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Heparin-Promoted Cellular Uptake of the Cell-Penetrating

Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single
Li-Chun Hung,,# Ingjye Jiang,,# Chien-Jung Chen,,# Jia-Yin Lu, Yi-Fen Hsieh, Ping-Hsieh Kuo,
Yi-Lin Hung,, Lily Hui-Ching Wang,, Margaret Dah-Tsyr Chang,*,, and Shih-Che Sue*,,

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan

Instrumentation Center, National Tsing Hua University, Hsinchu 30013, Taiwan

Department of Medical Science, National Tsing Hua University, Hsinchu 30013, Taiwan

Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan
S Supporting Information

ABSTRACT: A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from

human eosinophil cationic protein has been recently designated as a potent cell-
penetrating peptide. A model system containing peptide, glycan, and lipid was
monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-
penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC)
lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles
for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less
eect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane
insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it
signicantly improved the heparin-binding ability. GBPECP bound either heparin or lipid
in the presence or absence of the other ligand indicating that the peptide has two
alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are
for GAG binding. We developed a molecular model showing that the two eects synergistically promote the penetration. The
loss of either eect would abolish the penetration. GBPECP has been proven to enter cells through macropinocytosis. The GBPECP
treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be
modulated by GBPECP internalization. The intracellular penetration of GBPECP leading to inhibition of epithelial cell migration
and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides.
Therefore, GBPECP shows substantial potential as a novel delivery therapeutic through rapid and eective internalization and
interference with cell mobility.

I ntracellular delivery is one of the issues of greatest concern

for pharmaceutical agents. Biological membranes hinder the
entry of substances with low permeability into the cell. A few
cargoes further complicate the interaction between the
molecules and the membrane.
We have recently identied a multifunctional CPP from
successful agents have been designed to transport drugs or human eosinophil cationic protein, ECP (also known as human
drug-loaded pharmaceutical carriers into the cytoplasm through ribonuclease 3, hRNase3). The peptide has substantial heparan
the endocytic pathway.1 An emerging strategy to overcome the sulfate (HS), heparin (HP), and lipid binding activities.5,6 It
membrane barrier is the use of cell-penetrating peptides also exhibits epithelial cell targeting,6 antimigration,7 anti-
(CPPs). CPPs are generally small in size and contain less invasion,7 antiangiogenesis,7 and immunomodulatory8 activ-
than 30 amino acids.2 When CPPs are tethered on cargo, they ities. ECP interacts with cell surface glycosaminoglycans
deliver the payload into cells. Thus, attaching membrane- (GAGs), particularly HS. GAGs are anchored on the cell
impermeable therapeutics to CPPs provides a solution for surface as a signaling co-receptor, enhancing receptorligand
intracellular delivery. Many natural and synthetic peptides have interactions that mediate cell growth, motility, and the immune
been reported to have cell-penetrating activities.3 These response.9 Among the various GAGs, HS has been identied in
peptides shared some common features, such as positively all animal tissues, and HS chains are attached to a core protein
charged amino acids and hydrophobic residues, and some were
characterized as amphipathic peptides.4 However, dierent Received: September 29, 2016
CPPs still have varying hydrophobicity, charges, and Accepted: December 12, 2016
amphipathicity. The sizes and chemical properties of dierent Published: December 12, 2016

2016 American Chemical Society 398 DOI: 10.1021/acschembio.6b00864

ACS Chem. Biol. 2017, 12, 398406
ACS Chemical Biology Articles

Figure 1. GBP sequences and cell binding and cellular uptake activities. (A) Peptide sequences of GBPECP, GBPEDN, and GBPECP(W4R). (B) A549
lung cancer cells were incubated with FITC, FITCGBPECP, FITCGBPEDN, and FITCGBPECP(W4R) for 1 h at 4 C. The cells were subjected to
ELISA. (C) A549 cells were incubated with 5 M TMR, TMRGBPECP, TMRGBPEDN, and TMRGBPECP(W4R) for 1 h at 37 C. After the
treatment, the cells were trypsinized, suspended in PBS, and subjected to ow cytometry. The level of bound TMR signal was set as negative control
(black line). The intensities of TMRGBPECP, TMRGBPEDN, and TMRGBPECP(W4R) bound to A549 cells were represented by green, brown, and
cyan lines, respectively. (D) A549 cells were treated with 5 M TMR, TMRGBPECP, TMRGBPEDN, and TMRGBPECP(W4R) for 1 h at 37 C, and
the cellular uptake was examined using uorescence microscopy: red, TMR-labeled GBPECP; blue, DAPI in nucleus. The white scale bars in the
panels represent 20 m.

to form heparan sulfate proteoglycans (HSPGs). As a penetration activities in normal epithelium cells. GBPECP exerts
consequence of binding to a receptor, HS can recruit natural signicant cell-binding and penetration activities, whereas
receptor ligands to promote downstream bioactivities and also GBPEDN exhibits quite poor activities. We swapped the GBPEDN
induce crosstalk among various receptor families.10 sequence for the GBPECP sequence and prepared GBPECP
Three HS binding regions have been identied in human variants, such as GBPECP(R3Q) and GBPECP(W4R), to determine
ECP, 34RWRCK38, 73RSRFR77, and 101RPGRR105, and the rst the signicance of the individual positions. In GBPECP(R3Q),
binding region possessed the most signicant binding activity. replacement of Arg3 with Gln3 caused an 7080% reduction
A peptide (32NYRWRCKNQN41) containing the RWRCK in both cell binding and penetration activities. Surprisingly,
motif and 2 and 3 residue extensions of the N- and C-termini, substitution of a basic Arg residue at position 4 enhanced the
respectively, has been characterized as the minimal sequence cell binding activity by 5-fold but caused a signicant loss in
required for sucient cell penetration. The peptide is known as cellular internalization. The improved cell binding did not
a GAG-binding peptide of ECP (GBPECP).11 Interestingly, induce higher penetration activities in the case of GBPECP(W4R).
GBPECP did not contain the canonical heparin-binding motifs Although previous reports have suggested that Arg and Trp
BBXBX and BBBXXBX, where X represents hydrophobic or are important for increasing cell uptake eciency of CPP, the
uncharged residues and B represents basic residues.12 Another exact molecular pathways remain controversial.1315 Because
GAG-binding peptide from human eosinophil-derived neuro- cell surface binding and internalization are usually correlated,
toxin (EDN, also known as hRNase2) consists of a canonical GBPECP, GBPEDN, and GBPECP(W4R) constitute a great model
heparin-binding motif with the sequence NYQRRCKNQN and for elucidating CPP cellular uptake mechanisms. In this study,
is known as GBPEDN. The two peptides only dier in two GBPECP, GBPEDN, and the derivative GBPECP(W4R) were rst
residues at positions 3 and 4, which are Arg3 and Trp4 in characterized to determine their abilities to modulate epithelial
GBPECP and Gln3 and Arg4 in GBPEDN. The two-residue cancer cell migration and invasion, and the tendency was
dierence is responsible for dierences in the cell binding and correlated with the results established in normal epithelial cells.
399 DOI: 10.1021/acschembio.6b00864
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A model system containing the peptide, glycan, and lipid was

monitored by nuclear magnetic resonance (NMR) and
uorescence spectroscopy to determine cell penetration
mechanism. A heparin octasaccharide fragment with a
dodecylphosphocholine (DPC) lipid micelle was titrated into
the peptide solutions, and we evaluated the roles of individual
residues in promoting membrane penetration. Our data showed
that two separate binding sites for heparin and lipids are
integrated on GBPECP. The two eects synergistically promote
GBPECP penetration. This study provides the structural basis for
the mechanism by which CPP penetrates cells through a HS-
mediated process. Therefore, we establish a new framework for
using the unique structural motif RWRCK to assist in future
CPP designs.


Cell Surface Binding Activities of the GBPs. We
previously evaluated cell binding and penetration activities of
GBPECP and GBPEDN using normal human epithelial BEAS-2B
cells and Chinese hamster ovary (CHO) cells.6 To realize the
commonality, we further tested the eect on human epithelial
cancer cells. The abilities of N-terminal uorescein isothiocya-
nate (FITC)-labeled GBPs to bind the cell surface were
examined by incubating A549 lung cancer cells with the
peptides at 4 C. FITCGBPs bound the A549 cancer cells in a
concentration-dependent manner because the uorescence
intensity was linearly correlated with the peptide concentration
(Figure 1B). GBPECP(W4R) displayed the highest cell binding
intensity, which was approximately 34-fold higher than the
other peptides, whereas GBPECP was the second strongest
binder, with a slightly higher binding activity than GBPEDN. The
binding activities seemed to correspond to the number of basic
residues as GBPECP(W4R) contained 4 basic residues and the
other two peptides only contained three. The tendency of Figure 2. GBPs inhibit A549 cell migration and invasion. (A) A549
GBPECP and GBPEDN to bind to the epithelial cancer cell cells were treated with GBPECP, GBPEDN, and GBPECP(W4R) before the
surface was well correlated with previous results in BEAS-2B cells were seeded in the top chamber of the Transwell membranes.
and CHO cells.6 After an 18-h incubation at 37 C, the cells that migrated through the
Transwell lters were estimated by counting the number. (B) A549
Cellular Uptake of the GBPs. Because tetramethylrhod-
cells were treated with GBPECP, GBPEDN, and GBPECP(W4R) before the
amine (TMR)-conjugated GBPs showed greater cell pene- cells were added to the top chamber of Matrigel-coated Transwell
tration activity, TMR was alternatively used to label the N- membranes. After a 24-h incubation at 37 C, the cells that migrated
termini of the GBPs for cellular uptake experiments, which through the Transwell lters were estimated by counting the number.
were assessed by uorescence-activated cell sorting (FACS) The data represent the means SD (standard deviations) of three
and uorescence microscopy. TMRGBPECP displayed sig- independent experiments: *P < 0.05; **P < 0.01 compared with the
nicant internalization (Figure 1C). If we dened TMR control.
GBPECP internalization as 100%, GBPEDN and GBPECP(W4R)
exhibited 23% and 21% decreases in internalization, respec- concentration of 12.5 M, only 20% inhibition was detected
tively. In addition, only GBPECP acted as a cell-penetrating (Figure 2A). Because abnormal expression of cell surface
peptide, as shown in the uorescence microscopy image HSPGs also contributed to tumor cell growth and invasion,17
(Figure 1D). Again, the observation corresponded to the Transwell invasion assay was further employed to investigate
previous result.6 The strong cell surface binder, GBPECP(W4R), the inhibitory eects of the GBPs. The upper chambers of the
did not exhibit a greater cell-penetrating activity. The cell Transwell were coated with Matrigel to mimic extracellular
surface binding and cellular uptake properties appeared to be matrix, and the A549 cells were separately pretreated with 1, 5,
uncoupled in GBPECP(W4R). and 12.5 M GBPs. The invasion of A549 cells was signicantly
Inhibition of Cell Migration and Invasion by GBPs. inhibited by GBPECP in a dose-dependent manner, with
HSPGs modulate cell migration by interacting with growth reductions of 19%, 43% and 58%, respectively (Figure 2B).
factors or chemokines and drive cells to migrate toward specic However, GBPEDN and GBPECP(W4R) only reduced invasion by
stimuli.16 Because our GBPs have already been shown to bind 20% or less. These results clearly showed that GBPECP was the
cell surface HSPGs,6 we suspect that cancer epithelial cell strongest inhibitor of migration and invasion of A549 cells.
migration and invasion would be modulated accordingly. As Although GBPEDN and GBPECP(W4R) contain canonical heparin-
expected, in vitro Transwell migration assay indicated that A549 binding motifs with the sequences QRRCK and RRRCK,
cell migration was signicantly inhibited from 20% to 60% by 1 respectively, their abilities to modulate cell migration and
to 12.5 M GBPECP (Figure 2A). Meanwhile, GBPEDN and invasion are relatively weak. GBPECP, with the unique motif
GBPECP(W4R) showed mild inhibition, and even at the highest RWRCK, eectively inhibited cell migration and invasion.
400 DOI: 10.1021/acschembio.6b00864
ACS Chem. Biol. 2017, 12, 398406
ACS Chemical Biology Articles

Because cellular uptake activity is a critical factor to dierentiate However, we cannot detect Arg side chain H (fast exchange in
GBPECP from GBPEDN and GBPECP(W4R), the cell penetration neutral pH condition) and H (line broadening after binding)
mechanism of GBPECP was further investigated. while H, H, and H only produced small perturbations
Peptide Characterization by NMR. The 1H and 13C (Figure 4). Therefore, backbone signals were the best
NMR signals of GBPECP, GBPEDN, and GBPECP(W4R) were indicators for reporting this binding. We estimated the binding
assigned (Figures S1 and S2), and we listed the nal constant Kd by tting the titration curves (Figure S4). Only
assignments in Supporting Table S1. Backbone chemical shifts GBPECP(W4R) showed typical titration curves, and the binding
are correlated with backbone dihedral angles. This correlation was saturated after a molar ratio of 1:1. The Kd values estimated
allows us to predict the secondary structures using the chemical for Arg5 and Lys7 were 7 and 9 M, respectively. GBPEDN did
shift dierences and the predened values for random coil, not reach saturation under our experimental conditions, and a
namely, = observed randomcoil. We employed the values nearly linear response was observed in the dose-dependent
derived from 13C and 13C and predicted that when the (C titration experiment (Figure S4). This result implied that
C) value is negative, it represents a -strand, and when GBPEDN had a weaker heparin-binding activity. The estimated
the value is positive, it represents an -helix.18,19 Based on this Kd value was 80 M. GBPECP revealed more than one set of
parameter, the three peptides generally showed random coil resonances in the initial and middle titration points, such as the
structures, as the (C C) values were generally small examples observed at a molar ratio of 1:1 (Figure 4B, upper
(Figure 3). The result matched the observations from CD panel). The presence of multiple resonances implied that
GBPECP adopted dierent conformations. The exchange
between the dierent conformations led to severe peak
broadening, thus hampering accurate measurement of the
chemical shifts. The presence of multiple conformations
reected the binding selectivity of GBPECP toward heparin
sequences because the heparin octasaccharide with a dened
length contained structural inhomogeneity of more than 10
types of sequences. Nevertheless, the apparent Kd value was
estimated as 39 and 42 M for Arg5 and Lys7, respectively,
which was stronger than GBP EDN but weaker than
NMR Titration of the Eects of the Membrane on
GBPECP. We prepared micelle formulations using a lipid
mimetic DPC and titrated them into the GBP solutions.
Only GBPECP showed perturbations upon the DPC titration
(Figure S5). This result was correlated with the less cell
penetrating activities of GBPEDN and GBPECP(W4R).5 The
binding-induced perturbation of GBPECP is depicted in Figure
Figure 3. Predictions of the secondary structures of the GBPs. The 5. The NH resonances of Arg3, Cys6, and Asn8 showed
predictions of secondary structures are plotted using the value of signicant perturbations (Figure 5A). In the presence of the
(C C) versus the residue number. A negative value of heparin octasaccharide, DPC also induced similar downeld
(C C) represents a -strand; a positive value represents an
-helix. Three peptides show weak secondary structural propensities in
shifts in GBPECP/HP complex (Figure 5B), suggesting that the
solution. GBPECP has a greater value for Trp4. presence of heparin did not change the lipid-binding mode.
The same conclusion was derived by analyzing 1H13C HSQC
spectra, which showed that the GBPECP/HP complex still had
experiment (Figure S3). However, there was an exception for the ability to bind DPC (Figure S6). Notably, the presence of
GBPECP W4, which exhibited a signicant negative value of heparin increased the sensitivity of Trp4, Arg5, and Lys7 in that
1.7 ppm. The Trp4 residue showed local -strand tendency in the chemical shift perturbations were increased by 2-fold.
solution, presumably due to bulky and hydrophobic side chain. Tryptophan NMR Spectra and Fluorescence. The
This property might account for better dispersion in its 1H1H hydrophobic tryptophan residue, Trp4, is the major dierence
TOCSY spectrum (Figure S1). among the three peptides. We examined tryptophan side chain
NMR Titration of the Eects of Heparin on GBPs. uorescence and NMR Trp4-H signal. Fluorescence spectros-
Heparin octasaccharide was titrated into the GBP solutions in a copy determined whether the indole ring is buried in a
stepwise manner, and the titrations reached a nal molar ratio hydrophobic environment.22 During the DPC titrations,
of 1:2. We depicted the NH chemical shift perturbations of the intrinsic tryptophan uorescence emission was enhanced and
nal titrations (Figure 4). The titration induced signicant appeared to be blue-shifted in the presence and absence of
perturbations at Arg5 and Lys7 in GBPECP, indicating that these heparin (Figure 6A, insets), indicating that the indole ring was
residues had major eects from binding, whereas Arg3 inserted into the hydrophobic region of DPC. Interestingly, the
possessed less eect. In GBPEDN, Arg5 and Lys7 were also resulting uorescence intensities of GBPECP and heparin-bound
the dominant residues. Meanwhile, Arg4 became sensitive to GBPECP were not dierent, suggesting that heparin binding did
the titration. Notably, heparin induced comparable scales of not change the local polarity of the Trp residue. This result
perturbations in GBPECP and GBPEDN. In GBPECP(W4R), Arg5 matches the observation that the side chain NMR signals of
dominated the binding and displayed the greatest perturbation Trp 4 are not sensitive to the presence of heparin. There is no
while Arg4 and Lys7 had comparable secondary eects. Arg3 carbohydrate/aromatic stacking interaction23 to promote the
still had less eect. The participation of Arg could be assessed interaction. In addition, the corresponding uorescence
by monitoring side chain chemical shift perturbations.20,21 titration curves also indicated that there was no preference
401 DOI: 10.1021/acschembio.6b00864
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Figure 4. Heparin-induced chemical shift perturbations in the GBPs. (A) Heparin was titrated into GBP solutions in a [peptide]/[HP] molar ratio of
1:2. The chemical shift perturbations of NH were recorded. (B) Representative area of the TOCSY spectra for the titration experiments. The
TOCSY spectra were recorded at dierent [peptide]/[HP] molar ratios of 1:0, red; 1:1, green; and 1:2, blue.

Figure 5. DPC-induced chemical shift perturbations in GBPECP. (A) DPC-induced perturbation proles of GBPECP in the presence (blue) or absence
(red) of heparin. (B) Representative region of the TOCSY spectra of GBPECP titrated with DPC in the absence (upper panel) or presence (lower
panel) of heparin. The TOCSY spectra were recorded at [peptide]/[DPC] molar ratios of 1:0, black; 1:10, red; and 1:20, blue.

for membrane binding under both conditions (Figure 6A). The GBPECP bound DPC (Figure 7B). In the presence of heparin,
penetration eect was conrmed by much larger chemical shift the diusion coecient was even smaller, indicating the
perturbation in the Trp4-H signal (Figure 6B). Thus, GBPECP formation of a larger ternary GBPECP/HP/DPC complex.
has an alternative-binding site for DPC. The presence of The diusion coecients reached similar values at high DPC
heparin did not change this interaction with the lipid. concentrations, as most of the GBPECP or GBPECP/HP complex
Diusion Coecient and Lipid Micelles. We employed had already bound to DPC (Figure 7B). In addition, the
the diusion NMR technique to evaluate the eect of GBPECP presence of GBPECP signicantly changed the formation of
on DPC micelles. Perdeuterated DPC was used to eliminate the DPC micelles. DPC acts as a monomer at low concentrations
DPC 1H signal and determine the diusion coecient of and starts to aggregate into a micelle when the concentration is
GBPECP. Alternatively, regular DPC with 1H signal was used to higher than the critical micelle concentration (CMC). The
detect the diusion coecient of DPC. GBPECP displayed a process could be monitored using the diusion coecients.
consistent diusion coecient of approximately 26.5 1011 The CMC of free DPC could be empirically estimated from the
m2/s at GBPECP concentration from 0.5 to 5 mM. DPC intersection of two extreme segments of the curve using a linear
micelles decreased the diusion coecient, indicating that regression analysis.24 The value was estimated to be
402 DOI: 10.1021/acschembio.6b00864
ACS Chem. Biol. 2017, 12, 398406
ACS Chemical Biology Articles

Figure 6. Tryptophan side chain spectra of GBPECP. (A) Intrinsic uorescence spectra. Titration curves of the maximum uorescence intensity of
GBPECP versus the DPC concentration in the absence or presence of heparin. The inset in the lower right corner is the uorescence curves of 10 M
GBPECP without (red) and with 20 mM DPC (blue); the inset in the upper left corner represents the uorescence curves of 10 M GBPECP and 50
M heparin without (red) and with 20 mM DPC (blue). (B) TOCSY spectra of Trp4 H. The molar ratio of [GBPECP]/[HP] used in the
experiment was 1:2 mM, with increasing amounts of DPC: 0, 6, 10, and 20 mM (from top to bottom).

Figure 7. Diusion coecients of DPC and GBPECP. (A) The diusion coecient of DPC is plotted as a function of the DPC concentration: DPC
alone (black) and in the presence of GBPECP (red). (B) The diusion coecient of GBPECP is plotted as a function of the DPC concentration:
GBPECP alone (red) and in complex with heparin (black). The concentrations of GBPECP and heparin used in the diusion experiment were 1 and 2
mM, respectively.

approximately 4 mM (Figure 7A). When GBPECP was present represents a new framework for designing potent CPPs in
in the solution, stable micelles only formed at higher DPC the future. We propose a schematic model to depict the cell
concentrations. The estimated CMC increased to 7 mM. The penetration mechanism of GBPECP (Figure 8). As a co-receptor,
presence of GBPECP disrupted the formation of micelles. HSPGs are indispensable for the cell entry of GBPECP.
Model of GBPECP Penetration. Previous studies have Positively charged residues, Arg5 and Lys7, have substantial
shown that the guanidinium group of Arg rather than Lys or roles in recognizing the HS fragments, whereas the lipid-
His is important for cell penetration.2527 Furthermore, several binding residue Trp4 acts as an anchor to aid peptide docking
tryptophan residues spaced throughout the sequence of a on the lipid surface. Meanwhile, the residue further disrupts the
polyarginine peptide (i.e., RWRRWRRWRRWR) further compactness of the membrane and promotes ecient cell
increased cellular internalization eciency.28,29 In this study, entry. These two eects appeared to synergistically promote
the identied RWRCK sequence motif containing only one GBPECP penetration and further modulate the microenviron-
tryptophan and three basic residues appeared to be very ment of the cell surface, including reducing the migration and
eective at inducing cellular internalization. This peptide invasion of epithelial cells.
403 DOI: 10.1021/acschembio.6b00864
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by which GBPECP exerts its antimigration and anti-invasion

functions is currently unclear, the mechanisms modulating the
turnover of endocytosis receptors and cell surface molecules
will be investigated in future studies.

Peptides, Heparin Octasaccharide, and DPC. All GBPs,
including the FITC- and TMR-labeled versions, were synthesized by
Genemed Synthesis Inc. The purity of these GBPs was evaluated by
analytical high-performance liquid chromatography and shown to be
greater than 95% (w/v). The GBP sequences were conrmed using
Figure 8. A schematic model of the mechanism by which GBPECP mass spectrometry. The heparin-derived fragment corresponding to
interacts with heparin and the membrane lipids on the cell surface. the octasaccharide was used in this study. To prepare heparin
octasaccharide, low-molecular-weight porcine intestinal heparin
(Sigma-Aldrich Corporation) was digested with Flavobacterium
Roles of Individual Residues. Chargecharge interactions heparinase I (EC; Sigma H-2519), and the depolymerized
are the primary driving force for the heparin binding activity of heparin oligosaccharides were separated on a gel ltration Bio-Gel P-
GBPs30 because heparin is a highly negatively charged 10 column (Bio-Rad Laboratories). 32 The prepared heparin
molecule.31 Here, the Arg5 and Lys7 residues are more octasaccharide contained structural inhomogeneity that more than
responsible for heparin binding because their NMR resonances 10 types of sequences were detected in the preparation. To mimic a
displayed the most substantial perturbations. However, Arg3 hydrophobic environment, DPC was used to prepare lipid micelles.
was less sensitive to heparin, and we concluded that Arg3 is less DPC and perdeuterated DPC were purchased from Avanti Polar
important. The result corresponds well with previous Lipids Inc. and Cambridge Isotope Laboratories Inc.
A549 Lung Cancer Cells and Cell Culture. A549 lung cancer
observation that the replacement of Arg3 with Gln3 in the cells were purchased from ATCC and cultured in T-75 asks with
GBPECP variant GBPECP(R3Q) only reduced cell binding and Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-
penetration by 20%.6 Similarly, among the four basic residues in Aldrich) supplemented with 10% (v/v) heat-inactivated fetal bovine
GBPECP(W4R), Arg3 still exhibited a reduced response compared serum (FBS) (Gibco, Invitrogen) and 1% (v/v) PSA (penicillin,
with Arg4, Arg5, and Lys7 (Figure 4), and Arg4 was more streptomycin, and amphotericin). The cells were incubated in a
sensitive in the binding assay. The heparin-binding enhance- humidied atmosphere with 5% CO2 and 95% air at 37 C. For
ment did not entirely translate into the cell penetrating ability. subcultures, the medium in the cultured dish was removed, and the
Intriguingly, GBPECP(W4R) exhibited the strongest binding cells were washed with 10 mL of Ca2+/Mg2+-free PBS, detached with 1
anity for A549 cells, but it did not penetrate the cells. mL of trypsin/EDTA [0.05%/0.02% (w/v)] (Biochrom AG) for 5 min
at 37 C, and suspended in 9 mL of fresh serum-containing medium to
Based on the NMR and uorescence data, we concluded that inactivate trypsin. Subsequently, 1020 L of the cell cultures
Trp4 and lipids directly interacted and that the tryptophan side containing approximately 1 106 cells were seeded. The cells were
chain was inserted into the hydrophobic core of the membrane. transferred to a new T-75 ask containing a total of 12 mL of
The presence of Trp4 also changed the uidity of the lipids and prewarmed medium. The cells were used after 23 days.
prohibited micelle formation. In addition, among the three Cell Surface Binding Assay. The binding of FITCGBPs to
peptides, GBPECP containing Trp4, displays better secondary A549 cells was assayed using a cell-based ELISA. Approximately 2
structure properties, as reected in the improved dispersive 104 cells were seeded in each well of a 96-well black plate and cultured
resonance in the NMR spectra. Although chemical shift at 37 C for 24 h. The 96-well black plate was washed with 200 L of
information and CD spectra mainly indicated random-coil PBS and preblocked with 100 L of 2% (w/v) BSA/PBS per well for 1
h at 4 C. The black plate was washed with 200 L of ice-cold PBS.
properties, we still observed that Trp4 tended to form a weak FITC-conjugated peptides were diluted to the desired concentrations
local -strand in solution, partially due to the bulky indole ring. in PBS and added to each well. The plate was incubated on ice for 1 h.
Because of the weak -strand tendency, the Trp4, Cys6, and A549 cells were treated with 1, 2.5, 5, 10, and 12.5 M FITCGBPs.
Asn8 residues might preferentially remain on one side of After the binding was complete, each well was washed with 100 L of
GBPECP, and Arg5 and Lys7 are shifted toward the other side PBS. The FITC uorescence intensity was measured using a
(Figure 8). The result corroborated the observation that Cys6 uorescence spectrophotometer (Wallac Victor II, PerkinElmer)
and Asn8 of GBPECP were sensitive to the presence of DPC. with excitation and emission wavelengths of 485 and 521 nm,
GBPECP in Modulating Cell Migration and Invasion. respectively.
GBPECP targeted rapidly proliferating cells, including epithelial Cell Penetration Assay. Approximately 3.0 105 A549 cells were
seeded in each well of six-well plates and cultured in the RPMI 1640.
tumor cells with elevated HS expression on the cell surface.7 After 24 h of culture, each TMRGBP was dissolved in FBS-depleted
Therefore, GBPECP might show potential in the design of RPMI 1640, and 5 M peptide solutions were added to individual
delivery vectors and therapeutic supplements. GBPECP, which wells and incubated for 1 h at 37 C. Each well was treated with 0.05%
contains the RWRCK motif, inhibited A549 cell migration and trypsin and 0.02% EDTA and suspended in PBS to harvest the cells.
invasion across the membrane indicating that not only cell- The uorescent intensities of the cell samples were measured using a
binding but also cell-penetrating activity is essential for resisting FACSCalibur ow cytometer (BD Biosciences) and excitation and
cancer cell migration and invasion. Likewise, cell penetration emission wavelengths of 488 and 515545 nm, respectively. The
could be converted into cellular responses to prevent cell relative internalization of each peptide was reported as the mean
migration and invasion. Furthermore, GBPECP anchored on the uorescent signal for 10 000 cells.
Fluorescence Microscopy. Approximately 5.0 103 A549 cells
cell surface, followed by internalization via micropinocytosis, were cultured on coverslips in RPMI 1640 for 24 h. The cells were
which is correlated with lipid rafts.6 This study deciphers how a incubated with 5 M TMRGBPs for 1 h at 37 C to examine cellular
CPP penetrates the cell through individual specic residues by uptake. The cells were washed with PBST (1 PBS + 0.05% (v/v)
attaching to the negatively charged HS and subsequently Tween-20) twice, xed with 4% (w/v) paraformaldehyde, and rinsed
causing membrane instability. Although the signaling cascade twice with PBS. The coverslips were mounted in Vectashield antifade

404 DOI: 10.1021/acschembio.6b00864

ACS Chem. Biol. 2017, 12, 398406
ACS Chemical Biology Articles

mounting medium with DAPI (Vector Laboratories). Images were = 10 M/20 mM or [GBPECP]/[heparin]/[DPC] = 10 M/50 M/20
acquired using an Axiovert 135 inverted uorescence microscope (Carl mM.
Cell Migration and Invasion Assay. Cell migration activity was
assessed using a Transwell-24 plate preinserted with 8-m pore size
polyethylene terephthalate lter membranes (BD Falcon Cell Culture

S Supporting Information

Insert System). Approximately 2 104 A549 cells were suspended in The Supporting Information is available free of charge on the
200 L of serum-free RPMI 1640 and pretreated with 1, 5, and 12.5 ACS Publications website at DOI: 10.1021/acschem-
M GBPs for 30 min at 37 C, after which the cells were seeded on bio.6b00864.
the upper compartment. For the invasion assay, Matrigel Matrix NMR assignments of GBPs, HSQC spectra, CD spectra,
(Corning Inc., 0.125 g/mL, 15 L) in buer was added to the upper
NMR titration curves of residues Arg5 and Arg7, TOCSY
chamber of each Transwell. After the Matrigel Matrix has completely
dried, the cells were seeded on the top chamber. Approximately 5 and HSQC spectra of complexes with heparin, and
104 A549 cells were suspended in 200 L of serum-free RPMI 1640 chemical shifts for the GBPs (PDF)
and pretreated with 1, 5, and 12.5 M GBPs for 30 min at 37 C,
respectively. The lower compartment contained 300 L of RPMI 1640
containing 5% FBS (v/v), which was used as the chemoattractant.
After an 18 h (migration) or 24 h (invasion) incubation at 37 C, the
Corresponding Authors
cells that migrated to the lower surface of the membrane were xed *E-mail:
with 4% (v/v) paraformaldehyde for 15 min. The membrane was *E-mail:
stained with a Hoechst 33342 uorescent staining solution (1:700) ORCID
(Thermo Fisher Scientic Inc., catalog no. 62249) for 15 min. The Shih-Che Sue: 0000-0002-2169-0157
numbers of migrated cells were counted in 5 randomly selected
microscopic elds using an inverted microscope (Olympus CK40, Author Contributions
Artisan Technology Group). L.C.H., I.J., and C.-J.-C. contributed equally to this work.
NMR Experiments. All NMR samples were prepared in NMR Funding
buer (20 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 14.3 This work was supported by Ministry of Science and
mM -mercaptoethanol, and 10% (v/v) D2O, pH 6). All NMR spectra
were acquired on Bruker AVIII 600 or Bruker AVIII 850 NMR Technology (MOST), Taiwan (Grants 103-2627-M-007-007,
spectrometers at 298 K. Traditional and nonuniform sampling (NUS) 104-2627-M-007-006, 105-2321-B-007-007, and 105-2627-M-
methods were used, and the spectra were processed using the 007-004), National Tsing Hua University (Grants
NMRPipe33 and hmsIST34 programs. The spectra were analyzed using 105N527CE1, 105N524CE1, and 105N183DE1), and Ministry
SPARKY.35 The backbone and side-chain resonance assignments were of Economic Aairs, Taiwan (Grant 104-EC-17-A-22-1310).
obtained from a combination of 2D NMR spectra, including 1H1H Notes
COSY, and TOCSY,3638 and ROESY3739 using the sequential
The authors declare no competing nancial interest.

assignment method. Two-dimensional 1H13C HSQC4042 spectra
were acquired from the natural abundance 13C nuclei using the NUS
NMR Titrations. The titration-induced chemical shift perturbations The authors are grateful to the NMR facility at National Tsing
were monitored by TOCSY. The initial GBP concentrations ranged Hua University, to the Core Facility for Protein Structural
from 0.55 to 1.2 mM. For the triple molecule interactions, 1.2 mM Analysis at Academia Sinica, and to the National Synchrotron
GBPECP was mixed with heparin octasaccharide at a 1:2 molar ratio, Radiation Research Center.

and DPC was added stepwise to the GBPECP/heparin solution to
prepare dierent molar ratio samples until the nal [GBPECP]/
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406 DOI: 10.1021/acschembio.6b00864

ACS Chem. Biol. 2017, 12, 398406