Pyroglutamate aminopeptidase 1 may be an indicator of cellular inflammatory response as revealed using a sensitive long-wavelength fluorescent probe

Article abstract of DOI:10.1039/c6sc00951d

Pyroglutamate aminopeptidase 1 (PGP-1) can remove pyroglutamic acid from the N-terminus of a polypeptide, including some important anti-inflammatory proteins. Detecting the change and distribution of cellular PGP-1 in an inflammation process would be helpful to better understand the role of this enzyme. However, no report has been found on this subject, mainly due to the lack of a proper research approach. Herein, we develop such a new method by preparing a sensitive long-wavelength fluorescent probe combined with confocal fluorescence imaging. The probe, consisting of l-pyroglutamic acid and cresyl violet, exhibits high selectivity and sensitivity for PGP-1 under physiological conditions. With this probe, the up-regulation of PGP-1 in LO-2 cells under the stimulation of Freund's incomplete adjuvant and lipopolysaccharide (two main immunopotentiators) is revealed for the first time, and this up-regulation is also observed in typical phagocytic RAW264.7 cells, as evidenced by western blot and inhibition assays. Studies on the distribution of PGP-1 in cells using our probe showed that most PGP-1 is located in the cytoplasm, which is further supported by an immunofluorescence assay. Moreover, the inflammatory response induced by the immunopotentiators in either RAW264.7 or LO-2 cells is confirmed by measuring tumor necrosis factor alpha (a common inflammatory factor). The above findings indicate that cellular inflammation is accompanied by an increase in PGP-1, and PGP-1 may serve as a new indicator of cellular inflammatory response.

Full text of DOI:10.1039/c6sc00951d

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ARTICLE
Pyroglutamate Aminopeptidase 1 May be an Indicator of Cellular
Inflammatory Response as Revealed by a Sensitive Long-
Wavelength Fluorescent Probe†
Received 00th January 20xx,
Accepted 00th January 20xx
Qiuyu Gong, Lihong Li, Xiaofeng Wu and Huimin Ma*
DOI: 10.1039/x0xx00000x
Pyroglutamate aminopeptidase 1 (PGP-1) can remove pyroglutamic acid from the N-terminus of a polypeptide, including
some important anti-inflammatory proteins. Detecting the change and distribution of cellular PGP-1 in an inflammation
process would be helpful to better understand the role of the enzyme. However, no report has been found on this subject,
mainly due to the lack of a proper research approach. Herein, we develop such a new method by preparing a sensitive
long-wavelength fluorescent probe combined with confocal fluorescence imaging. The probe, consisting of L-pyroglutamic
acid and cresyl violet, exhibits high selectivity and sensitivity to PGP-1 under the physiological conditions. With this probe,
the up-regulation of PGP-1 in LO-2 cells under the stimulations of freund’s incomplete adjuvant and lipopolysaccharide
(two main immunopotentiators) is revealed for the first time, and this up-regulation is also observed in typical phagocytic
RAW264.7 cells, as evidenced by western blot and inhibition assays. Studies on the distribution of PGP-1 in cells by our
probe showed that most of PGP-1 locates in the cytoplasm, which is further supported by immunofluorescence assay.
Moreover, the inflammatory response induced by the immunopotentiators in either RAW264.7 or LO-2 cells is confirmed
by measuring tumor necrosis factor alpha (a common inflammatory factor). The above findings indicate that the cellular
inflammation is accompanied by the increase of PGP-1, and PGP-1 may serve as a new indicator of cellular inflammatory
response.
www.rsc.org/
nm) but also high selectivity and sensitivity to PGP-1 under the
physiological conditions. Using the probe, the up-regulation of
Introduction
PGP-1 in LO-2 cells under the treatment of
immunopotentiators is revealed for the first time, and this up-
regulation is also found in typical phagocytic RAW264.7 cells,
as confirmed by western blot and inhibitor experiments.
Interestingly, studies on the distribution of PGP-1 in cells by
our probe reveal that most of PGP-1 may locate in the
cytoplasm, and this is further verified by immunofluorescence
assay. In addition, the inflammatory response induced by
immunopotentiators in either RAW264.7 or LO-2 cells is
validated by measuring the change of tumor necrosis factor
Pyroglutamate aminopeptidase 1 (PGP-1, EC 3.4.19.3) is a type
of enzyme that cleaves the peptide bond of pyroglutamic
acid linked to the N-terminal end of a protein, including some
important anti-inflammatory proteins like immunoglobulin.1-5
On the other hand, inflammation, a protective response that
involves immune cells, blood vessels, and molecular mediators,
plays
a
significant role in many diseases,6 on which
considerable effort has already been expended.7-9 However, it
is unknown whether PGP-1 has any relationship with a
inflammatory process in cells. Without doubt, detecting the
change and distribution of cellular PGP-1 would be helpful to
understand this issue. To the best of our knowledge,
nevertheless, no report has been found on this subject, mainly
due to the lack of a proper research approach. Herein, we
develop such a new method by preparing a sensitive long-
wavelength fluorescent probe combined with confocal
alpha (TNF-
α a common inflammatory factor). Below we
report these results.
Results and discussion
Spectroscopic properties and analytical performance of probe 1
On the basis of our previous experience,10,11 we first prepared
fluorescence imaging. The probe (
cresyl violet and L-pyroglutamic acid (as a recognition moiety),
exhibits not only long analytical wavelengths ( ex/em = 585/625
1; Fig. 1A), consisting of
1
(Fig. 1A) as a sensitive long-wavelength fluorescent probe for
PGP-1. Cresyl violet with long-wavelength feature was chosen
as a fluorescent skeleton, because substitution of its amino
group usually leads to complete fluorescence quenching,11b
which would favor low background signal and thus sensitive
λ
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: mahm@iccas.ac.cn
detection. As shown in Fig. 1A, probe
1 can be readily
prepared by treating cresyl violet with the protected L-
pyroglutamic acid, and then removing the protecting group in
† Electronic supplementary information (ESI) available: Experimental section and
supporting figures. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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the presence of trifluoroacetic acid (Electronic Supplementary reaction were determined to be 120 µM and 0.25 µM/s.
Information, ESI ). The obtained probe was well characterized Moreover, inhibitor experiments with iodoacetamide (Fig. S12)
†
by NMR and mass spectral analyses (Figs. S1-S4, ESI
†
).
also supported that the fluorescence off-on response of the
probe arises from the enzymatic action of PGP-1.
A
N
O
OH
Detection of up-regulation of PGP-1 in living cells under the
stimulation of immunopotentiators
HATU, DIPEA
cresyl violet
O
N
H
O
O
NH2
N
O
N
(H3C)3C
O
O
C(CH3)3
O
O
Next, we explore whether PGP-1 has a relationship with an
inflammatory process in cells by detecting the change and
intermediate (10% yield)
distribution of cellular PGP-1 with probe
1 combined with
N
O
N
O
CF3COOH
PGP-1
confocal fluorescence imaging. As shown in Fig. 2, the
fluorescence intensity of LO-2 cells treated by
immunopotentiators [freund’s incomplete adjuvant (FIA), or
lipopolysaccharide (LPS)]14 gradually increases with increasing
the immunopotentiator concentration in the range of 0 -
0.25% (v/v) FIA (images B1-E1, and Fig. 2F1) and 0 – 1 µg/mL
LPS (images B2-E2, and Fig. 2F2), respectively. This
fluorescence increase reflects the up-regulation of intracellular
PGP-1 concentration, which is clearly confirmed by western
blot analysis (see Fig. 2G1 and 2G2). Studies on the effect of
incubation time of FIA or LPS revealed that the intracellular
fluorescence intensity, representing the PGP-1 concentration,
rises in a time-dependent manner (see images B1-E1 and panel
N
NH2
H2N
O
NH2
H
NH
O
1
(80% yield)
cresyl violet
B
C
0.12
90
60
30
0
100
50
0
2
1
2
0.08
0.04
0.00
1
0.0 0.4 0.8 1.2 1.6
C
(
ꢁ
g/mL
)
600
650
700
750
300 450 600 750
Wavelength (nm)
Wavelength (nm)
Fig. 1 (A) Synthesis of probe 1 and its reaction with PGP-1. (B)
Absorption spectra of 1 (5 µM) before (curve 1) and after (curve 2)
reaction with PGP-1 (2 µg/mL) at 37 °C in 6.7 mM PBS (pH 7.4). The
inset shows the corresponding color change. (C) Fluorescence
response of 1 (5 µM) to PGP-1 at different concentrations (from
bottom to top: 0 - 5 µg/mL). The inset shows the plot of ∆F versus
the PGP-1 concentration. λex/em = 585/625 nm.
G1 in Fig. S13 in ESI† for FIA; images B2-E2 and panel G2 in Fig.
S13 for LPS). Moreover, this rise in intracellular PGP-1 is
further supported by both the inhibitor experiments (images
F1 and F2 in Fig. S13; Fig. S14) and western blot assay (Fig. S14).
It should be pointed out that the fluorescence of the reaction
system of probe 1 with PGP-1 is scarcely affected by FIA or LPS
itself (Fig. S15). In addition, under the same conditions we also
observed the up-regulation of PGP-1 in HepG2 cells (Fig. S16),
possibly suggesting a universal phenomenon of PGP-1 increase
in inflammatory cells.
Spectroscopic properties and analytical performance of
probe 1 were then studied in detail. As shown in Fig. 1, probe 1
itself, with a maximum absorption at 490 nm, exhibits
extremely low background fluorescence (quantum yield, <
0.01). Under the optimized conditions (reaction for 30 min at
C1
D1
E1
B1
A1
37 °C and pH 7.4; Figs. S5 and S6 in ESI†), however, the probe
produces a good linear fluorescence off-on response to PGP-1
in the concentration range of 0.05 – 1.75 µg/mL (inset of Fig.
A2
B2
C2
D2
E2
1C), with an equation of ∆F = 47.6
0.995), where ∆F is the difference of fluorescence intensity of
after and before reaction with PGP-1. The detection limit12 is
determined to be 5.6 ng/mL PGP-1, which is more sensitive
than that (14.6 ng/mL; Fig. S7, ESI ) of a commercial probe
× C (µg/mL) – 1.65 (R =
1
0.6
0.4
0.2
0.0
G1 PGP-1
GAPDH
F1
1.2
0.9
0.6
0.3
0.0
***
†
***
***
***
(note that the commercial probe is unsuitable for cell imaging
due to its rather short wavelengths, λex/em = 340/440 nm).
Notably, the probe also exhibits rather high selectivity and
good biocompatibility (Figs. S8 and S9). It is worth noting that
the absorption and fluorescence spectra from the reaction
system accord well with those from cresyl violet.11 This
***
***
B1
G2
C1
PGP-1
D1
E1
***
B1
F2
C1
D1
***
E1
***
1.2
0.9
0.6
0.3
0.0
0.4
0.2
0.0
GAPDH
***
suggests that the enzymatic cleavage reaction of
1 by PGP-1
***
D2
***
occurred and caused the release of cresyl violet (Fig. 1A) with a
fluorescence quantum of 0.51, which was further verified by
mass spectral analysis (m/z = 262.0973 [M]+; Fig. S10).
According to Michaelis-Menten equation (Fig. S11),12,13 the
corresponding Michaelis constant (Km) and the maximum of
the initial reaction rate (Vmax) for the present enzymatic
B2
C2
E2
B2
C2
D2
E2
Fig. 2 Fluorescence images of PGP-1 in LO-2 cells. (A1, A2) LO-2 cells
only. (B1–E1) The cells were pretreated with FIA at varied
concentrations (0, 0.05, 0.1, 0.25%, respectively) at 37 °C for 24 h,
and then incubated with the probe (5 μM) for 30 min. (B2–E2) The
2 | J. Name., 2012, 00, 1-3
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cells were pretreated with LPS at varied concentrations (0, 0.2, 0.5, Location of PGP-1
ARTICLE
1.0 μg/mL, respectively) at 37 °C for 16 h, and then incubated with
Interestingly, we found that the fluorescence mainly appears
the probe (5 μM) for 30 min. Scale bar, 20 μm. (F1, F2) Relative
in the cytoplasm of the inflammatory cells, which may result
pixel intensity measurements (n = 3) from the above corresponding from the dominant distribution of PGP-1 in the cytoplasm. To
ascertain this, we performed immunofluorescence assay (a
technique that utilizes fluorescent-labeled antibodies to detect
specific target antigens).17 As shown in image C in Fig. 4, the
blue area (image A), representing cell nucleus, scarcely
overlaps with the green area (image B) that reflects the
location of PGP-1, which suggests that most of PGP-1 indeed
distributes in the cytoplasm. All the above results indicate that
FIA and LPS can induce the up-regulation of intracellular PGP-1,
and our probe can be used to visualize not only such increase
of PGP-1 but also the distribution of PGP-1 in cells, which
however cannot be realized by western blot assay.
images by using software ImageJ. The pixel intensities from images
E1 and E2 are defined as 1.0 (*** p<0.001, as compared with the
control, two-sided Student’s t-test). (G1, G2) The changes of PGP-1
in the above corresponding cells determined by western blot (the
molecular weight of PGP-1 is determined to be 23 kDa;
glyceraldehyde-3-phosphate dehydrogenase, GAPDH, as a protein
standard).
In order to further verify this phenomenon, RAW264.7 cells,
a typical phagocytic cell line,15 were also treated under the
same conditions. As can be seen from Fig. 3, the fluorescence
intensity of RAW264.7 cells treated by FIA or LPS gradually
C
A
B
increases
with
increasing
the
immunopotentiator
concentration (images B1-E1, and Fig. 3F1 for FIA; images B2-
E2, and Fig. 3F2 for LPS). The fluorescence increase indicates
the rise of intracellular PGP-1 concentration, which is again
proved by western blot analysis (Fig. 3G1 and 3G2). Therefore,
it is suggested that cells, whether normal, phagocytic, or non-
phagocytic,15b,16 exhibit the PGP-1 up-regulation in an
inflammation process, indicating the universality of this
behavior.
Fig. 4 Immunofluorescence images of LO-2 cells. (A) The cells were
stained by 4',6-diamidino-2-phenylindole (a nucleus-specific dye).
The blue fluorescence area represents the cell nucleus. (B) The cells
were stained by fluorescein isothiocyanate that was linked to the
antibody of PGP-1. The green fluorescence area, mainly appearing
in the cytoplasm, represents PGP-1 in the cells. (C) The overlapped
image of images A and B. Scale bar 20 μm.
C1
E1
D1
B1
A1
Measuring the inflammatory response
C2
E2
D2
B2
A2
Finally, the inflammatory response induced by the
immunopotentiators in either RAW264.7 or LO-2 cells was
studied by measuring a common inflammatory factor of TNF-
α
,
whose up-regulation reflects the cellular inflammatory
response enhancement.9c,18 As can be seen from Fig. 5, TNF-
increases with the increase of FIA or LPS, implying the
1.2
0.9
0.6
0.3
0.0
1.2
0.8
0.4
G1 PGP-1
GAPDH
F1
***
α
***
***
enhancement of the inflammatory response. All the above
findings indicate that PGP-1 may serve as an indicator of
cellular inflammatory response. The exact mechanism
underlying the up-regulation of intracellular PGP-1 needs to be
investigated in future research, but a possible reason may be
due to the fact that PGP-1 can cleave the N-terminal
pyroglutamic acid in some important anti-inflammatory
proteins like immunoglobulin.
***
***
**
0.0
0.9
B1
F2
C1
D1
***
E1
***
B1
G2
C1
D1
E1
***
E2
1.2
0.9
0.6
0.3
0.0
PGP-1
GAPDH
0.6
0.3
0.0
**
***
***
TNF-α
TNF-α
0.9
0.6
0.3
0.0
B
A
0.9
0.6
0.3
0.0
GAPDH
GAPDH
B2
C2
D2
E2
B2
C2
D2
***
***
***
**
***
***
Fig. 3 Fluorescence images of PGP-1 in RAW264.7 cells. (A1, A2)
RAW264.7 cells only. (B1–E1) The cells were pretreated with FIA at
varied concentrations (0, 0.05, 0.1, 0.25%, respectively) at 37 °C for
24 h, and then incubated with the probe (5 μM) for 30 min. (B2–E2)
The cells were pretreated with LPS at varied concentrations (0, 0.2,
0.5, 1.0, respectively) for 16 h, and then incubated with the probe
(5 μM) for 30 min. Scale bar, 20 μm. (F1, F2) Relative pixel intensity
measurements (n = 3) from the above corresponding images. The
pixel intensities from images E1 and E2 are defined as 1.0 (**
p<0.01, *** p<0.001, as compared with the control, two-sided
Student’s t-test). (G1, G2) The changes of PGP-1 in the above
corresponding cells determined by western blot.
0
0.05
C
0.1
0.25
0
0.2
C
0.5
1.0
(
ꢁ
g/mL
)
(%)
1.2
0.8
0.4
0.0
TNF-α
GAPDH
TNF-α
D
C
0.9
0.6
0.3
0.0
GAPDH
***
***
***
***
***
***
0
0.05
C
0.1
0.25
0
0.2
C
0.5
1.0
(
ꢁ
g/mL
)
(%)
Fig. 5 Western blot analysis of TNF-α. (A, B) The change of TNF-α in
LO-2 cells under treatment of FIA (0-0.25%) and LPS (0-1.0 μg/mL),
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11 (a) Q. Q. Wan, Y. C. Song, Z. Li, X. H. Gao, H. M. Ma, Chem.
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test).
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3183.
,
Conclusions
In conclusion, we have developed a new fluorescent PGP-1
probe, which exhibits good selectivity and high sensitivity.
Using the probe combined with confocal fluorescence imaging,
the up-regulation of intracellular PGP-1 in inflammatory LO-2
and RAW264.7 cells has been revealed, which is further
confirmed by western blot. The distribution of PGP-1 in the
cytoplasm is also successfully imaged. Importantly, by
measuring an inflammatory factor of TNF-α, we propose for
the first time that PGP-1 may serve as a new indicator of
cellular inflammatory response. Besides, it is believed that
with our probe more biological functions of PGP-1 may be
revealed.
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Acknowledgements
We are grateful for the financial support from the 973 Program
(no. 2015CB932001 and 2015CB856301), the NSF of China (no.
21535009, 21275147, 21435007, 91432101 and 21321003),
and the Chinese Academy of Science (XDB14030102).
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