Detection of Tumor Invasive Biomarker using a Peptamer of Signal Conversion and Signal Amplification

Article abstract of DOI:10.1021/acs.analchem.5b04423

Inspired by the structural and functional features of proteins in cell signaling, a switchable peptide is designed in this work. This switchable peptide is named a "peptamer," and it can react to ligand binding with conformational change and activation/deactivation of catalytic ability. The peptamer is constructed by elaborately integrating several different peptide motifs with targeting and catalytic abilities. Thus, targeted binding of the peptamer to an integrin can be regulated by a synthetic ligand. Moreover, the conformational rearrangement of the peptamer induced by both integrin and the synthetic ligand can resolve in altered affinity of the peptamer for a catalytic cofactor, cupric ion. This leads to greatly contrasted efficiency of catalysis in the presence/absence of integrin. This distinct switching on/off of catalytic activity also enables a bioassay of tissue integrin expression in clinical samples of thyroid carcinoma. Experimental results reveal that the detected integrin level parallels the state of lymph node metastasis. Therefore, this simple peptide model may help to understand the structural reconfiguration of proteins involved in cellular signal transduction, as well as to provide a new means to assess protein activity under pathological conditions such as cancer. (Figure Presented).

Full text of DOI:10.1021/acs.analchem.5b04423

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Article
Detection of Tumor Invasive Biomarker using Peptamer
of Signal Conversion and Signal Amplification
Hao Li, Weiwei Li, Fengzhen Liu, Zhaoxia Wang, Genxi Li, and Yannis Karamanos
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04423 • Publication Date (Web): 03 Mar 2016
Downloaded from http://pubs.acs.org on March 9, 2016
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Analytical Chemistry
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Scheme 1. (a) Design of the peptamer. (b) The peptamer conformation and affinity for cofactor ion can be
regulated variously by the interactions with ligands.
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Fig. 1 Isothermal titration calorimetry (ITC) results corresponding to the different types of peptamer-ligand
interactions designed in Scheme 1. In each of the above results, the top row is the raw data of power versus
time (in minutes), while the bottom row is the corresponding integrated enthalpy versus the molar ratio of
titrant vs titrand. The titrants vs titrands are respetively: (a1\') Cucurbituril (Q8) vs Peptamer (Pep), (a1)
Q8 vs Pep, (b1\') Pep-2Q8 vs Integrin (Int), (b1) Pep-Q8 vs Int, (b2) Pep vs Int, (c1\') Cu (II) vs Pep-2Q8-
Int, (c1) Cu (II) vs Pep-Q8-Int, (c2) Cu (II) vs Pep-Int, (c3) Cu (II) vs Pep.
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Scheme 2. Integrin bioassay developed based on the peptamer. (a) assay procedure, (b) scheme of the
reaction of catalytic signal amplification.
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Fig. 2 Quantitative analysis of integrin using the peptamer-based bioassay. (a) Square wave
voltammograms (SWVs) of DAP showing the gradual increase of signal response with integrin concentration.
(b) Peak currents in (a) plotted as a function of integrin concentration. Inset is the linear range and the
corresponding formula obtained via regression analysis. The error bars represent standard deviation from
average (n=3).
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Fig. 3 SWVs of DAP showing the specificity of the assay. All control species are of 100 nM.
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Fig. 4 Box chart to show the distribution of the detected concentration of integrin in patients of thyroid
carcinoma, grouped by (a) T stage, (b) N stage and (c) M stage. For each sample, the detected integrin in
cancerous tissue is normalized as the fold of increase compared with the adjacent normal tissue.
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Detection of Tumor Invasive Biomarker using Peptamer of
Signal Conversion and Signal Amplification
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Hao Li†, Weiwei Li†, Fengzhen Liu‡, Zhaoxia Wang‡, Genxi Li†, §,*, Yannis Karamanos∥
†. State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life
Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, China.
‡. Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China.
§. Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai, China.
∥. Laboratoire de la Barrière Hémato-encéphalique, Faculté des Sciences, Université d'Artois, rue Souvraz SP18,
62307 Lens Cedex, France.
ABSTRACT: Inspired by the structural and functional feature of proteins in cell signaling, a switchable peptide is
designed in this work. This switchable peptide is named as a “peptamer” and it can react to ligand binding with
conformational change and activation/deactivation of catalytic ability. The peptamer is constructed by elaborately
integrating several different peptide motifs with targeting and catalytic abilities. Thus targeted binding of the peptamer to
integrin can be regulated by a synthetic ligand. Moreover, the conformational re-arrangement of the peptamer induced by
both integrin and the synthetic ligand can resolve in altered affinity of the peptamer for a catalytic cofactor, cupric ion.
This leads to greatly contrasted efficiency of catalysis in the presence/absence of integrin. This distinct switching on/off of
catalytic activity also enables a bioassay of tissue integrin expression in clinical samples of thyroid carcinoma.
Experimental results reveal that the detected integrin level shows parallel with the state of lymph node metastasis. So,
this simple peptide model may help to understand the structural reconfiguration of proteins involved in cellular signal
transduction, as well as to provide a new means to assess protein activity under pathological conditions such as cancer.
Introduction
both a structural component and a regulatory factor of the
cell skeleton17, while the pentameric recognition sequence
can specifically block integrin from interactions with the
tumor stroma, thereby therapeutically modulating or
halting tumor invasion. This pentameric sequence shows
the highest affinity when constrained in a loop18, so in our
design the affinity is designed to be controllable through
Investigation of the functional structures of proteins is
of fundamental importance to the current understanding
of protein pathological functions1-6. For example, in the
development of cancer, tumor promoting effects can be
realized through structural rearrangements induced by
various cancerous and pro-metastatic cellular signals7-10
.
the formation/deformation of
the loop.
This
Specifically, the interactions with messengers or protein
ligands force the active site to adopt a different
conformation, leading to effects such as altered catalytic
activity or changed affinity towards effectors11-12. So, it can
be said that the process of signal transduction is mainly
realized by the flexible motifs in the protein active centers,
such as structurally reconfigurable peptide loops1314-16. To
apply such “smart” peptide loops in protein detection, we
propose in this work to model the principal bioactivity of
proteins in cell signaling (Scheme 1) on a “peptamer” that
can change conformation in response to the binding of
analytes. “Peptamer” is here defined as a synthetic peptide
sequence that can recognize certain targets and can change
its conformation to bind with the target, similar to the
recently developed nucleic acid-based “aptamer”.
formation/deformation of loop is in turn controlled by the
interaction with an artificial regulatory factor, cucurbituril
(Q8) (Scheme 1b), and both the concentration of Q8 and
the sequence of interactions are designed to affect
formation/deformation of the loop. Such spatiotemporally
distinct aspects of protein/ligand interactions are
molecular mechanisms usually employed by the cell
signaling processes for pinpoint regulation of protein
functions19. Moreover, each of these sequential
interactions with the regulatory factors of different
concentrations can either stabilize or destabilize the loop,
while the final conformation adopted depends on the sum
effect of all such factors. This is a process to integrate
signal20, or the mechanism enabling the crosstalk of
various signaling pathways and eventually leading to the
formation of cell signaling network. Finally, as a result of
conformational rearrangement, the peptide may show
The major functional motif of this peptamer is a
pentameric recognition sequence of integrin. Integrin is
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enhanced or hindered ability of cofactor (cupric ion) was buffered with 4 mL 10 mM PBS pH 7.4. Signal
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binding (Scheme 1b), resulting in correspondingly
augmented or diminished catalytic activity. In this
designed process of peptide/ligand interactions, the
relative abundance of each ligand, their affinity towards
the peptamer, and most importantly, the interaction with a
first ligand resulting in changed affinity of the peptamer
for a subsequent ligand, are all properties21-23 manipulated
by the cell signaling network to adapt protein functions to
specific biochemical niches. Using ligand binding to fine-
tune the targeting and catalytic abilities of peptides, the
development of this peptamer may help to improve the
protein-targeted bioanalytical strategies.
response of the generated 2,3-diaminophenazine (DPA)
was then recorded in the buffered reaction solution. These
electrochemical measurements were carried out on a
CHI660D Potentiostat (CH Instruments) with
a
conventional three-electrode system: the electrode
immobilized with peptide as the working electrode, a
saturated calomel electrode (SCE) as the reference
electrode and a platinum wire as the counter electrode.
Isothermal titration calorimetry (ITC) measurements were
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conducted using
a MicroCal ITC200 System (GE
healthcare life sciences). The titration was conducted at
25 °C. The titration schedule consisted of 38 consecutive
injections of 1 μL with at least a 120 s interval between
injections. Heats of dilution, measured by titrating beyond
saturation, were subtracted from each data set. All
solutions were degassed prior to titration. The data were
analyzed using Origin 7.0 software.
Method
Chemicals and Reagents Peptide probes (NH2-GH-G-
K(-F-NH2)-GGG-VRGDF-GGG-K(-F-NH2)-HK-CONH2,
NH2-GH-G-K(-MUA)-G-K(-F-NH2)-GGG-VRGDF-GGG-
K(-F-NH2)-HK-CONH2, Figure S1) were manufactured by
Shanghai Science Peptide as lyophilized powder,
purity>90%, For the interface probe (shown as the second
sequence), 11-mercaptoundecanol was incorporated into
their sequences for surface immobilization of these probes.
Human recombinant integrin αVβ6 was purchased from
R&D Systems. Powder of the peptides was dissolved with
10 mM phosphate buffer solution (PBS) (pH 7.4). The
target integrin was reconstituted with 10 mM PBS, pH 7.4
Redistilled water for the preparation of all the solutions
was produced with a Milli-Q purification system, the
resistance of 18 MΩ·cm was achieved to guarantee the
purity. Cancerous tissue samples of patients of thyroid
carcinoma were obtained from Nanjing Drum Tower
Hospital, the Affiliated Hospital of Nanjing University,
after elected consent by the local ethical committee. Upon
receiving the samples, they were directly sliced on ice to 1
mm3, followed by fractioned using a nuclear extraction kit
of Abnova, the cellular fraction thus obtained was 100×
diluted and immediately brought to detection procedures.
Results and discussion
The detailed design of the peptamer is shown in Scheme
1. The peptamer is designed to have three distinct motifs
(Scheme 1a): the integrin affinity motif is at the center,
flanked by two cucurbituril (Q8) affinity motifs, and at the
two ends is the Cu(II) binding and catalytic motif split in
two halves. The peptamer may have a variety of
interactions with the ligands (Scheme 1b). Conformational
change of the peptamer could be directed by Q8
concentration and facilitated by integrin binding. If Q8 is
abundant (leftmost column of Scheme 1b), each of the two
Q8 affinity motifs can be saturated by Q8, then the
peptamer could remain in a linear conformation. The
linear form will have greatly reduced affinity towards
integrin. In the meantime, even if the loop form is induced
by integrin binding, the peptamer will still have the two
halves of the cupric ion binding motif too separated to
effectively act as a single motif, leading to reduced affinity
towards the cofactor Cu (II). On the contrary, if Q8 is of
low abundance (the column second to the left), the two Q8
affinity motifs of the peptamer could be zipped together
by one molecule of Q8. The integrin affinity motif located
between will then be constrained into a loop, the most
appropriate conformation for integrin binding. Binding of
integrin may further stabilize this constrained
conformation, then the two halves could be in close
Electrode Treatment and Modification These steps
were essentially the same as previously reported24. Briefly,
the electrode was reacted with 5 μM peptide in 10 mM PBS
(pH 7.4) at 4 °C for 16 h, followed by being dipped in 1 μM
9-mercaptononanol for 3 h. To the surface of this electrode
was added a solution of proper amount of Q8, after
incubation, the electrode was then gently washed with
ddH2O and ready for protein detection.
Protein Detection Firstly, standard or clinical samples
were incubated with the peptide modified electrodes at
ambient temperature for proper time. After that, the
electrode was gently rinsed sequentially with Q8 and
ddH2O. To generate readout signal, the electrode was first
dipped in 60 μM CuCl2 briefly, after gentle rinsing with
ddH2O, it was then transferred into a 1 mM HCl solution (1
mL) containing 0.1 mg/ml ο-phenylenediamine (OPD).
The solution was then placed in 50 °C water bath for 30
min. After being cooled to room temperature, this solution
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Scheme 1. (a) Design of the peptamer. (b) The peptamer conformation and affinity for cofactor ion can be regulated variously by
the interactions with ligands.
vicinity of each other, and could act as an effective binding
site of the cofactor Cu (II). In these processes, the affinity
for both integrin and the cofactor ion can be regulated by
the abundance of Q8. If the peptamer is allowed to
interact with them without the pre-treatment with Q8
(the third column), both interactions can be expected to
be in a different state. The final formation of the effective
Cu (II) binding motif is also the cumulative effect of both
Q8 and integrin binding, whether they tend to enhance or
offset the conformational change induced by each other.
Without the interactions with these two ligands, the
peptamer will remain in a linear form very difficult to
recognize the cofactor ion (the rightmost column). So, the
ligand binding-induced conformational change may lead
to altered affinity towards the cofactor cupric ion and the
corresponding activation/de-activation of catalytic ability.
most telling features showing the regulatory effect of each
ligand on the final peptamer conformation and the ability
of cofactor binding. First, using proper amount of Q8 to
enhance peptamer binding with integrin, as well as the
final Cu (II) binding (Fig. 1, a1~c1), highest affinity towards
integrin and cupric ion can be observed (Table S1, sub-
table b1, c1). Specifically, 1:1 binding between Q8 and
peptamer is first observed on Figure 1, a1, corresponding to
two Q8 affinity motifs of the peptamer locked together by
one molecule of Q8. Meanwhile, negative entropy
accompanies this interaction (Sub-table a1), indicating a
enthalpy-driven process with conformational change of
the peptamer which might be assigned to the forming of a
loop for subsequent integrin binding. This binding shows
high affinity not too far away from that of the 5-mer
therapeutic cyclic peptide “cilengitide” (2.5 nM of
cilengitide as compared with 7.3 nM of the proposed
peptide),25 with almost no entropy penalty, suggesting an
optimized looped structure of the targeting sequence
already formed via Q8 binding. Second, if surplus amount
of cucurbituril is employed to keep the peptamer in a
linear form upon integrin binding, reduced affinity
towards both integrin and Cu (II) can be observed (Fig. 1,
The above designed peptamer and its conformational
response to the ligands can be examined by investigating
the thermodynamic aspects of the peptamer-ligand
interactions (Fig. 1). For each interaction, a characteristic
thermo-titration plot can be obtained. Among essential
thermodynamic parameters that can be deduced from the
plot (Table S1), the affinity and entropy of binding are
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Fig. 1 Isothermal titration calorimetry (ITC) results corresponding to the different types of peptamer-ligand interactions
designed in Scheme 1. In each of the above results, the top row is the raw data of power versus time (in minutes), while the
bottom row is the corresponding integrated enthalpy versus the molar ratio of titrant vs titrand. The titrants vs titrands are
respetively: (a1') Cucurbituril (Q8) vs Peptamer (Pep), (a1) Q8 vs Pep, (b1') Pep-2Q8 vs Integrin (Int), (b1) Pep-Q8 vs Int, (b2) Pep
vs Int, (c1') Cu (II) vs Pep-2Q8-Int, (c1) Cu (II) vs Pep-Q8-Int, (c2) Cu (II) vs Pep-Int, (c3) Cu (II) vs Pep.
a1’~c1’, Table S1, sub-table b1’, c1’). Specifically, surplus
amount of Q8 can first result in a 2:1 binding between Q8
and peptamer with positive entropy consistent with the
fact that this interaction is mainly driven by hydrophobic
force (sub table a1’). Upon integrin binding, the bending of
the peptamer put two bulky Q8 molecules in proximity,
resulting in structural hindrance and negative entropy, as
well as reduced affinity (sub table b1’). As the control, if
the interaction with Q8 is omitted, direct interactions with
integrin and Cu (II) ion both have very slight increase of
binding strength compared with the case of surplus Q8
(Fig. 1, b2~c2, Table S1, sub-table b2, c2), while the
unstructured peptamer, without any interactions with Q8
or integrin, show almost no affinity towards the cupric ion
(Fig. 1, c3, Table S1, sub-table c3). According to these
results, the design of this peptamer is successful, thus its
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Scheme 2. Integrin bioassay developed based on the peptamer. (a) assay procedure, (b) scheme of the reaction of catalytic signal
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amplification.
surplus Q8 is incubated with the sensing interface to open
all the integrin-free peptamers, while the integrin-bound
peptamers will resist this effect via the strong peptamer-
integrin binding and these peptamers can bind with Cu
(II). The cupric ion complexed by these integrin-bound
peptamers can then be used for catalytic signal
amplification to generate readout signal positively related
to integrin concentration in the samples.
To better realize this design, the essential step for signal
conversion, i.e., the blocking of peptide loop re-opening
by integrin binding, is first investigated by ITC. As shown
Fig. 2 Quantitative analysis of integrin using the peptamer-
based bioassay. (a) Square wave voltammograms (SWVs) of
DAP showing the gradual increase of signal response with
in Fig. S1a, the locked-up peptamer, in the absence of
integrin concentration. (b) Peak currents in (a) plotted as a
captured integrin, can interact with Q8 at a 1:1 ratio, in an
function of integrin concentration. Inset is the linear range
entropy-driven manner, indicating that these peptamers
and the corresponding formula obtained via regression
can be relieved by surplus Q8 from the entropically
analysis. The error bars represent standard deviation from
unfavorable locked form. In the presence of captured
integrin (Fig. S2b), no evident interaction is observed,
average (n=3).
final conformation and affinity for cofactor ion can reliably
represent the upstream interactions with protein and
other ligands.
indicating that integrin binding with the Q8-locked
peptamer can effectively stabilize this structure to negate
the trend of the re-opening induced by excessive Q8.
Meanwhile, the interaction of Cu (II) with Pep-Q8 in the
absence of integrin has also been investigated (Fig. S3).
The open Pep-Q8 has no evident interaction with Cu (II)
(Fig. S3a), leading to negligible background (curve A in Fig.
S3c). On the other hand, the closed Pep-Q8 can have
interaction with Cu (II) (Fig. S3b and curve B of Fig. S3c),
but it can be opened by surplus Q8 as shown in Fig. S2a. So
following the detection procedure in Scheme 2, low
background can be obtained.
Employing this peptamer, an integrin assay with high
sensitivity suitable for tissue sample analysis can be
developed (Scheme 2). The peptamer pre-immobilized on
the electrode surface can be locked-up in the looped form
by binding with proper concentration of Q8. This as-
prepared sensing interface can then be used for capturing
target integrin in standard or biological samples, while the
strong binding strength of this looped form can greatly
facilitate the sensitivity in diluted samples. To
quantitatively convert integrin binding to signal readout,
For a balanced best performance of the proposed assay,
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Also, as shown in Fig. S8c, free Cu ion has no evident
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interference to the signal readout. Under these optimal
conditions, the catalytically generated signal response
increases proportionally to the logarithm of integrin
concentration from 0.01 nM to around 0.5 μM (Fig. 2). The
average standard deviation of all repetitive measurements
is within 5%, showing an acceptable reproducibility.
Besides, several control species have been employed to
examine the specificity of this method. As can be seen in
Fig. 3, only background level of response can be observed
for these species, so the specificity is satisfactory.
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Fig. 3 SWVs of DAP showing the specificity of the assay. All
control species are of 100 nM.
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As an essential component of cell skeleton, integrin
connects the framework of the cells to the extra-cellular
matrix (ECM). In the process of tumor invasion, as well as
other tissue remodeling processes such as embryonic
development, the expression and activity of integrin are
frequently regulated to direct and facilitate cell
migration26. In a range of cancers, it has been reported that
integrin over-expression is connected with tumor
invasiveness and integrin may serve as a biomarker of
malignant transformation26-28. Here, employing the above
designed peptamer and the developed biosensing method,
integrin abundance in several clinical samples of thyroid
carcinoma is assayed, with the results being grouped
respectively according to the T (tumor), N (node) and M
(distant metastasis) stages of the cases examined (Fig. 4).
Under the grouping based on T stage and M stage, the
samples show no evident connection with the advancing
of cancer (Fig. 4a,c), but the expression seems to be, to
some extent, correlated with N stage, or the situation of
lymph node metastasis (Fig. 4b). This can be explained
considering the biological function of integrin in
regulating cell connection with ECM. As a matter of fact,
breaking through the tissue barriers such as the ECM and
the basal membrane is the necessary step of tumor
filtration and metastasis. In this process, the up-regulation
of integrin can indicate a frequent reconfiguration of
cellular connection with the ECM, so integrin over-
expression can usually be detected on the front edge of
invasion, as well as sites of early metastasis located closely
to the primary tumor, such as the sentinel lymph nodes.
other important steps of the assay method have been
optimized. First, the proper concentration of Q8 to
induceloop-formation of the peptamer is investigated. As
is shown in Fig. S4, the amount of looped peptamer
formed by different concentrations of Q8 is evaluated by
the signal readout of cupric ion complexed by the looped
peptamers. While insufficient Q8 cannot turn all the
surface-immobilized peptamers into the looped form,
excessive Q8 may keep the peptamer in a linearform,
resulting in reduced signal readout. For the accuracy of
detection, the optimized Q8 concentration (60 μM) is
selected for all the following experiments. The incubation
time for this step has also been optimized (Fig. S5), and 40
min incubation time is known to required to convert all
the surface immobilized peptamers to the closed form.
Since the catalytic activity of cupric ion is exploitable for
signal amplification, the response of the product of
catalytic signal amplification is recorded for all the
following experiments as the final signal readout. Using
this type of signal readout, as well as the biosensing
interface prepared under the above optimized conditions,
the interaction with the target integrin is found to
approach saturation in 40 min (Fig. S6). Finally, the
concentration of Q8 required to re-open the surplus
peptamers is studied, and 40 μM is known to be sufficient
to open most of the surplus peptamers on the electrode
surface (Fig. S7). In addition, the concentration of Cu (II)
has also been optimized (60 μM), the experimental result
of which has been shown in Fig. S8a, b.
Fig. 4 Box chart to show the distribution of the detected concentration of integrin in patients of thyroid carcinoma, grouped by
(a) T stage, (b) N stage and (c) M stage. For each sample, the detected integrin in cancerous tissue is normalized as the fold of
increase compared with the adjacent normal tissue.
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Conclusion
Analytical Chemistry
(10) Monecke, T.; Dickmanns, A.; Ficner, R., Febs Journal 2014,
281, 4179-4194.
1
In this work, a peptamer is designed to model the
structural feature of protein observed in cell signaling on
a simplified peptide sequence. This peptamer can react to
the presence and the concentration of the target protein
as well as other ligands by the formation/de-formation of
a specific conformation. In the meantime, its temporally
defined interaction with a series of ligands can stabilize
this conformation for the forming of a stable peptide-Cu
(II) complex that can show catalytic activity. Using this
peptamer, an interface electrochemical bioassay method
is established for the analysis of integrin expression.
Moreover, this method can also be successfully applied in
the detection of integrin in clinical tissue samples of
thyroid carcinoma. The detected integrin level has a
parallel with the state of lymph node metastasis,
indicating the active role of integrin in promoting tumor
invasion. So, these results may point to the prospective
use of the proposed peptamer in clinical analysis of
biomarker in the future.
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ASSOCIATED CONTENT
Supporting Information Supporting data on the design
principle as well as optimization of various experimental
conditions. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Corresponding author at State Key Laboratory of
Pharmaceutical Biotechnology, Department of Biochemistry,
Nanjing University, Nanjing 210093, China. Fax: +86 25
83592510. E-mail address: genxili@nju.edu.cn.
(26) Zhang, Z. Y.; Xu, K. S.; Wang, J. S.; Yang, G. Y.; Wang, W.;
Wang, J. Y.; Niu, W. B.; Liu, E. Y.; Mi, Y. T.; Niu, J., Clin. Oncol.
2008, 20, 61-66.
(27) Bendas, G.; Borsig, L., Inter. J. Cell Biol. 2012, 2012, 676731-
676731.
ACKNOWLEDGMENT
This work is supported by the National Natural Science
Foundation of China (Grant Nos. 21235003, 21327902, J1103512,
J1210026).
(28) Oxboel, J.; Binderup, T.; Knigge, U.; Kjaer, A., Oncol.
Rep. 2009, 21, 769-775.
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