Affinity-Guided Oxime Chemistry for Selective Protein Acylation in Live Tissue Systems

Article abstract of DOI:10.1021/jacs.7b07339

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Full text of DOI:10.1021/jacs.7b07339

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Article
Affinity-guided oxime chemistry for selective
protein acylation in live tissue systems
Tomonori Tamura, Zhining Song, Kazuma Amaike, Shin
Lee, Sifei Yin, Shigeki Kiyonaka, and Itaru Hamachi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07339 • Publication Date (Web): 15 Sep 2017
Downloaded from http://pubs.acs.org on September 15, 2017
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Affinity-guided oxime chemistry for selective
protein acylation in live tissue systems
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Tomonori Tamura,1 Zhining Song,1 Kazuma Amaike,1 Shin Lee1, Sifei Yin2, Shigeki Kiyonaka1
and Itaru Hamachi1,3
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1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, JAPAN
2Magdalene College, University of Cambridge
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3CREST(Core Research for Evolutional Science and Technology, JST), Sanbancho, Chiyodaku,
Tokyo, 102-0075, JAPAN
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Correspondence should be addressed to I.H. (ihamachi@sbchem.kyoto-u.ac.jp).
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Abstract
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Catalyst-mediated protein modification is a powerful approach for the imaging and engineering
of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine
(DMAP) (AGD) chemistry as an efficient protein modification method using a catalytic acyl
transfer reaction. However, because of the high electrophilicity of the thioester acyl donor
molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target
protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To
overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows
more specific and efficient protein modification. In this method, a highly nucleophilic
pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The
ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer
reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We
demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry,
can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently
than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane
proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise
quantification of diffusion coefficients in the protein’s native environment. Furthermore, the
excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the
selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are
highly complicated tissue samples.
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Introduction
The methodology to selectively label proteins of interest (POIs) with synthetic probes
in live cells or tissue samples is becoming increasingly important for the investigation of the
structure, function, dynamics, and localization of individual proteins in their native
environment.1 A wide variety of chemical strategies for the selective labeling of proteins have
been developed in the last two decades.1,2 Catalyst-mediated protein labeling is one of the most
powerful and successful of these strategies in chemical biology. For example, the copper
(I)-catalyzed azide-alkyne cycloaddition reaction, known as “click chemistry”, is now the most
widely used bioorthogonal reaction for protein bioconjugation.3 Other transition metal catalysts,
including palladium, rhodium, ruthenium, and iridium, have also proven to be useful for
modifying proteins with excellent chemoselectivity both in vitro and in bacterial cells.4 However,
despite several successful examples,5 transition metal catalyst-mediated protein labeling in
cellular contexts often suffers from cytotoxicity derived from high concentrations of the metal
catalyst, which have hampered biological applications in the more sensitive and complicated
mammalian cells. In addition, most of these methods, in principle, require the incorporation of
bioorthogonal reactive handles into POIs as coupling substrates via genetic engineering, because
it is impossible to selectively modify unengineered natural proteins. This requirement might
potentially interfere with the physiological conditions in living systems. Some recent studies
have reported that protein ligand-tethered transition metal catalysts can selectively modify native
proteins in crude biological samples.6 However, few reports have demonstrated the use of
transition metal-mediated affinity labeling in live-cell contexts.7
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As an alternative to transition metals, organocatalysts are also promising for use in
protein labeling.8 We have previously reported an organocatalyst-based protein labeling method,
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termed affinity-guided 4-dimethylaminopyridine (DMAP) (AGD) chemistry (Figure 1a).9 In this
strategy, the affinity ligand–DMAP conjugate selectively binds to the target protein, and the
DMAP moiety facilitates an acyl transfer reaction from thiophenyl ester acyl donors containing a
reporter tag to the nucleophilic group of an amino acid (e.g. the ε-amino group of Lys and
phenol group of Tyr) in the vicinity of the ligand-binding pocket of the POIs. Using this
proximity-driven protein modification, we achieved the selective fluorescence labeling of
membrane receptors endogenously expressed on living cell surfaces.9c,9e However, several issues
regarding the biocompatibility and bioorthogonality of AGD chemistry still remain to be
challenging (Figure 1a): (1) Basic pH conditions (pH > 8) are necessary for efficient labeling
because of the low nucleophilicity of DMAP at neutral pH. (2) Undesired (non-catalytic)
acylation to non-target proteins inevitably occurs in crude biological environments because of
the inherently high electrophilicity of thioester acyl donors. Thus, the reaction is usually carried
out at low temperature (~4 °C) to minimize non-specific labeling. (3) Thioester acyl donors are
readily decomposed by esterases in biological samples, which requires high concentrations of
reagents. These shortcomings have hampered the application of AGD chemistry to more
complicated and delicate biological samples, such as live tissues. Therefore, the development of
a new acyl donor/organocatalyst pair applicable for affinity-guided protein labeling is desirable.
Here, we describe the use of pyridinium oxime as a nucleophilic acyl transfer catalyst
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for proximity-driven protein labeling with a mild electrophilic N-acyl-N-alkylsulfonamide acyl
donor under physiological conditions (Figure 1b). This new catalytic system, called AGOX
(affinity-guided oxime) chemistry, allowed more efficient labeling of natural proteins in test
tubes and cell lysates than AGD chemistry. AGOX chemistry was also applicable to the selective
labeling and fluorescent imaging of endogenous cell-membrane proteins in live cells, providing a
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tool for the precise evaluation of diffusion coefficients in their native environments. Furthermore,
the improved biocompatibility and bioorthogonality enabled the selective modification of an
endogenous neurotransmitter receptor in acutely prepared mouse brain slices.
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Figure 1 Schematic illustration of protein chemical labeling by (a) affinity-guided DMAP
(AGD) chemistry and (b) affinity-guided oxime (AGOX) chemistry. DMAP,
4-dimethylaminopyridine; NASA, N-acyl-N-alkyl sulfonamide; POI, protein of interest; Pr,
probe; Lg, ligand; Nu, nucleophilic amino acid.
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Results and discussion
1. Design and in vitro evaluation of nucleophilic oxime catalysts
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We initially investigated the potential of pyridinium aldoxime (PyOx) derivatives as
new candidates for acyl-transfer catalysts for our affinity-guided strategies. PyOx compounds
are important antidotes for reactive organophosphate poisoning owing to their strong catalytic
ability to hydrolyze the O-phosphorylated serine of acetylcholinesterase (AChE).10 PyOx
compounds have reported pKa values close to neutral pH (7–8.5), and the corresponding oximate
ions exhibit a high nucleophilicity compared with conventional oxygen-based nucleophiles with
similar basicity.11 In addition, O-acylated oximes are known to be good acyl-transfer reagents.12
Given these properties, we envisioned that a ligand-tethered PyOx derivative would be able to
activate a weakly electrophilic acyl donor at neutral pH and transfer the acyl group to a
nucleophilic residue on a target protein. This method may also reduce undesired background
signals arising from non-catalytic labeling.
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To test this hypothesis, we compared the nucleophilicity of PyOx with DMAP and a
benzaldoxime (pKa ~10)13 at neutral pH (pH 7.2, 37 °C). The hydrolysis of p-nitrophenylacetate
(PNPA), a model electrophile, catalyzed by 4-pyridinium aldoxime derivative 1, DMAP, or
benzaldoxime 2 was spectroscopically monitored (Figure 2a, Figure S1).14 As shown in Figure
2b, compound 1 promoted hydrolysis more efficiently than DMAP. On the other hand, 2 showed
no catalytic effect, indicating that the lower pKa value of PyOx 1 is essential for the high
nucleophilicity. The second order rate constant (k2) was determined to be 2.14 ± 0.08 M-1s-1 for 1
and 0.237 ± 0.001 M-1s-1 for DMAP (Figure 2c), indicating that the nucleophilicity of
4-pyridinium aldoxime 1 is 9-fold higher than that of DMAP under neutral aqueous conditions.
We next examined the protein-labeling capability of the PyOx group conjugated to a
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protein ligand. FKBP12 was employed as the target protein,9c and thus a ligand-tethered PyOx
reagent 3 containing SLF (synthetic ligand of FKBP12) was designed and synthesized (Figure
2d). The linker length of 3 between the PyOx moiety and SLF was referred to a previously
developed AGD catalyst for FKBP12 (SLF-tri DMAP 8, Figure S6).9c, 15
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In vitro labeling was performed by incubation of purified FKBP12 (5 µM, Mw:
11,914) with SLF-PyOx 3 and a fluorescein (FL)-tethered thioester acyl donor (FL-SPh 4)9
(Figure 2d) in buffer solution (pH 7.2) at 37 °C. As shown in Figure 2e, the molecular mass
corresponding to FL-labeled FKBP12 (Mw: 12,357) was observed by MALDI-TOF mass
spectrometry. No significant labeling occurred without 3 or in the presence of excess amount of
rapamycin, a competitive ligand, in the 4 h incubation, indicating that the labeling reaction was
driven by specific protein–ligand recognition. Thus, ligand-tethered PyOx can activate a
thioester acyl donor and efficiently transfer the acyl group to nucleophilic amino acids of
FKBP12 through a proximity effect.
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Figure 2 In vitro evaluation of the nucleophilicity of PyOx under neutral pH conditions. (a)
Catalytic hydrolysis of PNPA. PNPA, p-nitrophenylacetate; PNP, p-nitrophenol. (b) Time
courses of the hydrolysis of PNPA in the presence of DMAP (○), PyOx 1 (□), or benzaldoxime
(BenOx) 2 (◇). Reaction conditions: 200 µM PNPA, 20 µM catalyst, 50 mM HEPES buffer, pH
7.2, 37 °C. The reaction was monitored by the absorbance at 348 nm with a UV-Vis
spectrometer. (c) k2 values for the hydrolysis of PNPA with DMAP and PyOx 1. (d) Molecular
structure of SLF-PyOx 3 and FL-SPh 4. (e) MALDI-TOF mass analysis of FKBP12 labeling
with SLF-PyOx 3 and FL-SPh 4. Reaction conditions: 5 µM FKBP12, 5 µM SLF-PyOx 3, 50
µM FL-SPh 4, 50 µM rapamycin (Rap), 50 mM HEPES buffer, pH 7.2, 37 °C. ○, native
FKBP12 (M.W. 11,914); *, FL-labeled FKBP12 (M.W. 12,357).
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2. NASA derivatives as new acyl donors
We next explored more suitable acyl donors, than the previously used thioesters, which
may reduce the background reactions of undesired protein labeling in crude biological
environments. We focused on N-acyl-N-alkyl sulfonamide (NASA) as a unique acyl-donor
scaffold, which is used in solid-phase peptide synthesis as Kenner’s safety-catch linker (Figure
3)16. NASA was expected to have the following advantages over conventional acyl donors: (1) as
there is no NASA scaffold in biological systems, it should be less susceptible to enzymatic
degradation, and (2) the electrophilicity of the acyl group of NASA is able to be finely tuned by
the substitution of electron-withdrawing group (EWG) at aryl sulfonyl (R1) and/or N-alkyl (R2)
moieties. To optimize the reactivity of NASA acyl donors, we prepared five compounds 5a–5e
containing a biotin tag (Bt) and varied the EWGs at R1 and R2, including nitrile, nitro, and
fluoride groups (Figure 3). Non-catalytic (background) reactions of these acyl donors with
FKBP12 were initially evaluated. FKBP12 (5 µM) was mixed with Bt-NASA 5a–5e (50 µM) or
Bt-thioester acyl donor (Bt-SPh 6) (50 µM), followed by MALDI-TOF mass analysis. While no
labeling occurred even after 24 h for compounds 5a–5d, labeled peaks corresponding to
non-catalytic reactions were detected for 5e (~9%), and for Bt-SPh 6 (~100%) (Figure 4a). We
thus excluded the Bt-NASA 5e because of this higher reactivity.
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We then performed FKBP12 labeling with Bt-NASA 5a–5d catalyzed by SLF-PyOx 3.
As shown in Figure 4b, 4c and Figure S2, efficient labeling took place only with Bt-NASA 5a,
which contains a p-nitrophenyl group in both the aryl sulfonyl (R1) and N-alkyl (R2) positions.
This reaction was completely abolished in the presence of a competitive inhibitor (FK506),
demonstrating that the labeling was greatly facilitated by an affinity-driven proximity effect
(Figure S2). We also found that the labeling yield exceeded 100% (i.e. multiple labeling
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occurred) after 1 h (Figure 4b and 4c), which indicated that the PyOx-mediated FKBP12
labeling catalytically proceeded with the NASA acyl donor. Use of 5b–5d showed lower yields
and slower reaction kinetics (Figure 4b, 4c and Figure S3), probably because the
electrophilicity of these compounds was too weak. Given these results,
N-(4-nitrobenzyl)-N-(4-nitrophenyl) sulfonamide was selected as the optimal acyl donor for
PyOx-mediated protein labeling.
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The stability of the optimal NASA was evaluated by a hydrolysis assay (Figure 4d,
Figure S4, and Table S1). The half-life for autolysis in aqueous buffer (pH 7.2) was determined
to be 81 h for the FL-NASA 7 (Figure 3), which is longer than that of the FL-SPh 4 (t1/2 = 60 h).
More importantly, FL-NASA 7 was also stable even in the presence of an esterase (t1/2 = 55 h), in
contrast with the rapid hydrolysis of FL-SPh 4 (t1/2 = 0.5 h). Thus, we envisioned that the NASA
acyl donor should be suitable for protein labeling under crude biological conditions.17
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Figure 3 Molecular structures of NASA and thioester acyl donors. Bt, biotin; FL, fluorescein.
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Figure 4 Reaction properties of NASA acyl donors. (a, b) MALDI-TOF mass analysis of
FKBP12 labeling with Bt-NASA 5a–5e or Bt-SPh 6 in the absence (a) and presence (b) of
SLF-PyOx 3. Reaction conditions: 5 µM FKBP12, 50 µM acyl donor, 5 µM SLF-PyOx 3, 50
mM HEPES buffer, pH 7.2, 37 °C. ○, native FKBP12 (M.W. 11,914); *, single Bt-labeled
FKBP12 (M.W. 12,140); **, double Bt-labeled FKBP12 (M.W. 12,366). (c) Time courses of
reaction yields of FKBP12 labeling by Bt-NASA 5a–5d with SLF-PyOx 3, and Bt-NASA 5a or
Bt-SPh 6 with SLF-triDMAP 8. (d) Time course plots of the hydrolysis of FL-NASA 7 and
FL-SPh 4 in the absence (7, ˜; 4, p) or presence (7, ¢; 4, ¿) of porcine liver esterase (PLE).
Survival rates at several time points were calculated by the integration of the signal in HPLC
analyses (Figure S4), and fitted using a single-phase exponential decay model to obtain the
half-life for each acyl donor in aqueous buffer. (e) The crystal structure of the FKBP12-SLF
complex (PDB ID:1FKG)
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3. In vitro protein labeling using AGOX chemistry
The labeling method using the PyOx catalyst 3/NASA acyl donor 5a pair showed
sufficient (2 h, ~100 %) FKBP12 labeling under neutral pH (7.2), and the labeling yield and
kinetics was comparable to (or slightly better than) AGD chemistry with SLF-triDMAP 8
(Figure S6) and Bt-SPh 6 under the same condition (Figure 4c). On the other hand, the reaction
using SLF-triDMAP 8 and NASA 5a resulted in no labeled products under the same conditions
(Figure 4c). This contrast can be ascribed to the fact that the PyOx catalyst, but not the triDMAP
group, can efficiently activate the NASA acyl donor, highlighting the high nucleophilicity of
PyOx even at neutral pH. The pH dependence of AGOX chemistry is shown in Figure S7. The
labeling occurred around physiological pH (6.5−8.0), with an increased rate at higher pH,
suggesting that deprotonation of both aldoxime and certain nucleophilic amino acid side chains
may contribute to the accelerated acyl transfer reaction at the basic pH. The labeled products
were stable at 37 °C in the pH range (pH 6.5−8.0) at least for 30 h (Figure S8).
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The sites of FKBP12 labeled by the SLF-PyOx 3/FL-NASA 7 pair were identified by
conventional peptide mapping analysis. FL-labeled FKBP12 was digested with trypsin, and the
resultant peptide fragments were analyzed by HPLC and MALDI-TOF MS/MS. As shown in
Figure 4e, and Figure S9–S11, Lys44 (79%) and Lys52 (21%), which are both the nearest
lysine residues from the SLF ligand in the binding pocket (11.8 Å and 11.9 Å, respectively),
were modified with FL. This identification was strongly supported by the labeling of two
FKBP12 mutants (K44R and K52A), in which the labeling yields were substantially diminished
(Figure S12).
According to the affinity-guided catalyst strategy, the target protein for labeling can be
readily altered by switching the ligand part of the ligand-tethered PyOx. In carbonic anhydrase II
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(CAII) labeling, phenylsulfonamide (SA) was selected as the affinity ligand18 (Figure 5a) and
the effective in vitro labeling of CAII was performed by the pair of SA-PyOx 9 and FL-NASA 7
(Figure S13). We found that increasing the number of PyOx moieties (divalent-PyOx) in the
ligand-tethered catalyst, SA-diPyOx 10, enhanced the reaction rate like the example of AGD
chemistry (Figure S13).9c We also determined the amino acids of CAII modified with 10 and 7
(Figure S14). Notably, in addition to lysine (Lys169, 76%), a serine residue (Ser2, 24%) was
identified as an amino acid targetable by AGOX chemistry. The resultant Ser2 ester bond is
chemically stable at 37 °C in a buffer (pH 8.0) at least for 30 h (Figure S15).
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To compare the target selectivity of AGOX with AGD chemistry, we conducted CAII
labeling in cell lysates. A HeLa cell lysate containing CAII (0.5 µM) was incubated for 3 h with
PyOx catalysts (9 or 10) and FL-NASA 7, or SA-triDMAP 11 and FL-SPh 4. As shown in
Figure 5b, the diPyOx 10/NASA 7 pair selectively labeled CAII without noticeable background
(lane 5). In contrast, multiple bands attributed to non-specific (non-catalytic) labeling were
detected for the triDMAP 11/thioester 4 pair (lane 8). The target-selective labeling of AGOX
chemistry was also observed for FKBP12 labeling in cell lysate (Figure S16). Taken together, it
is clear that the newly-developed PyOx catalyst/NASA acyl donor pair exhibited higher labeling
efficiency and selectivity compared with AGD chemistry in cell lysates.
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Figure 5 CAII labeling in cell lysates. (a) Molecular structures of SA-PyOx 9, SA-diPyOx 10,
and SA-triDMAP 11 for CAII and CAXII labeling. (b) SDS-PAGE analysis by in gel
fluorescence imaging and Coomassie brilliant blue staining. HeLa cell lysate (protein
concentration: 1.0 mg/mL) was mixed with recombinant CAII (final concentration, 0.5 µM), and
incubated with acyl donors (5 µM) and catalysts (0.5 µM) in 50 mM HEPES buffer, pH 7.2,
37 °C. EZA, Ethoxzolamide.
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Having established the AGOX chemistry, we next applied this strategy to the chemical
modification of endogenous membrane proteins expressed on live cells. Membrane-bound
carbonic anhydrase XII (CAXII) was chosen as a target protein.19 Mono- (9) and diPyOx (10)
containing the SA ligand, and the less cell-permeable FL-NASA 7 were employed for CAXII
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labeling. A549 cells were incubated in culture medium containing catalyst (5 µM) and 7 (5 µM)
at 37 °C.20 The cells were subsequently washed with phosphate buffered saline (PBS), lysed and
analyzed by western blotting using anti-CAXII and anti-FL antibodies.21 As shown in lane 3 of
Figure 6a, a strong band corresponding to FL-labeled CAXII was observed with SA-diPyOx 10
and FL-NASA 7 after 1h incubation. This band was not detected when the labeling was
performed in the absence of 10 or in the presence of excess EZA (ethoxzolamide), a strong
inhibitor of CAXII. Time-course experiments showed that the reaction with SA-diPyOx 10 and
FL-NASA 7 reached a plateau at approximately 3 h, and the labeling yield was estimated to be
48% of the entire population of CAXII (Figure S17 and S18). Compared with diPyOx 10, the
labeling efficiency of mono-PyOx 9 was 2.5-fold lower (Figure S19). We also compared the
target selectivity and labeling efficiency of AGOX with AGD chemistry in live cells. As shown
in lanes 5–7 of Figure 6a, FL-SPh 4 promiscuously reacted with many proteins even in the
absence of the AGD catalyst, leading to more background signals. In contrast, such non-catalytic
reactions did not occur using FL-NASA 7, because of the well-controlled electrophilicity.
Moreover, the labeling yield of CAXII with PyOx/NASA was 1.2-fold greater than with AGD
chemistry (lanes 3 and 6 in Figure 6a). These results clearly verified that AGOX chemistry is
superior to our previously reported AGD chemistry,9 in terms of both selectivity and efficiency
of membrane protein labeling in live cells.
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Fluorescent live imaging of endogenous CAXII with the Oregon Green (OG)-NASA
acyl donor 12 strongly supported the above-mentioned western blotting results (Figure 6b).22
Strong fluorescence of OG-labeled CAXII was observed from the plasma membrane area of live
A549 cells by confocal laser-scanning microscopy (CLSM) (Figure 6c). This fluorescence was
not detected in the absence of catalyst or in the presence of a competitive inhibitor (EZA), which
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indicated that the fluorescence from the cell surface is predominantly attributed to OG-labeled
CAXII. Similar to CAXII, the endogenous folate receptor (FR) on live KB cells was selectively
labeled and visualized using 13 containing the methotrexate (MTX) ligand and OG-NASA 12
(Figure 6d and Figure S21).23
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Taking advantage of the selective incorporation of a fluorophore into endogenous
membrane proteins with a high signal-to-noise ratio, we sought to apply the AGOX labeling
method to quantitatively analyze the protein dynamics in their native environments. After protein
labeling using AGOX chemistry in live cells, fluorescence recovery after photobleaching
(FRAP) was conducted to evaluate the protein diffusion kinetics on the cell surface.24 The
fluorescence recovery was monitored at 37 °C after bleaching the fluorescence of OG-labeled
CAXII or FR on a selected area (diameter: 3.0 µm) of the cell membrane with a confocal laser
beam (Figure 6e). As shown in Figure 6f, typical recovery curves, because of the lateral
diffusion of the corresponding proteins, were obtained for OG-labeled CAXII on A549 cells and
FR on KB cells. The lateral diffusion coefficients (D) were determined, by fitting the raw data
according to the previously reported Soumpasis mathematical equation,31 to be (7.5 ± 1.4) ×10-2
µm2/s for CAXII and 0.11 ± 0.02 µm2/s for FR (Figure 6f). The obtained D value for FR was
consistent with the reported value for GFP-fused FR (ca. 0.06 µm2/s)25, validating our FRAP
experiment. There are no reports for the diffusion kinetics of CAXII. To the best of our
knowledge, this is the first report measuring the diffusion dynamics of endogenous FR and
CAXII in live cells. Overall, the combination of the PyOx-mediated labeling method with FRAP
analysis provides a powerful platform to investigate the diffusion dynamics of endogenous
membrane proteins in their native habitats with minimal perturbation.
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Figure 6 Specific labeling and dynamic analyses of endogenous membrane proteins using
AGOX chemistry in living cells. (a) Western blotting analysis of CAXII labeling. A549 cells
were treated with catalysts (5 µM) and acyl donors (5 µM) for 1 h at 37 °C in DMEM-HEPES
buffer (pH 7.4). After washing, the cells were lysed and analyzed by western blotting using
anti-FL and anti-CAXII antibodies. The band with a black arrow corresponds to CAXII and the
black dot (˜) corresponds to non-specifically-labeled bovine serum albumin (BSA) derived
from the culture medium. (b) Molecular structure of OG-appended NASA acyl donor 12. (c)
Confocal micrograph images of A549 cells labeled with SA-diPyOx 10 and OG-NASA 12.
A549 cells were treated with 10 (5 µM) and 12 (5 µM) for 1 h at 37 °C in DMEM-HEPES buffer
(pH 7.4) (left). As negative controls, the labeling reaction was performed without 10 (middle) or
in the presence of EZA (50 µM) (right). Scale bars, 20 µm. (d) Molecular structure of
MTX-PyOx 13 for FR labeling. (e) Representative images of a confocal FRAP experiment.
Endogenous FR on KB cells was labeled with MTX-PyOx 13 and OG-NASA 12 for 1 h at 37 °C
in culture medium. After washing the cells 3 times with DMEM-HEPES, FRAP experiments
were conducted. Diameter of bleach circle, 3.0 µm. Scale bars, 5 µm. (f) Kinetics of recovery for
OG-labeled FR (˜) in KB cells and OG-labeled CAXII (¢) in A549 cells at 37 °C. Each plot
shows the mean ± SD from 12–15 cells. The calculated D values were (7.5 ± 1.4) ×10-2 µm2/s for
CAXII and 0.11 ± 0.02 µm2/s for FR.
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5. Endogenous AMPAR labeling in brain tissues
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Finally, we applied the AGOX chemistry to label native neurotransmitter receptor
proteins located in brain hippocampal and cerebellar tissue slices, which are more complicated
and delicate biological samples than cultured cells (Figure 7a). Glutamate receptors are
endogenously expressed in the excitatory neurons of the brain and classified into several groups
depending on their ligand recognition properties and relevant functions. Among them, the
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors
(AMPARs), which are composed of heteromeric tetramers of four subunits (GluA1–GluA4), are
essential for synaptic plasticity and play crucial roles in memory formation. We selected the
AMPARs as target proteins in the brain slices and designed the corresponding PyOx catalyst 14
containing di-PyOx and 6-pyrrolyl-7-trifluoromethyl-quinoxaline-2,3-dione (PFQX) as a ligand
(Ki =170 nM) (Figure 7b).26
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Covalent FL-modification of AMPAR by the 14/7 pair was initially confirmed by a
labeling experiment using living HEK293T cells transiently expressing the GluA2 subtype, a
major subtype of the AMPARs in the brain (Figure 7c). Western blotting analysis showed that
selective labeling of AMPAR occurred and this was inhibited by the addition of NBQX (a
competitive antagonist of the AMPARs). The labeling sites on the AMPAR were identified using
a recombinant of the ligand-binding domain of GluA2 to be Lys470 and Lys677, both located at
the entrance of the ligand-binding pocket (Figure S22 and S23).
These results encouraged us to investigate chemical modification of endogenous
AMPARs in brain slices. Acutely prepared hippocampal slices from mice brains were incubated
in buffer containing PFQX-diPyOx 14 (1 µM) and FL-NASA 7 (1 µM) at 20 °C for 30 min. The
tissues were then washed with buffer and lysed, followed by western blotting analysis. As shown
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in Figure 7d, while a few bands attributable to off-target labeling were detected, a strong band
corresponding to FL-labeled AMPARs was predominant in the presence of PFQX-diPyOx 14
and FL-NASA 7.27 This band was completely abolished by co-incubation with NBQX, strongly
indicating selective labeling of AMPARs in hippocampal slices. It should be noted that AGD
chemistry failed to label AMPARs in brain slices because of heavy nonspecific background
labeling (Figure 7d). In the cerebellum, neurons expressing AMPARs are highly covered with
glial cells, and the expression level of AMPARs is less than in hippocampal neurons.28 Even in
these conditions, successful AMPARs selective labeling using the PyOx 14/NASA 7 pair was
able to be achieved in cerebellar slices (Figure S24). These results highlight the sufficient
bioorthogonality and broad utility of AGOX chemistry in complex tissue samples.
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Figure 7 Selective labeling of endogenous neurotransmitter receptors in brain slices. (a)
Schematic illustration of chemical protein labeling in brain slices by AGOX chemistry. (b)
Molecular structures of PFQX-diPyOx (14) and PFQX-triDMAP (15) for AMPAR labeling. (c)
Western blotting analysis of AMPAR labeling in HEK293T cells transiently expressing AMPAR
(HA-tagged GluA2). HEK293T cells transiently expressing AMPAR were treated with catalysts
(5 µM) and acyl donors (5 µM) in the absence or presence of 50 µM NBQX in serum free
DMEM. The cell lysates were analyzed by western blot using anti-FL or anti-HA antibodies. The
band with a black arrow corresponds to AMPAR and the black dot (˜) corresponds to
non-specifically-labeled BSA derived from the culture medium. (d) Western blot analyses of
AMPAR labeling in hippocampal slices. Hippocampal slices were treated with catalysts (1 µM)
and acyl donors (1 µM) in the absence or presence of 100 µM NBQX in ACSF buffer. The cell
lysates were analyzed by western blotting using anti-FL or anti-GluA2 antibodies.
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Conclusions
In conclusion, we have developed a new affinity-guided chemistry for protein labeling
through precisely controlling both the nucleophilicity of the catalyst and the electrophilicity of
the acyl donor. AGOX chemistry showed remarkably improved selectivity and bioorthogonality
as compared with AGD chemistry, which enabled the quantitative evaluation of endogenous
protein dynamics in their native environments and the selective labeling of a naturally occurring
neurotransmitter receptor in brain tissue slices.
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It should be pointed out that, on the other hand, the AGOX chemistry has still
limitations. In particular, this method is not applicable to intracellular proteins so far. This is
partially due to the low membrane permeability of the pyridinium cation moiety of the catalyst
(Figure S25). We are now working on developing an oxime catalyst with both a high membrane
permeability and a strong nucleophilicity. Besides, ligand-directed protein labeling approaches
generally need a specific ligand for POIs, which may limit the scope of targetable proteins.
However, compared with ligand-directed chemistry, such as ligand-directed tosyl (LDT)29 or
acyl imidazole (LDAI) chemistry26,30, the most significant advantage of the affinity-guided
catalyst strategy is that protein or peptide ligands, as well as small-molecules, can be employed
as target-recognition modules. This is because the affinity-guided catalyst can minimize the
protein (ligand)-attacked decomposition of labeling reagents. Therefore, a variety of protein and
peptide ligands (e.g. cytokines, toxins, complements, lectins, and small antibodies) may be used
in PyOx-conjugates, which not only expands the range of available target proteins, but also
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enables diverse applications of this method. Given the previous examples of AGD chemistry9d,9e
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for instance, the AGOX-mediated protein labeling should be more applicable to detect
protein-protein interactions such as antigen-antibody and lectin-glycoprotein interactions, as a
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powerful alternative to photo-crosslinking strategy. Such research are now underway in our
group.
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Materials and Methods
Synthesis.
The synthesis of reagents 4, 6 and 8 has been reported previously.9b, 9c All other synthetic
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procedures and compound characterizations are described in the Supporting Information.
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General materials and methods for the biochemical/biological experiments.
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Unless otherwise noted, all proteins/enzymes and reagents were obtained from commercial
suppliers (Sigma-Aldrich, Tokyo Chemical Industry (TCI), Wako Pure Chemical Industries,
Sasaki Chemical, Bio-Rad, or Watanabe Chemical Industries) and used without further
purification. UV-vis absorption spectra were acquired on a Shimadzu UV-2550
spectrophotometer. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) spectra were recorded on an Autoflex III instrument (Bruker Daltonics)
using α-cyano-4-hydroxycinnamic acid (CHCA) or sinapic acid as the matrix.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were carried out
using a Bio-Rad Mini-Protean III electrophoresis apparatus. Fluorescence gel images and
chemical luminescent signals using Chemi-Lumi one (Nacalai Tesque) or ECL Prime (GE
Healthcare) were acquired with an imagequant LAS4000 (Fujifilm). Cell imaging was performed
with a confocal laser-scanning microscope (CLSM, Olympus, FV1000) using 60× objectives,
and images were analyzed using accompanying ASW 3.0 Viewer software. Reversed-phase
HPLC (RP-HPLC) was carried out on a Hitachi LaChrom L-7100 system equipped with a
LaChrom L-7400 UV detector, 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. All runs used linear gradients of
acetonitrile containing 0.1% TFA (solvent A) and 0.1% aqueous TFA (solvent B).
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Determination of the rate constants of nucleophilic reaction with PNPA.
To a solution of PNPA (p-nitrophenylacetate) (30 µM) in PBS buffer (pH 7.4) was added a
significant excess of DMAP or PyOx 1 (6, 9, 12, 15 mM) at 37 °C. Reaction progress was
monitored by the absorbance at 348 nm or 400 nm with UV-vis spectrometer. The
concentrations of PNP (p-nitrophenol) at various time points were determined by the absorbance
at 400 nm using the molar absorption coefficient of PNP (ε400 nm = 8084 cm-1M-1).14 The
pseudo-first-order rate constants (kobsd) at different concentrations of DMAP or 1 were obtained
by fitting the data to a single-phase exponential growth model. These rate constants at various
concentrations of DMAP or 1 were fitted by linear regression to determine the second order rate
constants (k2).
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Preparation of recombinant FKBP12 (wild type, K44R and K52A)
Recombinant human FKBP12 (wild type) was obtained as described previously.29b The K44R
and K52A mutants of FKBP12 were generated by QuickChange II XL site-directed mutagenesis
kit (Agilent) using primers 5’-CTCCCGGGACAGAAACAGGCCCTTTAAGTTTATG-3’ and
5’-CATAAACTTAAAGGGCCTGTTTCTGTCCCGGGAG-3’
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5’-GTTTATGCTAGGCGCGCAGGAGGTGATCCGAG-3’
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5’-CTCGGATCACCTCCTGCGCGCCTAGCATAAAC-3’ for the K52A, with a pGEX-ST
plasmid encoding wild-type FKBP12 (a kind gift from Dr. Shirakawa) as a template. Plasmids
were verified by sequencing and transformed into BL21 DE3 codon plus RIL cells. The cells
were grown in LB media containing ampicillin at 37 °C to an optical density (660 nm) of 0.6, at
which time the expression of protein was induced by the addition of 1 mM IPTG. After growth
for an additional 5−7 h at 30 °C, the cells were harvested by centrifugation. The cell pellets were
resuspended in a lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM
PMSF, pH 8.0), and lysed by sonication. The proteins were purified from the soluble fraction of
the lysate using a glutathione-Sepharose column chromatography (GE Healthcare) and dialyzed
against 50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0. The GST tag in the fusion protein was
cleaved with SENP protease at 4 °C for 30 h. The resulting (tag-free) FKBP12 mutants were
purified by gel filtration chromatography on HiLoad 16/600 Superdex 75 prep grade (GE
healthcare, Little Chalfont, UK) with a flow rate of 0.8 mL/min using an ÄKTA purifier system
equilibrated with 50 mM HEPES, 100 mM NaCl, pH 8.0. The fractions were monitored at 280
nm, collected using Frac-920, and analyzed the purity by SDS-PAGE. The concentrations of
FKBP12 were determined by bicinchroninic acid (BCA) protein assay (Thermo).
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FKBP12 Labeling in vitro.
Purified FKBP12 (5 µM) was incubated with acyl donors (50 µM) in the presence of catalyst (5
µM) in HEPES buffer (50 mM, pH 7.2) at 37 °C. Control labeling reactions in the presence of
excess rapamycin or 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
(matrix: CHCA)
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CAII labeling in vitro.
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Human carbonic anhydrase II (CAII) was purchased from SIGMA-Aldrich, and used without
further purifcation. The concentrations of CAII were determined by absorbance at 280 nm using a
molar extinction coefficient of 54 000 M-1 cm-1 in 50 mM HEPES buffer (pH 7.4). CAII (10 µM)
was incubated with acyl donor 7 (100 µM) in the presence of catalyst 9 or 10 (10 µM) in HEPES
buffer (50 mM, pH 7.2) at 37 °C. Control labeling reactions in the presence of excess EZA (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. (matrix: Sinapic acid).
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CAII labeling in HeLa cell lysate.
HeLa cells (1×107 cells) were suspended in HEPES buffer (50 mM, pH 7.2) containing 1%
protease inhibitor cocktail set III (Calbiochem) and lysed by Potter-Elvehjem homogenizer at 4 °C.
The lysate was centrifuged at 16000 × g for 3 min, and the supernatant was collected. Protein
concentration was determined by BCA assay and adjusted to 1.0 mg/mL. This solution was
incubated with CAII (0.5 µM), acyl donor (5 µM) and catalyst (0.5 µM) for 3 h at 37 °C. The
reaction mixture was mixed with the same volume of 2×sample buffer (pH 6.8, 125 mM Tris–HCl,
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20% glycerol, 4% SDS, 0.01% bromophenol blue, 250 mM DTT) and incubated for 1 h at 25 °C.
The samples were subjected to SDS-PAGE, and the gel was analyzed by fluorescence gel imager
(LAS4000) and Coomassie Brilliant Blue (CBB) stain.
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Evaluation of stabilities of acyl donors.
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Acyl donor 7 or 4 (20 µM) and internal standard (Terephthalic acid, 500 µM) were incubated
with or without Pig liver esterase (PLE, 0.1 µM) in HEPES buffer (50 mM, pH 7.2) at 37 °C.
Samples at different time points were taken and analyzed by RP-HPLC.
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Peptide Mapping of the FL-Labeled FKBP12.
Recombinant FKBP12 (20 µM) was incubated with acyl donor 7 (50 µM) in the presence of
catalyst 3 (20 µM) in HEPES buffer (50 mM, pH 7.2) at 37 °C. After 4h, the labeled FKBP12 was
purified by size-exclusion chromatography using a TOYOPEARL HW-40F column and dialyzed
against HEPES buffer (50 mM, pH 8.0) with a Spectra/Por dialysis membrane (MWCO: 3,000). To
this solution, urea (at a final concentration of 2 M) and Trypsin (Trypsin /substrate ratio = 1/10
(w/w)) were added. After incubation at 37 °C for 20 h, the digested peptides were separated by
analytical RP-HPLC. The collected fractions were analyzed by MALDI-TOF MS (matrix: CHCA)
and the labeled fragment was further characterized by MALDI-TOF-TOF MS/MS analysis.
Chemical labeling of CAXII in A549 cells.
A549 cells (1.5 × 105 cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 units/ml), streptomycin
(100 mg/ml), and amphotericin B (250 ng/ml), and incubated under the hypoxic condition (<0.1%
O2) generated with AnaeroPack (Mitsubishi Gas Chemical Company) for 1 day. The cells were
then incubated in FBS-free DMEM containing catalyst (5 µM) and acyl donor (5 µM) at 37 °C. As
control experiments, the labeling was conducted in the presence of EZA (50 µM) or without
catalyst. For western blot analysis, after chemical labeling, the cells were washed three times with
PBS, and then RIPA buffer (pH 7.4, 25 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 1% Nonidet
P-40, 0.25% deoxycholic acid) was added containing 1% protease inhibitor cocktail set III
(Calbiochem). The lysed sample was collected and centrifuged (15,200 g, 10 min at 4 °C). The
supernatant was mixed with the same volume of 2×sample buffer and incubated for 1h at 25 °C.
The samples were subjected to SDS-PAGE and electro-transferred onto an Immun-Blot PVDF
membrane (Bio-Rad). The labeled CAXII was detected by chemiluminescence analysis using
rabbit anti-fluorescein antibody (Abcam, ab19491) and anti-rabbit IgG-HRP conjugate (Santa Cruz,
sc-2004). The anti-fluorescein antibody (Abcam, ab19491) was also used for the detection of
oregon-green labeled proteins. The immunodetection of CAXII was performed with a rabbit
anti-CAXII antibody (Cell Signaling, D75C6) and anti-rabbit IgG-HRP conjugate.
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Chemical labeling of FR in KB cells.
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KB cells were cultured in folate-free RPMI1640 (Gibco) supplemented with 10% FBS, penicillin
(100 units/ml), streptomycin (100 mg/ml), and amphotericin B (250 ng/ml), and incubated in a 5%
CO2 humidified chamber at 37 °C. For the endogenous FR labeling, KB cells (1.5 × 105 cells) were
washed three times with folate- and FBS-free RPMI1640. The cells were incubated in folate- and
FBS-free RPMI1640 containing catalyst (5 µM) and acyl donor (5 µM) at 37 °C. As control
experiments, the labeling was conducted in the presence of folate (50 µM) or without catalyst.
After labeling, the cells were washed three times with PBS, and then RIPA buffer was added
containing 1% protease inhibitor cocktail set III. The lysed sample was collected and centrifuged
(15,200 g, 10 min at 4 °C). The supernatant was mixed with the same volume of 2×sample buffer
and incubated for 1 h at 25 °C. The samples were analyzed by western blotting using
anti-fluorescein/oregon-green antibody, anti-FR antibody (Abcam, ab125030) and anti-rabbit
IgG-HRP conjugate.
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CLSM imaging
After chemical labeling as described above, the cells were washed 3 times with HBS (20 mM
HEPES, 107 mM NaCl, 6 mM KCl, 2 mM CaCl2, and 1.2 mM MgSO4 at pH 7.4). Cells imaging
was performed with a confocal microscopy (FV1000, Olympus). The fluorescence images were
obtained by excitation at 473 nm and simultaneous detection with 500 – 600 nm emission filter.
FRAP experiment
After labeling of CAXII or FR with oregon green dye as described above, the cells were washed 3
times with HBS. The fluorescence images were obtained by excitation at 473 nm and simultaneous
detection with 500 – 600 nm emission filter at 37 °C. Bleaching was performed with a circular spot
(diameter 3.0 µm) using the 488nm lines from a 40 mW argon laser operating at 100% laser power
for 3 msec. Fluorescence recovery was monitored at low laser intensity at 3 sec intervals. The
lateral diffusion coefficient (D) was determined by fitting the raw data according to the previously
described Soumpasis mathematical equation shown below: 31
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ꢀ
ꢀ
ꢀ
ꢀꢀ
2ꢀꢁ
ꢀꢀ
2ꢀꢁ
ꢀꢀꢁ
ꢀꢁꢂꢃd(ꢀ) = ꢀ
Iꢀ (
) + Iꢀ (
)
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where w is the radius of the beam.
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Chemical labelling of AMPARs in HEK293T cells.
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HEK293T cells were incubated in DMEM under 5% CO2 at 37 °C. Cells were transfected with
a pCAGGS plasmid encoding flop form of RNA-edited GluA2 (GluA2flop (R))26 using the
lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction and subjected to
labeling experiments after 18 h of the transfection. For chemical labeling, the cells expressing
GluA2 were washed three times with FBS-free DMEM and treated with catalyst (5 µM) and acyl
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donor (5 µM) at 37 °C in FBS-free DMEM and incubated for 1 h. As control experiments, the
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labeling was conducted in the presence of NBQX (50 µM) or without catalysts. The cells were
washed three times with PBS, and then lysed with RIPA buffer as described above. The samples
were analyzed by western blotting using anti-fluorescein antibody, anti-HA tag antibody (for
AMPAR detection, abcam, ab9110) and anti-rabbit IgG-HRP conjugate.
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Chemical labeling and western blot analysis of Hippocampal or cerebellar slices.
Hippocampus or cerebellum (200 µm thickness) was prepared from P14–21 ICR mice. The slices
were treated with catalyst (1 µM) and acyl donor (1 µM) in ACSF solution (125 mM NaCl, 2.5 mM
KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose and 100
mM Picrotoxin) at 20 °C for 0.5 h under 95% O2/5% CO2. As control experiments, the labeling
was conducted in the presence of NBQX (100 µM) or without catalysts. The slices were then
washed with ACSF solution three times and lysed with RIPA buffer containing 1% protease
inhibitor cocktail set III. The lysed sample was centrifuged (15,200 g, 10 min at 4 °C). The
supernatant was mixed with the same volume of 2×sample buffer and incubated for 1 h at 25 °C.
The samples were analyzed using anti-fluorescein antibody, anti-GluA2 antibody (abcam,
ab20673) and anti-rabbit IgG-HRP conjugate.
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Quantitative evaluation of cell-membrane permeability of oxime compounds.
HEK293T cells were incubated in DMEM under 5% CO2 at 37 °C. Cells were transfected with
a pFN21A plasmid encoding HaloTag-FKBP12 (purchased from Promega, clone catalog #
FHC02102) using the lipofectamine 2000 according to the manufacture’s instruction and subjected
to labeling experiments after 38 h of the transfection. The culture media were exchanged with
FBS-free DMEM containing oxime-appended substrates (5 µM) and incubated for 1.5 h at 37 °C.
After washing with media, the cells were incubated in FBS-free DMEM containing Halo-AcFL
(purchased from Promega) (2 µM) for 0.5 h at 37 °C. The cells were washed three times with PBS,
and then lysed with RIPA buffer as described above. The samples were analyzed by western
blotting using anti-fluorescein antibody, anti-FKBP12 antibody (Abcam, ab2918) and anti-rabbit
IgG-HRP conjugate.
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Acknowledgements
We thank Naoya Takahashi for preparation of FKBP12 mutants. We also thank Karin Nishimura
(Kyoto University) for experimental support of MS measurements. This work was supported by
Grant-in-Aid for Young Scientists (B) (15K17884) and The Kyoto University Foundation to
T.T., Grant-in-Aid for JSPS Research Fellow to Z.S. (15J07815) and K.A. (17J09635),
SUNBOR Grant from Suntory Foundation for Life Sciences and the Takeda Science
Foundation to S.K., and the Japan Science and Technology Agency (JST) Core Research for
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Evolutional Science and Technology (CREST) to I.H. This work was also supported by a
Grant-in-Aid for Scientific Research on Innovative Areas“Chemistry for Multimolecular
Crowding Biosystems”(JSPS KAKENHI Grant No. 17H06348).
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Competing financial interests
The authors declare no competing financial interests.
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