Discovery of highly reactive peptide tag by ELISA-type screening for specific cysteine conjugation

Article abstract of DOI:10.1016/j.bmcl.2017.05.069

We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabl

Full text of DOI:10.1016/j.bmcl.2017.05.069

Accepted Manuscript
Discovery of Highly Reactive Peptide Tag by ELISA-type Screening for Spe-
cific Cysteine Conjugation
Nobutaka Kurashige, Hirokazu Fuchida, Shigekazu Tabata, Shohei
Uchinomiya, Akio Ojida
PII:
S0960-894X(17)30567-X
http://dx.doi.org/10.1016/j.bmcl.2017.05.069
BMCL 25014
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 April 2017
18 May 2017
23 May 2017
Please cite this article as: Kurashige, N., Fuchida, H., Tabata, S., Uchinomiya, S., Ojida, A., Discovery of Highly
Reactive Peptide Tag by ELISA-type Screening for Specific Cysteine Conjugation, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.069
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Graphical Abstract
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Discovery of Highly Reactive Peptide Tag by ELISA-type Screening for Specific Cysteine
Conjugation
Nobutaka Kurashige, Hirokazu Fuchida, Shigekazu Tabata, Shohei Uchinomiya, Akio Ojida*
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku,
Fukuoka, 812-8582, Japan
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*Corresponding author:
Akio Ojida
E-mail address: ojida@phar.kyushu-u.ac.jp
Tel: +81-92-642-6596
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Abstract
We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation
of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a
19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the
rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface
of living cells.
Main text
The modification of proteins with a synthetic functional molecule is a versatile method to
facilitate the imaging and manipulation of a protein of interest (POI) in living cells. In the past
20 years, numerous selective protein labeling methods to covalently label POIs have been
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devised. Among them, the sequence-specific labeling of a short peptide tag fused to POIs has
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attracted much attention. The pioneering work of Tsien and co-workers led to the development
of the first covalent protein labeling method employing a genetically encodable short peptide
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tag (CysCysXXCysCys) and a biarsenical probe. More recently, several peptide tag-based
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protein labeling methods, including the oligohistidine tag (His6–10), oligoaspartate tag (Asp4),
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π-clamp (Phe-Cys-Pro-Phe), and CX10R7 (VTNQECCSIPM) tag , have been developed and
successfully applied to the non-enzymatic modification of proteins in vitro and in cellulo with a
synthetic probe. As different approach, various enzyme-mediated labeling methods of tag-fused
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proteins have also been developed. Compared with the enzymatic labeling, the non-enzymatic
protein labeling methods benefit from more flexibility in the labeling conditions and a probe
structure independent from the enzyme properties. Despite its versatility, development of
non-enzymatic protein labeling method and its application to protein analyses in cellulo is still
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, 4, 5
rare.
We report herein the discovery of a highly reactive peptide tag for selective protein
labeling. In this system, the Zn(II) complex binds to the aspartate-rich part of the tag through a
metal-ligand interaction and this complex formation induces selective cysteine conjugation to
afford a labeled tag-fused protein (Figure 1). Optimization of the peptide tag sequence by an
efficient enzyme-linked immunosorbent assay (ELISA)-type screening provided a highly
reactive DCP3 tag for the rapid conjugation of cysteine with the zinc complex 4-4Zn(II) bearing
a weakly reactive Michael acceptor. This tag-probe pair allowed us to visualize the tag-fused
protein on the surface of living cells by fluorescence imaging.
To generate a specific tag-probe pair with a strong binding affinity, we employed a short
aspartate-rich peptide (DDAADD) and a thiazole-containing binuclear zinc complex (Figure

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S1), both of which have been recently reported by us. To obtain a stronger binding affinity, we
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first designed a repeated aspartate-rich peptide tag (i.e., DDAADDAADDAADD) and the
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dimer-type tetranuclear zinc complex 1-4Zn(II). The binding affinity (K ) of this pair was
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−1
determined to be 1.2 × 10 M by fluorescence titration under neutral aqueous conditions (50
mM HEPES and 100 mM HCl, pH 7.2) (Figure S2). Screening for a suitable peptide tag
sequence started with the optimization of the spacer unit that connects the aspartate-rich binding
region and a reactive cysteine. We employed the series of the oligo-proline (n = 1 ~ 9) as a rigid
spacer unit to evaluate the effect of the spacer length on the cysteine conjugation reaction. The
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reaction of these peptide tags with 2-4Zn(II) bearing dimethylamino-crotonate as a Michael
acceptor was evaluated by ELISA-type screening. In this assay, the purified tag-fused maltose
binding proteins (tag-MBPs) were adsorbed onto the wells of a culture plate and treated with
the biotin-appended complex 2-4Zn(II) for an appropriate time. The labeled protein was then
quantified by colorimetric determination using streptavidin (SAv) conjugated with horseradish
peroxidase (HRP) (Figure S3). The results of the kinetic analysis of the cysteine conjugation
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reactions are summarized in Table 1.
depending on the number of the prolines introduced into the peptide tag (R2P0 ~ R2P9). That is,
an R2P3 tag possessing three prolines exhibited the highest reactivity (half-reaction time t1/2
15.6 min), while an R2P8 tag possessing eight prolines had the lowest reactivity (t = 198 min).
We found that the reactivity of tag-MBPs varied
=
1
/2
These data suggest that the spatial orientation between the peptide and the probe in the binding
complex can significantly affect the reaction efficiency. We next changed the amino acid(s)
flanking the cysteine at the N-terminal position of the peptide tag and evaluated their reactivities
(Table 2). As an initial setting, we designed R2P1 ~ R2P9 tags, which have two positively
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charged arginines, to enhance the reactivity of the cysteine by lowering its pK value. The
removal of one arginine (RCP3 tag) or the replacement of the two arginines with a neutral
threonine (TCP3 tag) decreased the reactivity (t1/2 = 48.9 and 40.6 min, respectively). A large
decrease of reactivity was also observed when replacing the two arginines with a tyrosine
(YCP3 tag, t = 72.7 min), a histidine (HCP3 tag, t = 72.7 min), or a proline (PCP3 tag, t =
1
/2
1/2
1/2
48.7 min). Conversely, the introduction of the negatively charged aspartate(s) greatly enhanced
the reactivity. That is, the half reaction times (t ) of tag-MBPs possessing the DCP3 and
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/2
D2CP3 tags were 2.9 min and 7.2 min, respectively, which are both significantly shorter than
that of the initial R2CP3 tag (t1/2 = 15.6 min). Other tags containing a negatively charged
glutamate (ECP3 and EACP3) also exhibited higher reactivities compared to the R2CP3 tag.
We speculate that these carboxyl residue(s) may serve as general acids to promote the
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protonation of the terminal amino group of the dimethylamino-crotonate unit and thereby
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increase its reactivity due to the inductive effect of the positively charged ammonium group.
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The reactivity of the previously developed CA6D4x2 tag (t = 77.6 min) is apparently lower
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than that of the optimized DCP3 tag (t = 2.9 min), clearly demonstrating the high reactivity of
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the new tag-probe pair.
We next examined the covalent labeling of the DCP3 tag-MBP in neutral aqueous solution
(50 mM HEPES and 100 mM NaCl, pH 7.2) and evaluated the labeling efficiency by in-gel
fluorescence analysis (Figure 2). For this purpose, we prepared the zinc complex 3-4Zn(II)
®
appended with the fluorescent dye Oregon Green . We also synthesized 4-4Zn(II), which has a
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more reactive pyridine-fused Michael acceptor compared to that of 3-4Zn(II). Figure 2 shows
the time-course of the covalent labeling reaction of the DCP3 tag-MBP by 3-4Zn(II) or
−
1 −1
4-4Zn(II). The labeling reaction with 3-4Zn(II) proceeded rapidly (k = 69.7 M s ) and was
almost complete within 60 min (t1/2 = 17.1 min). As expected, 4-4Zn(II) exhibited a higher
−
1 −1
reactivity than 3-4Zn(II). The second-order rate constant of 4-4Zn(II) (k = 317 M s ) is
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comparable to that of typical bioorthogonal reactions for protein labeling reported to date. In a
control experiment, the conjugation reaction scarcely occurred when the complex formation
between the tag-MBP and the probe was inhibited by a high concentration of inorganic
pyrophosphate (PPi, 10 mM) (Figures 2 and S4), clearly indicating that the cysteine conjugation
reaction was largely accelerated by the formation of the tag-probe binding complex.
The present protein labeling method was further applied to the fluorescent imaging of a
G-protein coupled receptor (GPCR) expressed on the surface of living cells. In this experiment,
bradykinin receptor type 2 (B2R) protein tethered with a DCP3 tag at the exoplasmic
N-terminus was transiently expressed in HEK293 cells and imaged. When the cells were treated
with the reducing agent tris-(carboxyethyl)phosphine (TCEP) to activate the cysteine residue of
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the DCP3 tag followed by labeling with 4 µM of 4-4Zn(II) (37 °C for 10 min), a clear
®
fluorescence emission from Oregon Green was detectable on the cell surface (Figure 3a). This
fluorescence emission remained even after repeatedly washing the cells with a PPi solution (2
mM in HEPES buffered saline), indicative of the irreversible labeling of 4-4Zn(II) on the cell
surface. It is noteworthy that this fluorescence image overlapped well with that of a B2R
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antagonist peptide appended with a Cy5 fluorophore. Conversely, negligible fluorescence was
observed on the cell surface when the labeling experiment was conducted in the presence of PPi
(2 mM) (Figure S5a), indicating that the labeling reaction is primarily driven by the selective
tag-probe interaction (Figure S5). The labeling experiment was also conducted with 3-4Zn(II)
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(Figure 3b, Figure S5b). However, the probe fluorescence on the cell surface was apparently
weaker than that of 4-4Zn(II). To clarify this point, we semi-quantitatively analyzed the
fluorescence intensity of the probe on the cell surface (Figure 3b) The data suggested that
4-4Zn(II) produced a fluorescence emission that was over 4-fold brighter than that produced by
3-4Zn(II), indicative of the usefulness of 4-4Zn(II) as a reactive probe in the selective labeling
of the tag-fused protein.
In conclusion, we have efficiently identified a highly reactive DCP3 tag by an ELISA-type
screening method. The high labeling selectivity of the DCP3 tag with a rationally designed zinc
complex was successfully exploited for the covalent labeling and visualization of the tag-fused
B2R protein on the surface of living cells. Although the use of the present labeling method is
basically limited to cell surface proteins, its highly efficient and selective labeling property
would provide opportunity not only for fluorescence imaging of POI on cell surfaces, but also
for single protein analysis using electron microscopy. Our research on this topic is ongoing.
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a
S
S
b
S
S
N
N
N
S
N
N
S
N
N Zn
II
N
II
N Zn
II
N
II
O Zn
O Zn
S
S
N
6Cl-
N
6
Cl
-
H
O
O
O
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O
H
N
H
N
H
N
H
N
H
N
H
N
O
Boc
N
N
H
N
H
N
H
N
H
OMe
NH
3
S
O
O
O
O
O
H
N
ZnII
N
ZnII
S
1
-4Zn(II)
S
2-4Zn(II)
O
ZnII
N
O
N
N
N
N
N
N
ZnII
N
S
N
S
N
S
S
N
S
S
N
S
S
S
N
ZnII
N
II
O
Zn
N
S
N
6
Cl
-
O
O
O
F
H
N
H
N
H
N
X
O
O
N
N
H
N
H
N
H
O
O
O
3
HOOC
F
O
N
S
3
-4Zn(II): X = CH
-4Zn(II): X = N
Z
O
n
II
N
N
II
N
OH
Zn
4
S
N
N
4
5
S
S
Figure 1. (a) Schematic illustration of specific cysteine conjugation using a tag-probe pair. (b)
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Structures of the Zn(II) complexes.
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Table 1. Reaction kinetics analysis of the covalent labeling of tag-MBPs in ELISA-type
screening.
-
1 a
Tag
Sequence
kobs (s )
266 ± 24
209 ± 27
383 ± 15
741 ± 46
t1/2 (min)
43.7
55.8
30.2
15.6
27.8
41.7
27.1
74.0
198
R2CP0 RRC-DDAADDAADDAADD
R2CP1 RRCP-DDAADDAADDAADD
R2CP2 RRCPP-DDAADDAADDAADD
R2CP3 RRCPPP-DDAADDAADDAADD
R2CP4 RRCPPPP-DDAADDAADDAADD
R2CP5 RRCPPPPP-DDAADDAADDAADD
R2CP6 RRCPPPPPP-DDAADDAADDAADD
R2CP7 RRCPPPPPPP-DDAADDAADDAADD
R2CP8 RRCPPPPPPPP-DDAADDAADDAADD
442 ± 145
270 ± 4
427 ± 15
178 ± 86
81 ± 46
R2CP9 RRCPPPPPPPPP-DDAADDAADDAADD 243 ± 51
48.8
3
4
5
6
(a) Data shown are the averages of three independent measurements ± standard deviation.
Table 2. Reaction kinetics analysis of the covalent labeling of tag-MBPs in ELISA-type
screening.
-
kobs (s )
1 a
Tag
Sequence
t1/2 (min)
15.6
48.9
40.6
72.7
72.7
48.7
2.9
R2CP3 RRCPPP-DDAADDAADDAADD
741 ± 46
242 ± 48
287 ± 30
160 ± 19
161 ± 123
238 ± 16
3936 ± 205
505 ± 2
RCP3
TCP3
YCP3
HCP3
PCP3
DCP3
RCPPP-DDAADDAADDAADD
TCPPP-DDAADDAADDAADD
YCPPP-DDAADDAADDAADD
HCPPP-DDAADDAADDAADD
PCPPP-DDAADDAADDAADD
DCPPP-DDAADDAADDAADD
DACP3 DACPPP-DAAADDAADDAADD
D2CP3 DDCPPP-DDAADDAADDAADD
22.9
7.2
1608 ± 26
787 ± 27
747 ± 7
ECP3
EACP3 EACPPP-DDAADDAADDAADD
E2CP3 EECPPP-DDAADDAADDAADD
CA6D4x2 CAAAAAADDDDGDDDD
ECPPP-DDAADDAADDAADD
14.7
15.5
21.1
77.6
550 ± 31
149 ± 1
7
(a) Data shown are the averages of three independent measurements ± standard deviation.
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a
b
1
.0
time (min)
FL
0
0
5
5
10 20 40 60 90
0
0
0
0
.8
.6
.4
.2
0
CBB
time (min)
FL
10 20 40 60 90
CBB
0
20
40
60
80
time (min)
1
2
Figure 2. In-gel fluorescence analysis of the covalent labeling of tag-MBP. (a) Fluorescence
(FL) and Coomassie brilliant blue (CBB) detection of the DCP3 tag-MBP labeled with
3-4Zn(II) (upper) or 4-4Zn(II) (lower). (b) Plot of the fluorescence band intensity of the DCP3
tag-MBP labeled with 3-4Zn(II) (ꢀ), 4-4Zn(II) (ꢁ), or 3-4Zn(II) in the presence of 10 mM
pyrophosphate (ꢀ). Data shown are the averages of three independent measurements ± standard
deviation.
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9
a
b
1.0
Oregon Green
C y5
0
.75
1
0
µm
10 µm
0
.5
D IC
Merge
0
.25
0
1
0
µm
10 µm
4-4Zn(II)
3-4Zn(II)
1
0
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1
1
1
1
1
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Figure 3. Covalent labeling of DCP3 tag-fused bradykinin receptor type 2 (B2R) on the surface
of HEK293 cells. (a) Fluorescence visualization of the DCP3 tag-fused B2R upon labeling with
4-4Zn(II) (upper left), Cy5-appended B2R antagonist (upper right), transmission image (lower
left), and the overlay fluorescence image (lower right). (b) Comparison of the labeling
efficiency of DCP3 tag-fused B2R with 4-4Zn(II) and 3-4Zn(II) based on the fluorescence
intensity on the surface of the cells. Data shown are the averages of three independent analyses
of fluorescence images ± standard deviation.
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Acknowledgements
We appreciate the technical support from the Research Support Center, Graduate School of
Medical Sciences, Kyushu University. A. O. acknowledges Takeda Science Foundation for its
financial support.
References
1. (a) Xue, L.; Karpenko, I. A.; Hiblot, J.; Johnsson, K. Nature Chem. Biol., 2015, 11, 917. (b)
Dean, K. M.; Palmer, A. E. Nature Chem. Biol., 2014, 10, 512. (c) Krall, N.; da Cruz, F. P.;
Boutureira, O.; Bernardes, G. J. L. Nature Chem., 2016, 8, 103.
2. (a) Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem. Int. Ed., 2013, 52, 4088. (b) Hinner, M.
J.; Johnsson, K. Curr. Opin. Biotech., 2010, 21, 766.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3. Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science, 1998, 281, 269.
4. (a) Takahira, I.; Fuchida, H.; Tabata, S.; Shindo, N.; Uchinomiya, S.; Hamachi, I.; Ojida, A.
Bioorg. Med. Chem. Lett., 2014, 24, 2855. (b) Uchinomiya, S.; Nonaka, H.; Wakayama, S.;
Ojida, A.; Hamachi, I.; Chem. Commun., 2013, 5022.
5. Nonaka, H.; Fujishima, S.; Uchinomiya, S.; Ojida, A.; Hamachi, I.; J. Am. Chem. Soc., 2010,
132, 9301.
6. Zhang, C.; Welborn, M.; Zhu, T.; Yang, N. J.; Santos, M. S.; Voorhis, T. V.; Pentelute, B. L.
Nature Chem., 2016, 8, 120.
7. Ramil, C. P.; An, P.; Yu, Z.; Lin, Q. J. Am. Chem. Soc., 2016, 138, 5499.
8. Rabuka, D. Curr. Opin. Chem. Biol., 2010, 14, 790.
9. Fuchida, H.; Tabata, S.; Shindo, N.; Takashima, I.; Leng, Q.; Hatsuyama, Y.; Hamachi, I.;
Ojida, A. Bull. Chem. Soc. Jpn., 2015, 88, 784.
10. (a) Singh, J.; Petter, R. C.; Kluge, A. F. Curr. Opin. Chem. Biol., 2010, 14, 475. (b) A.
Wissner, E.; Overbeek, M. F.; Reich, M. B.; Floyd, B. D.; Johnson, N.; Mamuya, E. C.;
Rosfjord, C.; Discafani, R.; Davis, X.; Shi, S. K.; Rabindran, B. C.; Gruber, F.; Ye, W.; Hallett,
A.; Nilakantan, R.; Shen, R.; Wang, Y.; Greenberger, L. M.; Tsou, H. J. Med. Chem., 2003, 46,
49.
11. Lutolf, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A. Bioconjug. Chem., 2001,
12, 1051.
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