Fluorescence amplified detection of proteases by the catalytic activation of a semisynthetic sensor

Article abstract of DOI:10.1039/c3cc42791a

A general approach to mimic the sensing scheme of allosteric enzymes is developed. Through the covalent labeling of a sulfonamide inhibitor to the enzyme HCAII via SNAP-tag protein, the enzyme is rendered inactive. Catalytic activation is triggered only when a protease is present to cleave the recognized peptide sequence. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc42791a

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Fluorescence amplified detection of proteases by the
catalytic activation of a semisynthetic sensor†
Cite this: Chem. Commun., 2013,
49, 6212
Po-Ming Shih, Tao-Kai Liu and Kui-Thong Tan*
Received 16th April 2013,
Accepted 21st May 2013
DOI: 10.1039/c3cc42791a
www.rsc.org/chemcomm
A general approach to mimic the sensing scheme of allosteric enzymes pseudosubstrate from the active site. To date, several allosteric
is developed. Through the covalent labeling of a sulfonamide inhibitor enzyme mimics have been developed by using the protein
to the enzyme HCAII via SNAP-tag protein, the enzyme is rendered engineering technique for the sensing application.6 However,
inactive. Catalytic activation is triggered only when a protease is one major drawback of using the protein engineering approach
present to cleave the recognized peptide sequence.
alone to create a biomimetic allosteric sensor is the limited
application of only genetically encoded polypeptides at the
To increase assay sensitivity in detecting ultralow concentration allosteric sites and as pseudosubstrates which narrow the
of analytes or biomarkers, enzyme-catalyzed signal amplifica- scope and versatility to construct better allosteric enzyme
tion using enzyme conjugates is a common tool in many mimic sensors.
bioanalytical methods.1 Enzyme catalysis provides several
Herein, we describe a rational design of a semisynthetic
advantages such as high substrate specificity and rapid catalysis sensor to mimic the allosteric sensing scheme of natural
to generate optical2 or electrical signals.3 A classical example of enzymes by covalently labeling a synthetic sulfonamide inhi-
an enzyme-catalyzed signal amplification is the enzyme-linked bitor to the enzyme human carbonic anhydrase II (HCAII)
immunosorbent assay (ELISA) which uses a covalently labeled (Fig. 1a). Previous semisynthetic approaches have demon-
enzyme–antibody conjugate to carry out specific analyte detec- strated the amplified detection of DNA,7 where the semi-
tion and signal amplification.4 Although the ELISA-type assays synthetic DNA sensors were prepared by attaching the activated
have been widely employed in many research and clinical synthetic inhibitor-nucleic acid molecules to the proteases
laboratories, shortcomings such as laborious operating proce- through nucleophilic amino acids (cysteine or lysine). However,
dures and extensive washing steps to remove the unbound many proteins are not amenable to this strategy due to multiple
enzyme-conjugate before initializing signal amplification have inherent cysteine or lysine residues.8 A site-specific, easy
limited their application to only in laboratories. Thus, the purification and quantitative protein labeling method is crucial
development of a new enzyme-catalyzed signal amplification for obtaining a useful semisynthetic sensor. In our allosteric
sensor which does not require extensive washing steps and has enzyme mimic sensor, covalent labeling of the synthetic
a straightforward operation protocol would be an important step sulfonamide inhibitor to the HCAII enzyme was achieved by
toward the fabrication of a point-of-care diagnostic device to
detect ultralow concentration of biomarkers.
In contrast to the enzyme–antibody conjugate detection
scheme, a more elegant sensing mechanism performed by
many natural enzymes to control complex cellular processes
is via allosteric regulation,5 in which a polypeptide pseudo-
substrate of the enzyme binds to the active site to inhibit the
enzyme activity. Enzyme activation takes place only when an
activator interacts with the allosteric site, typically the site
between the enzyme and the pseudosubstrate, to induce
a conformational change or cleavage event to remove the
Fig. 1 (a) Schematic representation of the semisynthetic sensor mimicking natural
Department of Chemistry, National Tsing Hua University, 101 Sec. 2,
allosteric enzymes for caspase-3 detection. (b) Molecular structure of BG-(C6)3-
paraSA 1. The synthetic molecule is used to modulate HCAII activity in the
presence or absence of caspase-3.
Kuang Fu Road, Hsinchu 30013, Taiwan, ROC. E-mail: kttan@mx.nthu.edu.tw
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc42791a
c
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introducing a self-labeling protein SNAP-tag into HCAII to
facilitate quantitative and site-specific labeling via a benzylguanine
(BG) moiety.9 It has been shown that SNAP-tag labeling with the
BG moiety is efficient with high reproducibility and requires only
simple purification to obtain the labeled semisynthetic sensor.10
As a proof-of-principle, we demonstrate this semisynthetic
design by applying the sensor to the detection of protease
activity of caspase-311 which is a key mediator and a well-
established cellular marker of apoptosis.12 The recombinant
sensor protein SNAP_DEVD_HCAII (Fig. 1a) is functionalized
with the covalent linkage of a synthetic sulfonamide inhibitor
BG-(C6)3-paraSA 1 (Fig. 1b) to the SNAP-tag protein via the
corresponding benzylguanine moiety (BG) whereupon the
sulfonamide inhibitor can bind intramolecularly to the active
site of HCAII. The amino acid residue sequence DEVD, a
Fig. 3 Relative fluorescence intensities obtained using different synthetic sulfon-
amide inhibitors. BG-(C6)3-paraSA 1 shows an average of 17-fold fluorescence
amplification after 45 minutes.
caspase-3 cleavage sequence, is incorporated between SNAP- with caspase-3 and FLDA only shows that caspase-3 cannot
tag and HCAII for caspase-3 recognition and proteolysis. In the hydrolyze FLDA directly. We also tried different acetate pro-
absence of caspase-3, the proteolytic sensor exists in a closed tected fluorophores, and found that FLDA is the most suitable
conformation with the HCAII activity strongly inhibited by the substrate for HCAII (Fig. S2, ESI†). Fig. 2b shows the time
sulfonamide inhibitor due to the high effective concentration course of the fluorescence intensity. The fluorescence amplifi-
in the intramolecular closed conformation.13 In the presence of cation results showed a gradual increase in fluorescence
caspase-3, the protease recognizes the DEVD sequence and between 0 and 45 minutes. These results indicate that the
cleaves the recombinant sensor protein into two fragments. enzyme activity can be modulated by covalently linking a
In this intermolecular case, the sulfonamide inhibitor exhibits synthetic inhibitor to the enzyme and fluorescence amplifica-
weaker inhibition to HCAII, which in turn can hydrolyze non- tion occurs upon treatment of a specific activator.
fluorescent fluorescein diacetate (FLDA) to generate strongly
In order to optimize the fluorescence signals, we synthesized
fluorescent fluorescein (FL). HCAII was chosen as the signaling a series of inhibitors with different linker lengths and sulfon-
enzyme because of its esterase function to hydrolyze FLDA.14 amides. When BG-NH2 2, a compound without a sulfonamide
In addition, numerous other sulfonamide inhibitors have been inhibitor, was labeled to the sensor protein, the semisynthetic
reported to inhibit HCAII esterase activity,15 which provide us sensor was not able to differentiate between the presence or
with the means to control the sensitivity of our sensor.
absence of caspase-3 (Fig. 3). We also studied the effect of
To test our allosteric enzyme mimic sensor, we incubated linker length on the fluorescence enhancement and found that
our semisynthetic sensor with caspase-3 for 1 hour at 37 1C BG-(C6)3-paraSA 1 showed the highest signal amplification with
followed by addition of FLDA to initiate fluorescence signal an average of 17-fold intensity enhancement. The dissociated
amplification. In the presence of caspase-3, we observed a SNAP-sulfonamide fragment can still block the HCAII activity,
dramatic fluorescence enhancement (by 17-fold) as compared though with weaker affinity as compared to the intramolecular
to the sensor without caspase-3 (Fig. 2a). The proteolysis of the sulfonamide. We generally observed residual HCAII activity of
DEVD peptide at the sensor by caspase-3 releases the sulfon- about 30% under our caspase-3 sensing conditions as compared
amide inhibitor from HCAII and activates the enzyme toward to the non-inhibitor modified SNAP_DEVD_HCAII (Fig. S3, ESI†).
the hydrolysis of FLDA and the generation of fluorescent FL. In Shortening the linker to BG-(C6)2-paraSA 3 and BG-(C6)-paraSA 4
the absence of caspase-3, the activity of HCAII was completely or introducing hydrophilic ethylene glycol chain BG-(EG)5-paraSA
inhibited by the intramolecular sulfonamide inhibitor, and 5 or BG-(EG)11-paraSA 6 does not provide significant fluorescent
only very weak fluorescence was observed. Negative control amplification. When the weaker meta-sulfonamide inhibitor
BG-(C6)3-metaSA 7 was employed to label the sensor protein, the
fluorescence was amplified by merely 2-fold. The lower fluores-
cent amplification of compounds 3–7 is attributed to the incap-
ability of the inhibitors to completely inactivate the enzyme. In
these cases, we generally observed higher background fluores-
cence in the absence of caspase-3. Therefore, the sensitivity of the
semisynthetic sensor depended mainly on the activity of liberated
HCAII upon caspase-3 cleavage for fluorescence amplification and
the level of background fluorescence associated with the strong
inhibition by sulfonamide inhibitors in the closed conformation.
A series of different concentrations of caspase-3 was used to
calibrate the detection range of the sensor. As the concentration
of caspase-3 increases, the resulting fluorescence is enhanced
due to the increase of catalytically active free HCAII (Fig. 4a).
Fig. 2 (a) Fluorescence spectra of the semisynthetic sensor in the presence or
absence of caspase-3 (CP-3). (b) Time course of the fluorescence intensity in the
presence of 5 ng mLÀ1 caspase-3. The error bar was calculated from three
independent experiments.
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The semisynthetic sensor is modular and can only be catalytically
activated when the target protein caspase-3 is present. This offers
a novel bioanalytical approach to avoid time-consuming and
laborious washing steps encountered in most of the enzyme-
catalyzed signal amplification assays. By genetically introducing
a self-labeling SNAP-tag protein into the enzyme, we have
established a general scheme to assemble an enzyme, a sensing
site and an inhibitor into one single functional unit to mimic
natural allosteric enzymes. Thus, our semisynthetic sensor
design not only provides a general approach to mimic allosteric
enzymes but also highlights the potential of semisynthetic
sensors in practical applications where sensitive and simple
detection methods are required.
Fig. 4 (a) Fluorescence spectra of the semisynthetic sensor with increasing
concentrations of caspase-3. (b) Linear relationship between the fluorescence
intensity and the concentration of caspase-3 within the range of 0.5 ng mLÀ1 to
5 ng mLÀ1. The error bar was calculated from three independent experiments.
We are grateful to the National Science Council (Grant
No.: 100-2113-M007-018-MY2) and Ministry of Education (Grant
No.: 101N2011E1), Taiwan (ROC), for financial support.
Notes and references
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