A near-infrared fluorescence probe for imaging of pantetheinase in cells and mice: In vivo

Article abstract of DOI:10.1039/d0sc04537c

Pantetheinase is an amidohydrolase that cleaves pantetheine into pantothenic acid and cysteamine. Functional studies have found that ubiquitous expression of this enzyme is associated with many inflammatory diseases. However, the lack of near-infrared fluorescence probes limits the better understanding of the functions of the enzyme. In this work, we have developed a new near-infrared fluorescence probe, CYLP, for bioimaging of pantetheinase by using pantothenic acid with a self-immolative linker as a recognition group. The probe produces a sensitive fluorescence off-on response at 710 nm to pantetheinase with a detection limit of 0.02 ng mL-1 and can be used to image the intraperitoneal pantetheinase activity in mice in vivo. Moreover, with the probe we have observed that pantetheinase is significantly increased in the tissues of mouse inflammatory models as well as in the intestines of mice with inflammatory bowel disease. Therefore, CYLP may provide a convenient and intuitive tool for studying the role of pantetheinase in diseases.

Full text of DOI:10.1039/d0sc04537c

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A near-infrared fluorescence probe for imaging of pantetheinase
in cells and mice in vivo†
Yuantao Yang,‡ab Yiming Hu,‡a Wen Shi*ab and Huimin Maab
xReceived 00th January
20xx,
Pantetheinase is an amidohydrolase that cleaves pantetheine into pantothenic acid and cysteamine.
Functional studies have found that ubiquitous expression of this enzyme is associated with many inflammation
diseases. However, the lack of near-infrared fluorescence probes limits the better understanding of the
functions of the enzyme. In this work, we have developed a new near-infrared fluorescence probe CYLP for
bioimaging of pantetheinase by using pantothenic acid with a self-immolative linker as a recognition group.
The probe produces sensitive fluorescence off-on response at 710 nm to pantetheinase with a detection limit
of 0.02 ng/mL, and can be used to image the intraperitoneal pantetheinase activity in mice in vivo. Moreover,
with the probe we have observed that pantetheinase is significantly increased in the tissues of mouse
inflammatory models as well as in the intestines of the inflammatory bowel disease mice. Therefore, CYLP may
provide a convenient and intuitive tool for studying the role of pantetheinase in diseases.
Accepted 00th January
20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Our group reported a fluorescent probe for imaging pantetheinase
activity in living cells.11 It was comprised of pantothenic acid as a
recognition moiety and cresyl violet as a fluorochrome, both of which
Introduction
Pantetheinase, mainly known as vascular non-inflammatory
molecule-1 (vanin-1), is highly expressed in many organs, such as
liver, intestine, kidney and lung.1 The initial inspections on
pantetheinase focused on its function in coenzyme A (CoA)
metabolism and lipid metabolism, since it can break down
pantetheine into cysteamine and pantothenic acid (vitamin B5), a
precursor of CoA.2 In recent years, many studies have intensely
elucidated the latent role of pantetheinase in relation to
inflammation and diseases. For example, the vanin 1 knockout mice
showed higher susceptibility to drug-induced hepatic injury and
decreased capacity to detoxify xenobiotics, suggesting the enzymic
were connected through acyl amide bond directly. However, the
analytical wavelength of the previous probe is relatively short and
unsuitable for in vivo imaging study due to the poor penetration
depth in biological matrix. After that, Li et al. developed a
bioluminogenic probe for pantetheinase by using aminoluciferin as
the bioluminophore;12 however, the costly transgenic mice are
needed. As is known, a near-infrared (NIR) fluorescence probe would
possess significant advantage in imaging pantetheinase activity in
vivo on account of the simple operation and deep tissue
penetration,13-15 but such an NIR probe has not been reported yet.
Herein we have developed the first fluorescent probe CYLA
towards pantetheinase with NIR analytical wavelengths (Scheme 1).
Initially, we constructed a probe (control; Scheme 1) by directly
coupling pantothenic acid to the fluorophore of the amino
hemicyanine, also expecting that the enzymatic cleavage of the acyl
amide bond would release the fluorophore and generate the NIR
fluorescence. Nevertheless, the control probe showed almost no
response toward pantetheinase (Fig. S11). The possible reason for
this phenomenon might be due to that the narrow substrate channel
in the enzyme was incompatible with the bulky fluorophore,
preventing the substrate from accessing the active center.16 To
facilitate the recognition moiety of the probe approaching the
catalytical site, a self-immolative linker, 4-aminobenzyl methyl(2-
(methylamino)-ethyl)carbamate, was inserted between the bulky
fluorophore and pantothenic acid to obtain the probe CYLA.17,18 To
our delight, this indirect construction strategy significantly improved
the analytical performance of the resulting probe, i.e., CYLA
exhibited outstanding selectivity and sensitivity toward
pantetheinase with NIR emission wavelength (> 700 nm). Therefore,
protective effects.3 Moreover, pantetheinase has displayed
a
promising capacity as disease marker since it is upregulated in the
early phase of kidney injury and inflammatory bowel diseases.4,5 On
the other hand, the pantetheinase’s inhibitor can attenuate the
oxidative stress and inflammation in both acute and chronic
intestinal diseases, implying pantetheinase as a pro-inflammatory
factor.6,7 The diverse and seemingly paradoxical results reflect the
poor understanding of the pantetheinase biofunction. Obviously, this
is closely related to the lack of more methods to analyze the activity
of pantetheinase in complex biological samples under physiological
and pathological conditions.8-10 Especially, the imaging method that
can be used to study the pantetheinase activity in vivo is still rare.
a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China. E-mail: shiwen@iccas.ac.cn
b University of Chinese Academy of Sciences, Beijing 100049, China.
†
Electronic supplementary information (ESI) available. See DOI:
10.1039/x0xx00000x.
‡ These authors contributed equally to this work.
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the fluorescence imaging of the pantetheinase activity could be be 0.02 ng/mL pantetheinase, which is even much loVwieewrAtrthicalenOtnhlinaet
achieved in vivo.
obtained with the bioluminescent probDeO. I:T10h.u10s,39/pDr0oSbCe04C53Y7LCP
constructed by coupling the recognition moiety through the self-
immolative linker showed the higher efficacy in the enzymatic
reaction compared to those probes from the direct strategy.
The specificity of CYLP was investigated by reaction with various
substances, including inorganic salts (KCl, MgCl2, CaCl2, ZnCl2, CuCl2
and FeCl3), small biomolecules [glucose, cysteine, lysine, glutamic
acid, aspartic acid and lipopolysaccharides (LPS)], and some enzymes
[superoxide dismutase (SOD), leucine amino peptidase (LAP) and
carbonic anhydrase]. The results (Fig. 1D) showed that CYLP
exhibited excellent selectivity for pantetheinase over the other
biosubstances tested, indicating the reliability of the detection.
O
N
O
O
O
OH
N
O
O
N
N
H
H
OH OH
O
pantetheinase
N
+
+
O
N
O
N
CYLP
NH
N
CyOH
O
O
H2
N
N
H
OH OH
pantothenic acid
O
O
HN
N
H
OH OH
pantetheinase
+
no reaction
O
N
control
A
Scheme 1 Structures of CYLP and a control, and their reaction with
pantetheinase.
B
b
100 ng/mL
50
3000
2000
1000
0
4000
2000
0
Results and discussion
Fluorescent response of CYLP to pantetheinase
10
0
a
0
50
100
150
700
750
800
850
t (min)
Wavelength (nm)
The fluorescence spectra of CYLP in the absence and presence of
pantetheinase are shown in Fig. 1. As is seen, CYLP (10 μM) itself has
almost no fluorescence in the NIR region (quantum yield < 0.1%);
upon reaction with pantetheinase (50 ng/mL), the system showed
40-fold fluorescence enhancement at 710 nm. Meanwhile, the
maximum absorption peak of the reaction system is red-shifted from
590 to 680 nm (Fig. S12). These observations suggested that the
reaction of CYLP with pantetheinase led to the release of the
fluorophore CyOH (quantum yield 21%), which was clearly verified by
the ESI-MS analysis with the generation of the mass peak of CyOH at
m/z = 412 [M]+ (Fig. S13).
C
D
3000
2000
1000
0
3000
2000
1000
0
0
1 2 3 4 5 6 7 8 9 1011121314151617
0
10 20 30 40 50
pantetheinase (ng/mL)
Fig. 1 (A) Fluorescence spectra of CYLP (10 μM) before (a) and after
(b) reaction with pantetheinase (50 ng/mL). (B) Fluorescence kinetic
curves of CYLP (10 μM) with pantetheinase at varied concentrations
(from bottom to top): 0 (control), 10, 50, 100 ng/mL. (C) Linear fitting
curve of fluorescence enhancement toward the concentration of
pantetheinase from 0−50 ng/mL. (D) Fluorescence responses of CYLP
(10 μM) to different species 1−17: (1) blank; (2) KCl (150 mM); (3)
MgCl2 (2.5 mM); (4) CaCl2 (2.5 mM); (5) ZnCl2 (100 μM); (6) CuCl2 (100
μM); (7) FeCl3 (100 μM); (8) glucose (1 mM); (9) cysteine (1 mM); (10)
lysine (1 mM); (11) glutamic acid (1 mM); (12) aspartic acid (1 mM);
(13) LPS (2 μg/mL); (14) SOD (2 μg/mL); (15) LAP (2 μg/mL); (16)
carbonic anhydrase (2 μg/mL); (17) pantetheinase (50 ng/mL).
The fluorescence kinetic study of CYLP reacting with different
concentrations of pantetheinase is shown in Fig. 1B. The probe
showed
a dose-dependent response toward the addition of
pantetheinase and the fluorescence reached an approximate plateau
in about 1 h, which is much faster than that from our previous
probe.11 Meanwhile, no significant fluorescence variation can be
observed in the absence of pantetheinase, suggesting the excellent
stability of CYLP. Furthermore, the Michaelis constant (Km) of CYLP
was determined to be 84 μM and the Kcat was 23000 s-1 by
Lineweaver-Burk plot, which exhibited high affinity and rapid
catalytic rate toward pantetheinase (Fig. S14).
Then the effects of pH ranging from 4 to 10 and temperature
between 25 °C and 42 °C were assessed (Fig. S15). The probe itself
kept consistently low fluorescence in the tested pH range, and the
reaction system displayed the maximum fluorescence enhancement
in the neutral media of about pH 7.4. On the other hand, modestly
elevated temperature (e.g., 37 °C) facilitated the enzymatic reaction
slightly compared to room temperature (about 25 °C). The above
observations indicated that the detection of pantetheinase by CYLP
can be performed under physiological conditions.
The fluorescence titration of CYLP with pantetheinase exhibited
a good linearity (Fig. 1C) in the concentration range of 0−50 ng/mL
pantetheinase, with an equation of ΔF = 68.5×[C (ng/mL)] (R = 0.99),
where ΔF is the fluorescence enhancement of CYLP at 710 nm with
and without pantetheinase. The detection limit was determined to
Fluorescence imaging of pantetheinase in living cells
To test whether probe CYLP can be used to image pantetheinase in
living cells, four cell lines were chosen as models. Firstly, the
cytotoxicity of CYLP was evaluated with HK-2 and HepG2 cells by
standard MTT assay, and no significant toxicity was observed for 10
μM CYLP with 24 h incubation, indicating the good biocompatibility
of the probe (Fig. S16).
In cell imaging, the cells (including B16, HK2, HepG2 and HeLa
cells) themselves displayed no fluorescence under the excitation
wavelength of 635 nm. After incubation with CYLP for 1 h, the cells
exhibited obvious fluorescence (Fig. 2). However, if we pretreated
the cells with the specific inhibitor of pantetheinase, RR6, the
fluorescence in the cells was largely suppressed (Fig. S17). Moreover,
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dose positive correlation inhibition can be observed for these cell a representative example shown in Fig. 3, at 50 min after
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DOI: 10.1039/D0SC04537C
lines, suggesting that the intracellular fluorescence may result from intraperitoneal injection, the experimental mouse showed a 2.5-fold
the activity of pantetheinase. In addition, RR6 itself was proved to brighter fluorescence than that in the control group with the
have no obvious influence on both the cell viability (Fig. S18) and the inhibitor, which indicated that CYLP can image the endogenous
fluorescence of the released fluorophore in cells (Fig. S19). On the pantetheinase in living animals.
other hand, these cell lines were subjected to Western blot analyses
A
B
to assess the pantetheinase level (Fig. S20), revealing that HepG2
cells displayed the most abundant pantetheinase, which is consistent
with the fluorescence imaging result. Besides, HK-2 and HeLa cells
had similar pantetheinase levels in Western blot assay, but they
showed obviously different fluorescence intensity, possibly due to
the different enzymatic reactivity or uptake efficiency of the probe in
different cells. Even so, the above data suggest that the probe is
capable of imaging the intracellular pantetheinase.
25000
CYLP
CYLP+RR6
12000
8000
4000
*
3500
Fluorescence
(counts/pixel)
0
10 20 30 40 50 60
t (min)
Fig. 3 (A) The representative images at 50 min of mice after the
intraperitoneal injection of CYLP. Left: mouse pretreated with RR6 as
negative control; right: mouse pretreated with only saline (NaCl
0.9%). The full images of the mice at different time are given in Fig.
S21. Scale bar, 2 cm. (B) Time dependent change of the fluorescence
in the mice captured at different time (n = 3 pairs of mice). * p<0.05,
two-sided Student’s t-test.
B16
HK-2
HepG2
HeLa
blank
Fluorescence imaging of pantetheinase in mice with inflammation
The inflammation, a common defense mechanism, can protect our
body from infection and injury by immune cells and the substances
they produced, including cytokines and reactive oxygen species
(ROS).19 However, abnormal ROS level would cause certain diseases.
Since pantetheinase can lead to the release of cysteamine, which can
further regulate oxidative stress of the cells, this enzyme has been
implicated in many inflammatory diseases by serving as a pro-
inflammatory factor or upregulated sera biomarker.1 However, the
in situ pantetheinase level in the inflammatory diseases has not been
studied due to the lack of suitable tools. Therefore, encouraged by
the analytical performance of CYLP, we further explored the probe’s
application in imaging the pantetheinase activity change in an
inflammation model. Namely, the hind limb inflammation of the
Kunming mice was made by injecting 200 μL of 1 mg/mL LPS into the
right hind leg of the mouse (Fig. 4A and Fig. S22). In the meantime,
an equal amount of saline was injected into the left leg as control.
After 14 h, CYLP (400 μM×20 μL) was injected into both the legs of
the mouse subcutaneously. Fluorescent images of mice were
captured at different time points (Fig. S22). As can be seen from Fig.
4A, the mouse has stronger fluorescence in the right hind leg than
the left hind leg. The analyses from 3 mice showed that the
fluorescence intensity of the inflammation leg was significantly
higher than that of the control leg and the raised pantetheinase was
confirmed by Western blot analysis (Fig. S23). Further, we conducted
a negative control experiment in which the inhibitor RR6 was
injected for 30 min prior to the administration of CYLP in the
inflammatory leg. As shown in Fig. S24, the RR6 pretreatment can
suppress the fluorescence of the inflammatory leg substantially (Fig.
S24). This also supports that the fluorescence enhancement is mainly
due to the pantetheinase activity.
+CYLP
Fig. 2 Confocal fluorescence images of different cells. The 1st row:
cells themselves (control); the 3rd row: the cells incubated with CYLP
(10 μM) for 1 h; the 2nd and 4th rows are the differential interference
contrast images of the corresponding cells. Scale bar, 50 μm.
Fluorescence imaging of pantetheinase in mice in vivo
Due to the excellent analytical performance of CYLP in vitro, we
subsequently applied the probe to fluorescent imaging of
pantetheinase activity in vivo. Pantetheinase’s primary function is
the recycling of pantothenic acid, an important precursor of CoA,
which is the key cofactor for fat metabolism. Besides, pantetheinase
is often expressed in organs with high CoA turnover, such as liver and
intestine. Hence, we attempted to image the endogenous
pantetheinase activity in these organs. Also, negative controls with
pantetheinase inhibitor RR6 were conducted.6 Specifically, Kunming
mice were intraperitoneally injected with saline or RR6 (30 mg/kg,
negative control) firstly. After
1 h, all mice were injected
intraperitoneally with CYLP (400 μM × 200 μL), and then their
abdominal fluorescence was imaged every 10 min. The fluorescence
increased in the mice of the experimental group continued and
became relatively slow in 50 min. Meanwhile, the fluorescence in the
mice of the control group did not show noticeable enhancement. In
Finally, we demonstrated the feasibility and practicability of probe
CYLP for imaging pantetheinase in inflammatory bowel disease (IBD)
model of mice. IBD is a chronic inflammatory syndrome of the
digestive tract, and pantetheinase has been regarded as
a
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therapeutic target or fecal biomarker in the disease.20 The IBD mice All animal care and experimental protocols complied with the
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were induced by dextran sulfate sodium (DSS) via drinking water for Animal Management Rules of the MiniDsOtrIy: 10o.f10H39e/aDl0thSC0o4f53th7Ce
8 days.21 Then, CYLP (400 μM × 100 μL) was administrated through People’s Republic of China and were approved by the Institute
the anus. As a result, the IBD mice displayed stronger fluorescence in of Process S12 Engineering, Chinese Academy of Sciences.
abdomen compared to the control mice (Fig. 4C). It should be
mentioned that the fluorescence appeared mostly in the small This work is supported by grants from the NSF of China (Nos.
intestines rather than in the injection point of the colon region (Fig. 21922412, 21675159, 21820102007, and 21775152) and Youth
S25), probably implying the upregulation of pantetheinase in this Innovation Promotion Association of CAS (2016027).
area. Thus, CYLP may be used as a convenient and intuitive tool to
illustrate the change of the pantetheinase activity in living animal
models.
Notes and references
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t (min)
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Fig. 4 (A) The representative image at 50 min of the mouse after the
administration of CYLP. Left leg: injected with saline as control; right
leg: injected with 200 μL of 1 mg/mL LPS as inflammation model. The
full images of the mouse at different time points are shown in Fig.
S22. Scale bar, 2 cm. (B) Time dependent change of the fluorescence
of the control leg and inflammation leg captured at different time (n
= 3 mice). * p<0.05, two-sided Student’s t-test. (C) The images of the
IBD mice and control mice after the administration of CYLP through
the anus into colon.
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Conclusions
In summary, by inserting a carbamate-based self-immolative
linker between the recognition group (pantothenic acid) and the
fluorophore (CyOH), we have developed CYLP as a new near-infrared
fluorescent probe for pantetheinase assay. The probe displays high
selectivity and sensitivity, with a detection limit of 0.02 ng/mL
pantetheinase, and has been used to image pantetheinase in cells
and mice. Moreover, with the probe, we demonstrate that the local
inflammation can indeed increase the pantetheinase level in the
tissue. We believe the probe CYLP may serve as an effective tool for
studying the biological functions of pantetheinase and related
diseases.
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Conflicts of interest
There are no conflicts to declare.
16 Y. L. Boersma, J. Newman, T. E. Adams, N. Cowieson, G.
Acknowledgements
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Chemical Science
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DOI: 10.1039/D0SC04537C
TOC
O
N
O
O
O
N
O
N
N
H
O
H
OH OH
near-infrared
sensitive
O
CYLP
N
+ pantetheinase
CYLP
700 750
800
850
Wavelength (nm)
A near-infrared fluorescence probe for detecting pantetheinase activity has
been used for imaging pantetheinase in mice with inflammatory bowel
disease.