Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts

Article abstract of DOI:10.1021/ja204422r

Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc.2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B₂ receptor (B₂R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B ₂R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research.

Full text of DOI:10.1021/ja204422r

ARTICLE
pubs.acs.org/JACS
Chemical Cell-Surface Receptor Engineering Using Affinity-Guided,
Multivalent Organocatalysts
Hangxiang Wang,† Yoichiro Koshi,† Daishiro Minato,†,‡ Hiroshi Nonaka,†,§ Shigeki Kiyonaka,† Yasuo Mori,†
Shinya Tsukiji,|| and Itaru Hamachi*,†,^
†Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura,
Nishikyo-ku, Kyoto 615-8510, Japan
^Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, 5 Sanbancho,
Chiyoda-ku, Tokyo 102-0075, Japan
Top Runner Incubation Center for Academia-Industry Fusion, Nagaoka University of Technology, 1603-1 Kamitomioka,
Nagaoka, Niigata 940-2188, Japan
S Supporting Information
b
ABSTRACT:
Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated
reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided
DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.;
Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows
specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of ‘multivalent’ DMAP
groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments
that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different
proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors
containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B2 receptor (B2R), a G-protein
coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B2R)-based
fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor
should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design
concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein
modification tools, providing new opportunities for organic chemistry in biological research.
’ INTRODUCTION
In this strategy, the ligand selectively binds to the target protein,
driving a chemical reaction of the reactive group with an amino
acid residue located on the protein surface through the proximity
effect. The utmost advantage of this so-called “affinity labeling” is
its applicability to specific modification of natural proteins with-
out genetic engineering and without orthogonal reactions.2ꢀ9
However, although several successful applications have been
reported, the potential of organic chemistry-based approach in
chemical protein labeling in cellular contexts has not yet been
fully explored. In particular, chemical reactions and strategies
The ability to decorate specific proteins in a highly selective
fashion with desired (unnatural) functionalities in cellular con-
texts is of great value in the study and control of living biological
systems. Previous efforts have mainly focused on the develop-
ment and use of engineered proteins in which a bioorthogonal
moiety, such as unique peptide/protein tags and azide and alkyne
groups, is genetically incorporated.1 Selective labeling of the
previously modified proteins is achieved in living cells using
bioorthogonal chemical or enzymatic reactions. Alternatively,
organic chemistry-based methods have also been developed,
which typically use synthetic labeling reagents consisting of a
specific protein ligand and a reactive (electrophilic) group.2ꢀ11
Received: May 14, 2011
Published: July 18, 2011
r
2011 American Chemical Society
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Figure 1. AGD catalyst-mediated selective chemical protein labeling. Schematic illustrations of (a) the basic strategy and (b) the present multivalent
DMAP system. Lg, protein ligand; Nu, nucleophilic amino acid.
that can be used for protein surface modification remain very
limited.
DMAP groups into the AGD catalyst structure. In in vitro ex-
periments, the multivalent AGD catalysts led to a significant
enhancement of reaction rate and efficiency in the labeling of
three different proteins: congerin II, a Src homology 2 (SH2)
domain, and FKBP12. Taking advantage of the improved labeling
properties, we carried out the covalent attachment of a synthetic
fluorophore to bradykinin B2 receptor (B2R), a G-protein coupled
receptor, and constructed a membrane protein (B2R)-based fluor-
escent biosensor on the surface of live cells. To our knowledge,
the ‘catalyst’ multivalency effect on enhanced reactivity is un-
precedented. The design concept presented here is expected to
facilitate further development of diverse catalyst-based protein
modification tools.
Catalysts have long played an essential role in organic synth-
esis, and thus hold promise as tools for chemical protein mod-
ification. However, the application of catalysts or catalyst-mediated
reactions to protein engineering has only recently begun to be
addressed.12ꢀ21 To date, no reports have demonstrated the use
of catalysts in selective chemical protein labeling in cellular
contexts. The difficulty of designing molecular catalysts with
high target selectivity and reaction efficiency that can function
toward structurally complex biopolymers such as proteins in
water-rich, heterogeneous environments represents a significant
impediment.
DMAP is a representative organocatalyst which has been
widely used for facilitating the acyl transfer reaction in a myriad
of organic transformations.22 We have recently shown that affinity-
guided DMAP (AGD) catalysts consisting of the DMAP group
and a saccharide ligand can be used for selective in vitro chemical
acylation of lectins in combination with optimized, thiophenyl
ester type of acyl donors (Figure 1a).23 In this chemistry, the
AGD catalyst selectively binds to the target protein, and the DMAP
moiety mediates the transfer of the acyl group from the acyl
donor to a nucleophilic amino acid located near the ligand-binding
pocket. Therefore, this prototype of AGD catalyst has the advan-
tages of target protein selectivity and reaction site specificity.
However, the direct extension of this approach to live-cell systems
was hampered by the slow reaction (see below). The develop-
ment of a new strategy to enhance the reaction efficiency was
necessitated.
’ RESULTS AND DISCUSSION
Design and in Vitro Evaluation of Affinity-Guided, Multi-
valent DMAP Catalysts. The original AGD catalysts have
proved to be applicable to selective protein (lectin) acylation,23
but suffered from slow reaction rates. We hypothesized that in-
creasing the number of DMAP catalysts within an AGD reagent
might accelerate the reaction rate and efficiency of protein labeling
(Figure 1b). This idea was inspired by the knowledge that
binding affinity and specificity can be significantly enhanced by
introducing multiple ligands into a molecule, known as the (ligand)
multivalency effect.24,25 First, to examine the validity of the multi-
valent catalyst strategy, we carried out in vitro labeling experi-
ments using three different purified proteins: congerin II, the Src
homology 2 (SH2) domain and FKBP12. AGD catalysts contain-
ing the corresponding specific ligand and a set of one, two, or
three DMAP groups were designed and synthesized: compounds
1ꢀ3 for congerin II (ligand: lactose26); 4ꢀ6 for the SH2 domain
Here, we describe an approach to remarkably improve the
protein acylation efficieny using the AGD chemistry. Our ap-
proach is based on the introduction of an increased number of
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Figure 2. Chemical structures of (a) AGD catalysts and (b) acyl donors used in this study. 1ꢀ3 for congerin II, 4ꢀ6 for the SH2 domain derived from
the p85 subunit of human phosphatidylinositol-3-kinase, 7ꢀ9 for FK506-binding protein 12 (FKBP12), and 10 and 11 for bradykinin B2 receptor
(B2R). Hyp, hydroxyproline.
(phosphotyrosine-containing peptide27); and 7ꢀ9 for FKBP12
(synthetic analogue of FK50628) (Figure 2a). We used pre-
viously optimized acyl donor 12 containing fluorescein23 in the
labeling experiments (Figure 2b). Labeling reactions were per-
formed by incubating protein (10 μM) with AGD catalyst (10 μM)
and acyl donor (50 μM) in buffer, and monitored by MALDI-
TOF mass spectrometry. As shown in Figure 3a and Figure S1, all
of the protein targets were predominantly modified in a ratio of
one fluorescein group per protein, irrespective of the DMAP
valency. In addition, in all cases, no significant labeling occurred
without AGD catalyst or in the presence of excess competitive
ligand (Figure S2), which indicates that a specific proteinꢀligand
interaction is required to drive the AGD-mediated protein acylation
reaction. Of particular importance is the enhanced reactivity of
multivalent AGD catalysts. As summarized in Figure 3b and
Table S1, in the congerin II labeling, the initial rate of labeling was
accelerated with the increase of the number of DMAP moieties;
by 3.6-fold for 2 (di-DMAP) and by 4.6-fold for 3 (tri-DMAP)
compared to 1 (mono-DMAP). In the case of the SH2 domain, a
more significant enhancement was observed (see also Figure 3c).
Compounds 5 (di-DMAP) and 6 (tri-DMAP) accelerated the
labeling reaction by 6.7-fold and 17.2-fold, respectively, compared to
4 (mono-DMAP). Similarly, 8 (di-DMAP) and 9 (tri-DMAP)
showed 4.7-fold and 11.5-fold enhancement of FKBP12 labeling,
respectively, relative to 7 (mono-DMAP) (see also Figure S3).
It is noteworthy that the labeling of FKBP12 using tri-DMAP
type 9 was very rapid and efficient, reaching approximately 90%
labeling yield within 15 min.
We also determined the chemical modification sites. Peptide
mapping analysis revealed that, in SH2 domain labeling, both the
monovalent AGD catalyst 4 and its trivalent type 6 mediated
specific acylation of a single residue, Lys379, which is an amino
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Figure 3. Improvement of chemical protein acylation efficiency by the DMAP multivalency effect. (a) Monitoring of the SH2 domain labeling processes
by MALDI-TOF MS. Purified SH2 domain (10 μM) was incubated with AGD catalyst 6 (10 μM) and acyl donor 12 (50 μM) in buffer (pH 8.0) at
25 ꢀC; b, unlabeled SH2 domain; 1, fluorescein-labeled SH2 domain. (b) Initial rates of protein labeling using a series of AGD catalysts and 12. Fold
numbers are shown on the bars. (c) Reaction kinetics of the SH2 domain labeling using mono-, di-, and trivalent AGD catalysts.
acid proximal to the ligand binding pocket (Figure S4). This
result indicates that the observed enhancement of labeling efficiency
in the tri-DMAP system was not caused by changes in labeling
pattern, such as the number and position of labeling sites. In the
case of congerin II and FKBP12, the labeling also occurred at
single sites: Tyr5123 and Lys44 (Figure S5), respectively. These
are each located near the ligand binding pocket of their proteins.
To explore the mechanism of the multivalent DMAP effect, we
first investigated whether the distance of the DMAP group from
the affinity ligand, which differed among the mono-, di-, and
trivalent catalysts, was related to labeling efficiency. Three different,
monovalent AGD derivatives 14, 15, and 16 (for the SH2
domain) were used, in which a single DMAP was placed in a
position corresponding to one of the three DMAP groups in
trivalent AGD catalyst 6 (Figure S6). They showed almost no
difference in labeling efficiency relative to the original type of
monovalent AGD catalyst 4. Next, we replaced one of the DMAP
groups in divalent AGD catalysts with a triethylamine (TEA)
derivative to examine the possible role of the second DMAP as a
base. AGD catalysts 17 (for the SH2 domain) and 18 (for FKBP12)
possessing one DMAP and one TEA group accelerated the
labeling reaction by 3.7-fold and 2.4-fold, respectively, compared
to the parental, monovalent AGD catalysts that do not contain
the TEA moiety (4 and 7, respectively) (Figure S7). The labeling
efficiencies observed with 17 and 18 were intermediate between
those with monovalent and divalent DMAP counterparts (4 and
5, and 7 and 8, respectively). Although the exact mechanism is
not yet fully understood, it is very likely that some DMAPs within
multivalent AGD catalysts act not only as an activator, but also
partially as a base to accelerate the acyl transfer reaction. Thus,
the activator DMAP increases the reactivity of the acyl donor via
formation of an N-acylpyridinium intermediate, and the DMAP
base may assist the deprotonation of other DMAP(s) and/or a
nucleophilic amino acid residue targeted for labeling (e.g., lysine
and tyrosine). Overall, incorporating a set of multiple DMAP
groups into an AGD catalyst was demonstrated to dramatically
enhance the efficiency of the protein acylation reaction.
receptor (B2R), a G protein-coupled receptor (GPCR), was chosen
as the first target protein. We used the B2R-selective antagonist
peptide, DArg0[Hyp3, DPhe7, Leu8]BK,29 as a specific ligand and
synthesized AGD catalyst 11 containing three DMAP groups
(Figure 2a). Human embryonic kidney (HEK) 293 cells trans-
fected with human B2R were used in the experiments. After
washing twice with HBS buffer, the cells were incubated in
the buffer with 11 (10 μM) and the cell-membrane impermeable
fluorescein-type acyl donor 12 (20 μM) at 37 ꢀC for 30 min.
Subsequently, the cells were washed to remove the catalyst and
unreacted acyl donor and observed by confocal laser scanning
microscopy (CLSM). As shown in Figure 4a, a strong fluores-
cence signal was detected in the periphery of the cells. To
examine the possibility of nonspecific labeling, we incubated
the cells with 11 and 12 in the presence of excess HOE 140
(Table S2),30 a strong inhibitor of B2R, or with 12 alone (no
catalyst). In neither case was fluorescence observed. Therefore,
it was concluded that the fluorescence detected was due to
fluorescein labeling of B2R at the cell surface. We also attempted
to modify B2R with a biotin tag using acyl donor 1323 (Figure 2b).
After performing the reaction as described above, the cells were
washed, lysed, and analyzed by Western blotting. Using anti-B2R
antibody and streptavidinꢀhorseradish peroxidase conjugate
(SAv-HRP), we detected only a single major band corresponding
to biotinylated B2R (Figure 4b). This band was not present when
the labeling was carried out in the presence of excess HOE 140 or
in the absence of 11. Taken together, these results verify that the
seven membrane-spanning B2R can be modified covalently and
selectively with synthetic functional molecules via 11-catalyzed
acyl transfer reaction. No significant change in the cell morphol-
ogy occurred throughout the labeling processes, indicating the
biocompatibility of this method.
Kinetic experiments showed that fluorescein labeling of B2R
reached a plateau at around 30 min, at which time the labeling
yield was estimated to be approximately 85% of the entire cell-
surface population of B2R (Figure 4c). In sharp contrast, the
mono-DMAP type of AGD catalyst (10) gave only a negligible
level of fluorescein labeling even after 60 min incubation. This
result demonstrates that the DMAP multivalency is essential to
achieve cell-surface B2R labeling. Furthermore, we confirmed
that the AGD-mediated modification did not impair the natural
Selective Chemical Labeling of B2R on the Surface of Live
Cells. Having proved the efficacy of multivalent AGD catalysts,
we were motivated to apply the strategy to chemical modification
of cell-surface proteins in the context of living cells. Bradykinin B2
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Figure 4. Specific labeling of cell-surface B2R on living cells using AGD chemistry. (a) Fluorescein labeling. HEK 293 cells transfected with B2R were
treated with trivalent AGD catalyst 11 (10 μM) and acyl donor 12 (20 μM) for 30 min at 37 ꢀC in HBS buffer (pH 8.0) (top). As negative controls, the
labeling reaction was performed in the presence of HOE 140 (10 μM) (center) or without 11 (bottom). After washing, the cells were observed by
CLSM. Scale bars, 10 μm. (b) Biotin labeling. Labeling reactions were performed as described in panel a using acyl donor 13. After washing, the cells were
lysed and analyzed by Western blotting. The arrowhead indicates B2R, which is observed as a broad band. (c) Comparison of the efficiency of the cell-
surface B2R labeling using monovalent and trivalent AGD catalysts (10 and 11, respectively). Labeling reactions were performed as described in panel a,
and the cells were washed at indicated time points and observed by CLSM. The labeling efficiency was determined as described in the Experimental
Section.
function of B2R. The agonist-induced increase in intracellular
Ca2+ concentration was measured using fura-2. Bradykinin-stimu-
lated Ca2+ signaling activity was essentially identical before and
after fluorescein labeling (Figure S8). In addition, the present
labeling method was applicable not only in buffer, but also in cell
culture medium both with and without serum (data not shown).
Construction of a GPCR-Based Semisynthetic Fluorescent
Biosensor. Owing to their critical roles in diverse physiological
and pathological processes, the GPCR family is now one of the
most important target classes for drug discovery.31 Few convenient,
quantitative and direct methods have been developed for the
evaluation of GPCRꢀligand interactions as a means of identifying
drug candidates. Given the success of fluorescent labeling of B2R
using AGD chemistry (see above), we sought to construct a
GPCR-based semisynthetic biosensor that responds to specific
ligands with ‘turn-on’ fluorescence (Figure 5a). Our strategy uses
the bimolecular fluorescence quenching and recovery (BFQR)
mechanism.9,32 Taking advantage of the straightforward removal
of AGD catalyst 11 by a simple washing procedure (Figure S9),
the fluorescein-modified B2R (Fl-B2R) in an unliganded state
was constructed on the surface of HEK293 cells. A B2R antag-
onist peptide ligand conjugated with the 4- dimethylaminophe-
nylazobenzene-4-carboxylic acid (DABCYL) quencher (19)
(1 μM) was added to the culture medium and induced a sig-
nificant decrease in the Fl-B2R-derived fluorescence in a typical
saturation behavior, generating a quenched state of Fl-B2R.
(Figure 5bꢀd and Figure S10). Subsequently, the addition of
HOE 140 (2 μM; reported KD for human B2R, 0.41 nM (ref 30))
resulted in a substantial recovery of fluorescence. In contrast,
almost no fluorescence change was observed by the addition of
saralasin,33 an antagonist of the angiotensin II receptor (Figure 5e
and Figure S11). These observations are fully consistent with the
proposed BFQR scheme shown in Figure 5a, and demonstrate
that the fluorescence recovery induced by HOE 140 was due to
the displacement of the noncovalently bound antagonistꢀ
DABCYL conjugate 19 from the Fl-B2R. This BFQR-based turn-
on fluorescence assay system allowed us to evaluate the (relative)
affinities of various ligands in a simple manner. B9430 (Table S2;
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Figure 5. Construction of B2R-based fluorescent biosensor on the basis of the BFQR mechanism. (a) Schematic illustration of the strategy. (b)
Fluorescence quenching and recovery of Fl-B2R observed by CLSM. Postlabeled HEK293 cells (top) were treated with DABCYL-appended B2R
antagonist 19 (1 μM) (center), which was followed by the addition of HOE 140 (2 μM) (bottom). Scale bars, 10 μm. (c) Quantitative evaluation of
fluorescence intensity changes of Fl-B2R and EGFP-B2R upon the addition of 19. (d) Fluorescence intensities (arbitrary units) along the white lines
shown in images in panel b. (e) Quantitative fluorescence detection of various B2R ligands with the Fl-B2R biosensor. To the quenched state of Fl-B2R
prepared as described in panel b was added each B2R ligand.
reported KD, 0.25 nM (ref 30)), another high-affinity antagonist,
potently restored fluorescence, and DArg0[Hyp3, DPhe7, Leu8]BK
(reported KD, 2.8 nM (ref 29)) induced a moderate but
significant change (Figure 5e). On the other hand, the Fl-B2R
biosensor did not respond to des-Arg9-BK and Lys-des-Arg9-BK
(Table S2; reported KD, 8.1 μM and >30 μM, respectively30),
both of which are low-affinity ligands. The relative order of affinity of
the ligands assessed using the BFQR system was in good
agreement with that reported previously. Therefore, we have
demonstrated that the fluorophore-labeled B2R can function as a
turn-on fluorescent biosensor for various antagonist candidates.
As an intriguing comparison, we also attempted to construct a
similar BFQR-based biosensor system using a conventional genetic
technique. The B2R was fluorescently labeled by fusing enhanced
green fluorescent protein (EGFP) to its N-terminus (EGFP-
B2R). HEK 293 cells expressing EGFP-B2R displayed fluores-
cence predominantly in the periphery of the cells (Figure S10).
However, in contrast to the above Fl-B2R system, no quenching
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of EGFP fluorescence took place upon the addition of 19 (Figure 5c
and Figure S10), suggesting that the distance between the
quencher and the EGFP moiety was too great. This highlights
the advantage of the present AGD-based method in which a small
fluorophore is installed close to the ligand binding pocket of the
target protein.
HEPES buffer (50 mM, pH 8.0) at 25 ꢀC. Control reactions in the
presence of excess lactose (100 equiv) or without catalyst were also
carried out. Aliquots at different time points were taken, and the labeling
yields were determined by MALDI-TOF MS (SA).
SH2 Domain Labeling. Recombinant human SH2 domain was
obtained as described previously.9 Purified SH2 domain (10 μM) was
incubated with acyl donor 12 (50 μM) in the presence of AGD catalyst
4, 5, or 6 (10 μM) in HEPES buffer (50 mM, pH 8.0) at 25 ꢀC. Control
labeling reactions in the presence of excess peptide ligand (100 eq,
AcHN-VDpYVPMLD-CONH2) or without catalyst were also carried
out. Aliquots at different time points were taken, and the labeling yields
were determined by MALDI-TOF MS (SA). Labeling reactions with
AGD derivatives 14ꢀ17 were performed under the same condition.
FKBP12 Labeling. Recombinant human FKBP12 was obtained as
described previously.8 Purified FKBP12 (10 μM) was incubated with
acyl donor 12 (50 μM) in the presence of AGD catalyst 7, 8, or 9
(10 μM) in Tris buffer (50 mM, pH 8.0) at 37 ꢀC. Control labeling
reactions in the presence of excess FK506 (10 equiv) or without catalyst
were also carried out. Aliquots at different time points were taken, and
the labeling yields were determined by MALDI-TOF MS (SA). Labeling
reactions with AGD derivative 18 were performed under the same
condition.
Peptide Mapping of the Fluorescein-Labeled SH2 Do-
main. Labeling was performed under the following condition: 17 μM
SH2 domain, 17 μM AGD catalyst 4 or 6, 85 μM fluorescein acyl donor
12 in 50 mM HEPES (pH 8.0) at 25 ꢀC. The labeled SH2 domain was
purified by semipreparative RP-HPLC. The fraction containing the
labeled SH2 domain was collected and dialyzed against HEPES buffer
(50 mM, pH 8.0) with a Spectra/Por dialysis membrane (MWCO: 3,000).
The resulting solution was concentrated using a Centricon Ultracel YM-
3 (Millipore). To this solution, urea (at a final concentration of 2 M) and
trypsin (trypsin/substrate ratio = 1/10 (w/w)) were added. After in-
cubation at 37 ꢀC overnight, the digested peptides were separated by
analytical RP-HPLC. The collected fractions were analyzed by MALDI-
TOF MS (CHCA) and the labeled fragment was further characterized
by MALDI-TOF MS/MS analysis.
’ CONCLUSION
Introducing multiple DMAP groups into AGD catalysts has
proven a powerful strategy to achieve efficient, selective chemical
acylation of proteins under mild and biocompatible conditions.
While the effect of ligand multivalency on molecular recognition
has been intensively studied,24,25 the ‘catalyst’ multivalency effect
on enhanced reactivity described here is, to our knowledge, un-
precedented. Besides the high target selectivity imposed by the
affinity guidance, the enhanced labeling efficiency induced by the
catalyst multivalency was essential for modifying and engineering
a specific membrane-spanning receptor with synthetic probes on
the live cell surface. In recent decades, chemists have developed a
variety of organocatalysts for environmentally benign chemical
processes.34 The present study demonstrates the first successful
application of organocatalysts to (selective) biopolymer mod-
ification in cellular contexts. In general, organocatalysts should be
less toxic and highly tolerant to a wide range of functionalities
found in biological environments. The catalyst multivalency along
with the affinity-guidance strategy may be of key importance in
utilizing organocatalysts for chemical biopolymer labeling, ima-
ging and engineering in the context of biological systems.
’ EXPERIMENTAL SECTION
Synthesis. All synthetic procedures and compound characteriza-
tions are described in the Supporting Information.
General Materials and Methods. Unless otherwise noted, all
proteins/enzymes and reagents were obtained from commercial suppli-
ers (Sigma, Aldrich, Tokyo Chemical Industry (TCI), or Wako Pure
Chemical Industries) and used without further purification. UVꢀvisible
spectra were recorded on a Shimadzu UV-2550 spectrophotometer.
SDSꢀpolyacrylamide gel electrophoresis (SDSꢀPAGE) and Western
blotting were carried out using a Bio-Rad Mini-Protean III electrophor-
esis apparatus. Analytical RP-HPLC was carried out on a Hitachi LaChrom
L-7100 system equipped with LaChrom L-7400 UV and L-7485
fluorescence detectors, and a YMC-Pack ODS-A column (5 μm,
250 ꢁ 4.6 mm) at a flow rate of 1.0 mL/min. UV detection was at
220 nm and fluorescence detection was at 515 nm with excitation at
480 nm. All runs used linear gradients of acetonitrile containing 0.1%
TFA (solvent A) and 0.1% aqueous TFA (solvent B). MALDI-TOF MS
spectra were recorded on an Autoflex II instrument (Bruker Daltonics)
using R-cyano-4-hydroxycinnamic acid (CHCA) or sinapinic acid (SA)
as the matrix. Cell imaging was performed with a confocal laser scanning
microscope (CLSM, Olympus, FV1000, IX81) equipped with a 100ꢁ,
NA = 1.40 oil objective. Fluorescence images were acquired using the
351 nm line of an argon laser for excitation of Hoechst 33342 (emission
detection range, 400ꢀ470 nm), the 488 nm line of an argon laser for
excitation of fluorescein and EGFP (emission, 500ꢀ600 nm) and the
633 nm line of a HeNe Red laser for excitation of Cy5 (emission,
645ꢀ745 nm). The chemiluminescence signal using Chemi-Lumi one
(Nacalai Tesque) was detected with a LAS 4000 imaging system (Fuji Film).
Congerin II Labeling. Recombinant congerin II was expressed in
Escherichia coli strain JM109/pTV-Con II and purified as previously
described.35 Purified congerin II (10 μM) was incubated with acyl donor
12 (50 μM) in the presence of AGD catalyst 1, 2, or 3 (10 μM) in
Peptide Mapping of the Fluorescein-Labeled FKBP12.
Labeling was performed under the following condition: 20 μM FKBP12,
20 μM AGD catalyst 9, 40 μM fluorescein acyl donor 12 in Tris buffer
(50 mM, pH 8.0) at 37 ꢀC. The labeled FKBP12 was purified by
semipreparative RP-HPLC. The fraction containing the labeled FKBP12
was collected and dialyzed against HEPES buffer (50 mM, pH 8.0) with
a Spectra/Por dialysis membrane (MWCO: 3,000). The resulting solution
was concentrated using a Centricon Ultracel YM-3 (Millipore). To this
solution, urea (at a final concentration of 2 M) and lysyl endopeptidase
(LEP) (LEP/substrate ratio = 1/10 (w/w)) were added. After incubation
at 37 ꢀC overnight, the digested peptides were separated by analytical
RP-HPLC. The collected fractions were analyzed by MALDI-TOF
MS (CHCA) and the labeled fragment was further characterized by
MALDI-TOF MS/MS analysis.
Construction of the Expression Plasmid for r7-FLAG-
Fused B2R. An oligo DNA fragment coding the FLAG tag sequence
was inserted into XhoIꢀBamHI sites of pBS-B2R (ref 36) to yield pBS-
FLAG-B2R. The sequences of the 50-phosphorylated DNA fragments
were as follows: forward (50-TCGAGGAAGCTTGGACTACAAG-
GACGATGACGACAAG-30), reverse (50-GATCCTTGTCGTCATC-
GTCCTTGTAGTCCAAGCTTCC-30). An oligo DNA fragment coding
the N-terminal sequence of R7 nicotinic acetylcholine receptor, a signal
sequence for plasma membrane transportation, was inserted into XhoI
site of pBS-FLAG-B2R to yield pBS-R7-FLAG-B2R. The sequences
of the 50-phosphorylated DNA fragments were as follows: R7-1(+)
(50-TCGAGCCACCATGGGCCTCCGGGCGCTGATGCTGTGG-
CTGCTGGCGGCGG-30), R7-2(+) (50-CGGGGCTCGTGCGCGA-
GTCCCTGCAAGGAGAGTTCCAAAGGAAGCTGTACAAGGA-30),
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R7-1(ꢀ) (50-TCGATCCTTGTACAGCTTCCTTTGGAACTCTCC-
TTGCAGGGACTCGCGCACGA-30) and R7-2(ꢀ) (50-GCCCCGCC-
GCCGCCAGCAGCCACAGCATCAGCGCCCGGAGGCCCATGG-
TGGC-30). An XhoIꢀEcoRI fragment of pBS-R7-FLAG-B2R was
inserted into the same digestion site of pCI-neo plasmid (Promega) to
yield pCI-R7-FLAG-B2R.
Construction of the Expression Plasmid for EGFP-Fused
B2R. An EGFP fragment was obtained by PCR from pEGFP-N1
(Clontech) using a pair of primers, forward (50-AGGATCCAATGGT-
GAGCAAGGGCGAGGAGC-30) and reverse (50-TGGATCCGCCT-
TGTACAGCTCGTCCATGCC-30), and subcloned into the BamHI
site of pBS-R7-FLAG-B2R to yield pBS-R7-FLAG-EGFP-B2R. An
XhoIꢀEcoRI fragment from pBS-R7-FLAG-EGFP-B2R was inserted
into pCI-neo plasmid (Promega) to yield pCI-EGFP-B2R.
Fluorescein Labeling of B2R and the BFQR Assay on the
Surface of HEK 293 Cells. HEK 293 cells stably expressing human
B2R fused to an R7-FLAG sequence were maintained in DMEM with
10% fetal bovine serum (FBS), penicillin (100 units/mL), and strepto-
mycin (100 μg/mL) at 37 ꢀC and 5% CO2. Before labeling, the cells
were washed twice with HBS buffer (107 mM NaCl, 6 mM KCl, 1.2 mM
MgSO4, 2 mM CaCl2, 11.5 mM glucose, 20 mM HEPES, pH 8).
Labeling was conducted in HBS buffer containing AGD catalyst 10 or 11
(10 μM) and acyl donor 12 (20 μM) for 30 min at 37 ꢀC. As control, the
labeling experiments in the presence of HOE 140 (10 μM) or in the
absence of catalyst 11 were also conducted. After labeling, the cells were
washed with HBS buffer three times to remove the unreacted acyl donor
and catalyst. The cells were then observed by CLSM. The BFQR assay
using quencher-ligand 19 was performed as described in the correspond-
ing figure captions.
To estimate the fluorescein labeling efficiency, we used fluorescein-
modified B2R ligand 20 as a fluorescent marker. After labeling and
washing procedures, the fluorescence intensity of the labeled, cell-surface
B2R (Flabel) was measured by CLSM. Subsequently, 20 was added to the
cell culture medium (at a final concentration of 1 μM) to stain the whole
cell-surface B2R, and the total fluorescence intensity (Ftotal) was
measured. The labeling efficiency was then determined by the eq 1:
with anti-B2R antibody and anti-rabbit IgG-HRP conjugate (both
Santa Cruz Biotechnology).
Evaluation of the B2R-Mediated Ca2+ Influx Activity. The
B2R-mediated Ca2+ influx activity was measured according to the method
previously reported.37 HEK 293 cells transiently expressing B2R were
plated onto poly-L-lysine-coated glass coverslips. B2R labeling was carried
out using AGD catalyst 11 and acyl donor 12 as described above. The
postlabeled cells were added with 1 μM Fura-2/AM (Dojindo) and
incubated at 37 ꢀC for 40 min in HBS buffer (pH 7.4, containing 2 mM
Ca2+). The coverslips were set in a perfusion chamber mounted on the
stage of the microscope. One micromolar bradykinin, the B2R agonist,
was applied to the cells by perfusion as a stimulus of B2R-mediated Ca2+
signaling. Fluorescence changes of Fura-2 inside the cells were recorded
and analyzed with a video image analysis of system (Aqua Cosmos;
Hamamatsu Photonics). As control, HEK 293 cells transfected with
pCI-neo vector were subjected to the Ca2+ measurement.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1ꢀS11, Tables S1ꢀS2,
b
and experimental details of the synthesis. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
ihamachi@sbchem.kyoto-u.ac.jp
Present Addresses
‡Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
§INAMORI Frontier Research Center, Kyushu University, 744
Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
’ ACKNOWLEDGMENT
We thank Dr. Tomohisa Ogawa (Tohoku University) for the
E. coli strain JM109/pTV-Con II. Y.K. and H.N. acknowledge the
Japan Society for the Promotion of Science Research Fellowships
for Young Scientists.
Labeling efficiency ð%Þ ¼ Flabel=ðFtotal ꢀ FlabelÞ ꢁ 100
ð1Þ
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