Seeing citrulline: Development of a phenylglyoxal-based probe to visualize protein citrullination

Article abstract of DOI:10.1021/ja308871v

Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.

Full text of DOI:10.1021/ja308871v

Communication
pubs.acs.org/JACS
Seeing Citrulline: Development of a Phenylglyoxal-Based Probe To
Visualize Protein Citrullination
Kevin L. Bicker,† Venkataraman Subramanian,† Alexander A. Chumanevich,‡ Lorne J. Hofseth,‡
and Paul R. Thompson*,†
†Department of Chemistry, The Scripps Research Institute, Scripps Florida, 120 Scripps Way, Jupiter, Florida 33458, United States
‡Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina,
Columbia, South Carolina 29201, United States
S
* Supporting Information
that can be used to diagnose and monitor the efficacy of
ABSTRACT: Protein arginine deiminases (PADs) cata-
lyze the hydrolysis of peptidyl arginine to form peptidyl
citrulline. Abnormally high PAD activity is observed in a
host of human diseases, but the exact role of protein
citrullination in these diseases and the identities of specific
citrullinated disease biomarkers remain unknown, largely
because of the lack of readily available chemical probes to
detect protein citrullination. For this reason, we developed
a citrulline-specific chemical probe, rhodamine−phenyl-
glyoxal (Rh−PG), which we show can be used to
investigate protein citrullination. This methodology is
superior to existing techniques because it possesses higher
throughput and excellent sensitivity. Additionally, we
demonstrate that this probe can be used to determine
the kinetic parameters for a number of protein substrates,
monitor drug efficacy, and identify disease biomarkers in
an animal model of ulcerative colitis that displays
aberrantly increased PAD activity.
treatments for these diseases. Additionally, the ability to detect
changes in protein citrullination rapidly in response to the
addition of a PAD inhibitor will facilitate the development of
such compounds.
Common methods for studying citrullination include the
color development reagent (COLDER) assay17−20 and a
commercially available anti-citrulline (modified) detection kit
(ACM kit; Millipore,; Billerica, MA).21 Although widely used
for studying small-molecule and peptide substrates of the
PADs, the COLDER assay has a relatively high limit of
detection (LOD) of ∼0.6 nmol, making it difficult to use with
less concentrated protein substrates. The ACM kit has also
proven useful but suffers from relatively high cost ($40/assay),
a lengthy two-day procedure, and specialized analysis. Although
other means of detecting citrullinated proteins have been
described, these are also antibody-based (e.g., the F95
antibody22 and citrullinated histones H3 and H4). In contrast,
the probe described herein is readily synthesized, show
excellent sensitivity, react with any citrulline bearing protein,
and can be used by most researchers because detection uses
reagents/instruments that are available in virtually all biochemi-
cally focused laboratories.
The design of the citrulline-specific probe described herein is
based on the chemoselective reaction that occurs between
glyoxals and either citrulline or arginine under acidic or basic
conditions, respectively [Figure 1A and Figure S1 in the
Supporting Information (SI)].23 Although Fleckenstein and co-
workers23 reported the derivatization of phenylglyoxal onto the
solid phase via the para position, this chemistry is not amenable
to the solution phase (Figure S2). Therefore, we focused on
synthesizing phenylglyoxal compounds with an azido group at
the meta position that could be reacted with alkyne-modified
rhodamine via the Cu-catalyzed azide−alkyne cycloaddition
reaction (Figure S3).24,25 The reaction between citrulline and
phenylglyoxal likely proceeds as previously suggested (Figure
S4).20,23
he protein arginine deiminases (PADs) catalyze the post-
T
translational conversion of peptidyl arginine to peptidyl
citrulline, a process commonly called deimination or citrullina-
tion.1,2 PAD activity is aberrantly increased in different
diseases,1,2 including rheumatoid arthritis (RA), ulcerative
colitis (UC), Alzheimer’s disease (AD), multiple sclerosis
(MS), lupus, Parkinson’s disease (PD), and cancer.1,3−8 For
example, increased PAD levels are observed in the RA
synovium,9 during the formation of neutrophil extracellular
traps (NETs) in colitis and lupus,10 within the brains of
patients with MS,11 in the joints of patients with osteoarthritis,9
in UC lesions,12 in the hippocampal extracts of AD patients,5
and in several carcinomas.6 Furthermore, the administration of
Cl-amidine, a potent PAD inhibitor,13 or a Cl-amidine
analogue14 has proven useful in decreasing disease severity in
animal models of UC, RA, and cancer.14−16
Although abnormally high PAD activity and protein
citrullination are observed in these and other diseases, the
exact role(s) and target(s) of these enzymes are still poorly
understood, in part because of a lack of chemical tools that can
be used to identify the proteins that are citrullinated within a
specific disease model. The ability to investigate the specific
target and role of PADs in these diseases will inevitably allow
for better, more tailored treatments and identify biomarkers
The probe selectivity against commonly reactive amino acids
was first determined. Under acidic conditions, Rh−PG reacted
selectively with citrulline, homocitrulline, and cysteine (Figure
S5). Using a peptide substrate containing both citrulline and
Received: September 6, 2012
Published: October 3, 2012
© 2012 American Chemical Society
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performed in 20% trichloroacetic acid (TCA) at 37 °C, it is
important to note that increased labeling temperatures and
times led to the formation of SDS-insoluble protein aggregates
and decreased gel loading (Figure S10). Also, quenching of
excess probe with citrulline after labeling reduced background
labeling of arginine residues upon resuspension of the protein
(data not shown).
To demonstrate that the Rh−PG probe detection method
compares favorably to the commercially available ACM kit, we
performed a comparative analysis of the two methods using
PAD4 autodeimination as the end point. PAD4, autodeimi-
nated for various amounts of time, was analyzed with Rh−PG
(Figure 2A, top) and with the ACM kit according to the
manufacturer’s instructions (Figure 2A, bottom). Silver staining
of the fluorescent gel was done to confirm equal protein
loading (Figure 2A, middle). Analysis of these data (Figure 2B)
indicated that the two techniques provide virtually identical
results. The rate of recombinant histone H3 citrullination was
also followed, and the two methods again showed essentially
identical results (Figure S11). These data indicate that the Rh−
PG method is comparable to commercially available techniques.
However, relative to the ACM kit, the Rh−PG labeling method
takes significantly less time (∼3 h vs ≥25 h), requires fewer
steps (6 vs 12), and involves simpler analysis (fluorescent
imaging vs Western blotting).
Figure 1. (A) The rhodamine−phenylglyoxal (Rh−PG) probe
chemoselectively labels citrulline over arginine at acidic pH. (B)
Schematic representation of the method used to monitor citrullination
with the Rh−PG probe. Specific arginine residues (green squares) of
histone H3 are converted to citrulline (blue diamonds) by PAD4.
Treatment of the citrullinated protein with the Rh−PG probe under
acidic conditions labels citrulline residues selectively. Subsequent
separation by SDS-PAGE and fluorescent analysis reveals citrullinated
proteins. An actual gel image is shown, demonstrating the selectivity of
Rh−PG.
Given the high sensitivity of Rh−PG, we next determined
whether this methodology could be used to quantify changes in
citrullination accurately by determining the steady-state kinetic
parameters for a protein substrate. Although we and others
have used the traditional COLDER assay to do this type of
analysis on protein substrates,26 it is difficult to obtain highly
accurate rates because of the limited sensitivity of the COLDER
assay and the high concentrations of proteins required for
accurate measurements of citrulline production. For the Rh−
PG kinetic experiments, we first monitored H3 citrullination as
a function of time to ensure that the kinetic parameters would
be determined under initial velocity conditions (Figure S12).
The Rh−PG fluorescence was converted to citrulline
concentration using a citrulline standard curve (see the SI for
details). Having identified a specific time that could be used to
obtain the initial rates, we used this method to determine the
steady-state kinetic parameters for the citrullination of histone
H3 by PAD4 (Figure 2C). The results of this analysis provided
cysteine, we showed that the thiohemiacetal generated between
Rh−PG and Cys is hydrolyzed at neutral pH, while the cyclic
system formed between Rh−PG and citrulline is not (Figure
S6). Since the gel-based screening method includes a
neutralization step prior to gel loading, the problem of cysteine
cross-reactivity is effectively negated; the selectivity versus Cys
under these conditions is ≥650-fold. After demonstrating that
our PG probe is capable of selectively reacting with either
arginine or citrulline in a pH-dependent manner (Figure S1),
we optimized the conditions for probe labeling, including the
ideal probe concentration (i.e., 100 μM; Figure S7), the ideal
labeling time (i.e., 30 min; Figure S8), and the probe
sensitivity/LOD [i.e., ∼10 ng (∼0.67 pmol) of citrullinated
histone H3 and ∼1 ng (∼12.7 fmol) of autodeiminated PAD4;
Figure S9]. The different LODs are due to differences in the
citrulline content of the two proteins. Since Rh−PG labeling is
a kcat/Km value of 4800
1100 M−1 s−1, which compares
favorably with our previously reported value of 3700 M−1 s−1.26
Figure 2. (A, B) The time course of PAD4 autodeimination is similar for the Rh−PG probe and commercially available antibody methods. (A) Rh−
PG probed fluorescent image (top) and Anti-Cit (Mod) Kit Western blot analysis (bottom) of autodeiminated PAD4. Silver staining (middle) shows
equal protein loading. (B) Plot of percent citrullination over time for each type of analysis, indicating that the two methods are comparable. (C) The
Rh−PG labeling method can also be used to determine the kcat/Km value of histone H3 citrullination by PAD4.
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Similar analyses performed on the PAD4- catalyzed deimina-
tion of histone H4 gave a value of 2800 200 M−1 s−1 (Figure
S13), which is also similar to the previously reported value.26 In
view of the poor sensitivity and high signal-to-noise ratios of
current methods for determining the rates of protein
citrullination (i.e., the COLDER analysis and ammonia
detection methods),8,27 the accuracy and excellent sensitivity
of our method (notably, the samples had to be diluted 60-fold
before analysis) make it a unique tool to study protein
deimination because it permits the determination of Km values
for high-affinity protein substrates and allows for the study of
proteins that are difficult to produce in large quantities.
We envisioned that a key advantage of this probe would be
its facile ability to visualize target engagement (i.e., decreased
citrullination upon inhibitor treatment) as well as the
detection/identification of disease-associated biomarkers. To
demonstrate its utility for these types of analyses, we evaluated
changes in protein citrullination using serum samples from our
previous study demonstrating that Cl-amidine, a PAD inhibitor,
reduces the disease severity in a mouse model of dextran
sodium sulfate (DSS)-induced UC.15 Citrullinated proteins
present in these samples were labeled with the Rh−PG probe
and separated by SDS-PAGE, and the fluorescent proteins were
imaged (Figure 3A). We subsequently analyzed whole-lane
fluorescence as a function of the addition of Cl-amidine (Figure
3B). Consistent with our previous studies, which used the
COLDER assay to measure changes in total protein
citrullination, analysis of the whole-lane fluorescence revealed
a significant (P < 0.01) reduction in fluorescence intensity for
the serum samples treated with Cl-amidine. Notably, selectivity
analysis indicated the Cl-amidine itself does not react with the
probe (data not shown). Unlike the previous analysis, which
measures only total protein citrullination, the Rh−PG method
allowed us to monitor the effects on specific proteins as
opposed to global serum citrullination. On the basis of visual
inspection of the fluorescent images, we identified five protein
bands within each lane (at 10, 25, 50, 70, and 100 kDa) that
appeared to show a marked change in protein citrullination as a
function of Cl-amidine. From a comparison of the fluorescence
intensities of these bands for the two groups, we observed that
four of the five protein bands showed statistically significant
decreases in citrullination in response to Cl-amidine (P < 0.05;
Table S1 in the SI). The only protein band analyzed that did
not show a significant decrease in response to Cl-amidine (P =
0.44) was the band at 10 kDa.
Given these findings, we hypothesized that direct correlations
between the citrullination of these proteins and the disease
severity may exist. To test this hypothesis, we compared the
fluorescence intensity data for the five proteins described above
to each of five different disease metrics obtained for the mice in
this study (i.e., colon length, weight difference, white blood cell
count, lymphocyte count, and inflammation score) (Table S2).
Correlation plots (Figure 3C and Figure S14) and correlation
coefficients (Figure 3D) were generated, and colon length (four
out of six significant correlations) and inflammation scores (five
out of six significant correlations) showed the highest number
of correlations between disease severity and citrulline level. It is
noteworthy that these two end points are the key indicators of
disease severity.15,28,29
Figure 3. (A) Fluorescent image of Rh−PG-labeled serum samples
from a mouse model of DSS-induced UC. (B) Analysis of the UC
samples indicated a significant decrease in protein citrullination in
response to Cl-amidine (*P < 0.05; **P < 0.01). (C) Correlation
between colon length and fluorescence of the protein band at 25 kDa.
(D) Table of correlation coefficients between various disease scores
and fluorescence intensities of specific protein bands.
correlation coefficients, and therefore the most relevant
correlations, exist between colon length and the levels of the
citrullinated 25 kDa and 70 kDa proteins (correlation
coefficients of −0.932 and −0.826, respectively; Figure 3C).
It is important to note that the average protein levels, as
analyzed by Coomassie staining, were the same for untreated
and Cl-amidine-treated samples of each of these protein bands,
indicating that the change in fluorescence was indeed due to a
change in citrullination. The 25 kDa band also showed the
Interestingly, the 10 kDa protein, the only protein with no
significant decrease in citrullination in response to Cl-amidine,
also showed no significant correlation to any of the disease
scores, so it essentially served as a negative control. The highest
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ASSOCIATED CONTENT
* Supporting Information
■
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Synthetic procedures, experimental details, and supporting
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AUTHOR INFORMATION
Corresponding Author
pthompso@scripps.edu
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work was provided by NIH (Grants
GM079357 to P.R.T. and CA151304 to P.R.T. and L.J.H) and
TSRI.
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