling of fusion regions.
Expression analysis of
FliC/TrxA-peptide fusion protein and Western blot analysis
After being induced with 100 mg/ml tryptophan under optimized
conditions for 6 h, the cultured bacteria were collected and the flagellin
extracted as described by Ibrahim et al [12], with some modifications.
Briefly, the flagellin protein was extracted by exposure of the bacterial cells
to 1 N hydrochloric acid at pH 2.0 for 20 min. Cellular debris was then
separated by centrifugation at 1500 g for 15 min and the flagellin
protein in the supernatant was collected. The pH of this supernatant solution
was adjusted to 7.2 with 1 N sodium hydroxide before incubation with the
polyvinylidene fluoride membrane.HepG2, L02, and SLC cells were lysed in the sodium dodecyl sulfate
sample buffer; proteins were separated on 8% polyacrylamide gels and transferred
to polyvinylidene fluoride membranes. Blots were incubated with 10 mg/ml
FliC/TrxA-peptide fusion proteins (supernatant as mentioned above in PBS/0.2%
Tween-20/5% skimmed milk) overnight at 4 ?C and washed with PBS/0.2% Tween-20.
Membranes were detected using mouse Anti-Thio antibody (1:5000 diluted) and
horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (1:2000
diluted). The final detection method is to add the horseradish peroxidase
substrate solution containing DAB (3,3′-diaminobenzidine) (1.5 mg/ml) and watch
for the appearance of coloured band on the nitrocellulose.
Results
Bioscreening
HepG2 binding-specific peptides were screened from the FliTrx random
peptide library containing 1.77108 primary clones. Induced bacterial
display peptides were counter selected with normal liver cell line L02 to
deplete healthy tissue-binding peptides. After incubation with L02 cells,
unbound bacteria were obtained and panned with HepG2 cells. Five rounds of
selection were carried out and the efficiency of selection was monitored by
comparing the number of recovered bacteria in each round. An ever-increasing
ratio of output/input (Table 1) indicated that the positive clones
specific for HepG2 were fully enriched, especially in the last round.After five subtract-screening rounds, 700 clones were picked from
the recovered bacteria in the last round for bacterial PCR to select clones
containing whole fusion protein. Approximately 28% (200/700) of the selected
clones containing the full-sized insert of 170 bp (Fig. 1) were selected
for further investigation. Each of the clones and the corresponding exogenous
sequence was given a sequential name from 1 to 200.
Confirmation of in vitro
binding by flow cytometry
The binding ability of individual clones was verified by flow
cytometry. To identify the specificity of selected clones for HepG2, the cell
line L02 served as the control. Ten of the 200 peptides had clearly higher
binding activity with HepG2 than with L02 (Fig. 2). They were chosen to
be sequenced.
Sequence and homology analysis
Among the 10 selected clones, seven peptide sequences were
identified due to repeated events (Table 2). Three clones, 11, 16, and 76,
included the same dodecamer peptide sequence. Two clones, 34 and 60, were
totally repeated sequences. Analysis of the amino acid sequences suggested that
individual clones might share some consensus groups at an identical position,
such as VA, EL, RI, AP, S-S, L-E, and W-E. In other words, there were some
high-frequency amino acids. But these sequences were different from
hepatocellular antigens or any other peptide or protein sequence available, as
confirmed by blast in various
protein databases such as SwissProt, PubMed, and the EMBL databases.
Characterization of
specificity of peptides
Additional flow cytometry analysis was carried out to further
determine the binding specificity of these seven peptides with tumor cell lines
SLC, MKN28, K562, Hela, MCF7, and HL60. Three clones (11, 12, and 34) showed
obviously lower binding activity with other tumor cell lines than with HepG2 (Fig.
3). We named these three peptides Hep1 (11), Hep2 (12), and Hep3 (34).
Furthermore, we identified their binding specificity with hepatocellular
carcinoma cell SMMC7721. Results showed that they could also bind to SMMC7221
cells, but the affinity was significantly lower compared with HepG2 (Fig. 4).
Prediction of the structure of
TrxA-peptide fusion protein
The amino acid sequences of Hep1, Hep2, and Hep3 were submitted to
AGADIR and the results revealed that both Hep1 and Hep2 peptides had a very
high helical potential [Fig. 5(a–c)]. This, in turn, implies that the dodecamer peptides might adopt a
coiled-coil structure. As Hep3 peptide showed a very low helical potential, it
was selected as a control for further structural and recognition investigation.As dodecapeptides were inserted within the active loop of TrxA, we
submitted the sequences of TrxA-peptide fusion proteins to the 3D-JIGSAW server
to further develop the TrxA-peptide structural model. The overall structure is
shown in Fig. 5(D,E). The fusion protein consisted of two regions; TrxA
had a central pleated b-sheet with five parallel and antiparallel b-strands, surrounded by
three a-helices. Dodecapeptides formed a stable protrusion from the active
site of TrxA, located at the beginning of a long a-helix. Hep1 formed a big
loop [Fig. 5(d)], Hep2 was
displayed as a b-sheet combined with one b-fold of TrxA overhanging from the TrxA region
[Fig. 5(e)], and Hep3
stuck out from the TrxA region [Fig. 5(f)],
forming two b-sheets by itself. The model showed that even Hep3 might not be a
simple linear structure in the fusion protein.Comparison with the secondary structure analysis showed that only
the structure of Hep3 in the fusion protein was affected by TrxA. Hep1 and Hep2
were also constrained by TrxA, but the tertiary structure was similar to the
secondary structure and not influenced by the fusion protein.
Identification of a novel
target on HepG2 cells
The flagellin protein was extracted for Western blot assay. The
Western blot analysis revealed that: (1) FliC/TrxA-peptide fusion proteins were
approximately 65 kDa; (2) no binding occured on L02 cell lysate or SLC cell
lysate with FliC/TrxA-peptide fusion proteins; and (3) three FliC/TrxA-peptide
fusion proteins bound to the same fragment of approximately 140 kDa of the
HepG2 cell lysate (Fig. 6).
Discussion
Targeting specific binding ligands on tumor cells is an efficient
way to improve early cancer diagnosis schemes and therapy. It has been reported
that cancer cells often display high levels of certain cell surface molecules,
such as tumor-associated antigens or tumor-specific antigens, that are sparse
on normal tissues, representing potential sites for delivery of toxic agents
[13,14]. Therefore, we carried out subtract-screening based on the FliTrx
bacterial peptide library to search for HepG2 cell-specific peptides. As HepG2
is a human hepatoma cell line derived from hepatic neoplasia, which still
retains numerous cellular functions typical of differentiated, normal
hepatocytes (such as synthesis of albumin, transferring, a1-antitrypsin,
fibrinogen, and certain other coagulation factors), it is widely accepted as a
valuable and informative model system for studying human hepatocyte function
[15,16]. Phage display libraries have been applied in ligand selections using
cultured cells, animals [17], or human patients [18], yielding peptides
specific for cell surface markers [19]. The principal advantage of display
library methodologies is that selection can be carried out in a native-like,
membrane-bound environment without prior knowledge of the target cell receptors
[20]. Peptide ligands generated using this method have been proved useful in
tumor diagnosis and target therapy [21,22]. The FliTrx system has been widely
used as a novel bacterial displayed peptide library for studying
protein-protein interactions involved in receptor-ligand binding,
enzyme-substrate specificity, and antibody-antigen recognition [8,23]. Cell surface proteins are frequently post-translationally modified,
including malignancy-specific modifications. Thus, peptides selected against purified
recombinant protein might not be able to access their targets on living cells
[24,25]. Here, for the first time, we described successful living cell
biopanning with this particular bacterial library. The data proved that
positive clones specific for HepG2 were fully enriched after subtract screening
and three HepG2-specific clones were selected from the peptide library.Although it is possible to screen peptide libraries to select peptidic
ligands that bind to previously uncharacterized molecules on the cell surface,
a problem in this approach is the risk of selecting clones that bind a whole
population of cell surface molecules, not the target molecule alone. To enhance
the selection of specific ligands from the phage-displayed peptide library,
subtractive approaches have been applied to eliminate potential selection of
clones that bind to irrelevant surface molecules [26–28]. In this case, we chose
the normal liver cell line L02 as the negative control and after five
subtract-screening rounds we obtained 200 clones containing the full-sized
fusion from 700 random clones. Of these, 10 clones showed obviously higher
affinity with HepG2 than with L02. Finally, seven different sequences were
identified on account of repeated cases. We carried out not first sequencing of
all random clones picked up from recovered bacteria, but bacterial PCR for
identification of correct construction of fusion regions after bioscreening.
The results proved that, with even the obvious enrichment of bacteria
specifically binding to HepG2, only 28% of clones containing full-size inserts
could be used for further binding ability assay. And only 5% of the clones
appeared to bind more effectively than the others, and were able to bind
specifically to hepatoma cells. Such a low positive rate was not what we aimed
for and the evidence revealed the meaning of identification of binding ability
of selected clones for deletion of non-specific peptides and avoidance of blind
random sequencing. The analysis of amino acid sequences in clones initially indicated
some consensus groups, such as VA, EL, RI, AP, S-S, L-E, and W-E, shared by
individual clones. However, after subsequent sequenced blasts in various protein databases, including SwissProt,
PubMed, and EMBL databases, these peptides turned out to be different from any
hepatocellular-specific antigens or any other peptide or protein sequences
available. Further flow cytometry assays distinguished three clones that showed
obviously higher affinity with HepG2 cells compared with other tumor cell
lines. They were named Hep1, Hep2, and Hep3, and they could also bind to
another liver carcinoma cell line SMMC7721, although the affinity was lower
compared with HepG2.Screening of protein databases allows determination of proteins with
homologies to selected peptide sequences. In that event, the matched proteins
are biologically relevant (can theoretically serve as ligands for the target
molecules). But, in our experiment, the consensus sequence of the selected
peptides did not bear any obvious similarity to the sequence of any
characterized protein in the databases. It is very possible that the selected
peptides mimic a complex binding epitope of potential ligands and, thus, cannot
be found in databases [29]. Therefore, we tried to investigate the
characteristics and gather biological information about these HepG2-specific
peptides.Variation in the FliTrx loop epitope sequence, the location of the
linear epitope sequence, and the non-epitope sequence composition indicated
that some different amino acid sequences are recognized by the same HepG2
cells. Bioinformatics analysis and prediction of the secondary structures of
peptides and tertiary structures of TrxA-peptide fusion proteins suggested
that: (1) three peptides isolated from the FliTrx library were constrained in
the fusion region, and formed different 3-D structures sticking out of the
fusion protein, which benefit their interaction with molecules; (2) the
structure of Hep3 was influenced by TrxA, however, the secondary and tertiary
structures of Hep1 and Hep2 showed no significant difference; and (3) the
structure character might be more important for peptide function and
recognition than the sequence of peptides. We propose, therefore, that the 3-D
structural model of TrxA confirmed and guaranteed the structure of peptides and
affected their affinity.As TrxA-peptide fusion proteins are more available than the
respective peptides, we attempted to verify the recognition between fusion
proteins and tumor cells using Western blot analysis. We used the flagellin
protein, so-called FliC/TrxA-peptide fusion proteins, to identify the tumor
cell lysate. Induced bacteria were incubated with cell lysate blot and
Anti-Thio positive bands were observed only on the HepG2 cell lysate. Selective
binding to the same band of HepG2 cell lysate occurred by different
FliC/TrxA-peptide fusion proteins but not by the negative control. These
results revealed that FliC/TrxA-peptide fusion proteins recognize a certain
molecule or subunit on HepG2 cells, but not TrxA or the peptide itself.
Furthermore, the fragment of approximately 140 kDa on HepG2 cells, which binds
to fusion proteins, was also different from the other known tumor antigens,
such as transferrin 95 kDa, fibrinogen 340 kDa, and a1-antitrypsin 54 kDa. The
fact that several peptide consensus groups were apparent among the sequences
isolated using the bacterial library suggests that these clones recognize
different cell surface receptors or epitopes on whole cells [30]. However, the
results suggested that different clones detected the same target molecule,
implying the existence of a possible protein or subunit of approximately 140
kDa on HepG2 cells. These three peptides are not repeated sequences and their
3-D structures are not identical, but they can bind the same target molecule on
tumor cells.A protein epitope can either be continuous or discontinuous. While a
continuous
(also called sequential or linear) epitope is a
sequential fragment from the protein sequence, a discontinuous
(also called conformational) epitope is composed of several
fragments scattered along the protein sequence and brought
together in spatial proximity when the protein is folded
[31]. Most epitopes are discontinuous, although they are often
composed of small continuous elements of the sequence. Most
linear epitopes of approximately five residues are involved, four residues
being involved in the binding [32], and the other replaceable residues
essentially fulfilling a spacer function. E (glutamin) and L (leucine) show a
high frequency among the amino acid sequences of Hep1, Hep2, and Hep3. Peptides
could recognize and bind the same epitope based on the folding and reversal of
the 3-D structure. Peptides selected using this methodology have moderate
biological activity towards their receptor molecules on cell surfaces. This
indicates that the peptides can serve as potential leads for the development of
diagnostic and therapeutic agents [33]. In summary, the development of new biological technology provides
powerful methods to identify biomarkers on tumor cells or tissues, to
consummate the tumor therapy methods and drugs, by specific delivery of drugs
in tumor cells. A major advantage of these techniques, especially the
combination-interdisciplinary method, is not affecting on innocent bystander
cells. The success encountered with the bacterial peptide library with
TrxA-dodecapeptides suggests that the approach described in this study might be
used to develop powerful and more specific peptides. Three FliC/TrxA-peptide
fusion proteins selected in this study (FliC/TrxA-Hep1, FliC/TrxA-Hep2, and
FliC/TrxA-Hep3) showed remarkable cell specificity of HepG2 cells. And all the facts
surely pointed toward the improvement of a novel target in HepG2 cells, as
there was a 140 kDa band detected by the FliC/TrxA-peptide fusion proteins.
Structural bioinformatic approaches could be a helpful methodology for further
investigation of target molecules on HepG2 cells and improvement of the
affinity of HepG2-specific peptides.
Acknowledgements
We thank Jingfang Shao, Yue Zhang, Jing
Yang, Zhihui Liang, Wei Feng, Xiaodan Jiang, Ping Xiong, and Yong Xu
(Department of Immunology, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan, China) for their help.
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