A simple but efficient electrochemical method to assay protein arginine deiminase 4

Sensors and Actuators B 227 (2016) 43–47

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A simple but efficient electrochemical method to assay protein arginine deiminase 4 Xixian Chen a , Yun Lv a,b , Yuanyuan Zhang c , Jing Zhao a,∗ , Lizhou Sun c,∗ a b c

Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, PR China Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210036, PR China

a r t i c l e

i n f o

Article history: Received 6 October 2015 Received in revised form 10 December 2015 Accepted 13 December 2015 Available online 17 December 2015 Keywords: Protein arginine deiminase 4 Electrochemical assay Inhibitor screening Citrullination [Ru(NH3 )5 Cl]2+

a b s t r a c t Nowadays, protein arginine deiminase 4 (PAD4) has become a potential therapeutic target for human diseases, especially in rheumatoid arthritis (RA) patients. In this paper, we have reported a simple but efficient electrochemical method to assay protein arginine deiminase 4 (PAD4) activities and screen its inhibitors. The electropositive peptide monolayer can prevent [Ru(NH3 )5 Cl]2+ from approaching to the electrode surface due to the strong electrostatic repulsion. After PAD4 catalyzes the citrullination of arginine within substrate peptide, the signal molecules can be much closer to the electrode surface for the reduced positive charges on the peptide monolayer, thereby leading to an obvious electrochemical response. By tracing the electrochemical response of [Ru(NH3 )5 Cl]2+ , our method has displayed satisfactory sensitivity and specificity toward PAD4 assay with a low detection limit of 3.5 pM. Moreover, the electrochemical response has been found to decrease with the addition of the potent PAD4 inhibitor Cl-amidine, suggesting the potential application of our method for the inhibitor screening in the future. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Citrullination is a kind of post-translational modification of arginine in vivo, which can be catalyzed by protein arginine deiminase (PAD) [1,2]. At present, there are five isozymes in PAD enzyme family, which have been designated as PAD 1, 2, 3, 4 and 6 [3–5]. Among them, PAD4 that can catalyze citrullination in histones H2A, H3, and H4 has drawn most of the attention. One of the main reasons is that the upregulation of PAD4 has been especially found in rheumatoid arthritis (RA) patients, which is believed to play causative role in the onset and progression of RA [6–9]. Besides, aberrant increase of PAD 4 has also been involved in multiple sclerosis, glaucoma and even cancers [10–12]. Therefore, with the deciphering the relation between PAD4 and different disease models [13,14], PAD4 has emerged as a potential therapeutic target for human diseases. Although the dysregulated PAD4-catalyzed protein citrullination has been commonly observed in many human diseases, the exact physiological and pathological functions of PAD4 have not been fully understood [5,15]. One of the biggest obstacles is lack of

∗ Corresponding authors. Tel.: +86 25 86663616/+86 21 66137541. E-mail addresses: [email protected] (J. Zhao), lizhou [email protected] (L. Sun). http://dx.doi.org/10.1016/j.snb.2015.12.050 0925-4005/© 2015 Elsevier B.V. All rights reserved.

the effective means for the accurate identification of PAD4. Up to now, quite a few methods have been reported to assay PAD as we know, such as colorimetry, SDS–PAGE and fluorescence [16–20]. The existing methods always suffer from several disadvantages, including low sensitivity, complex operation and high cost. Therefore, developing effective tools for PAD4 assay may not only help to reveal the specific target and role of PAD4 within the disease models, but also facilitate the screening of PAD4 inhibitors for the disease treatments. Nowadays, electrochemical technique has been developed into a powerful analytical approach in the biosensor fabrication, benefiting from the unique advantages of high sensitivity, simple operation and rapid response [21–26]. By taking the advantages of electrochemical technique in protein detection, we have herein firstly proposed a simple but efficient electrochemical method to assay the activities of PAD4 and screen its inhibitors. The positively-charged substrate peptide on the surface of gold electrode can strongly repel the approaching electrochemical signal molecule [Ru(NH3 )5 Cl]2+ . After PAD4-catalyzed citrullination of arginine within substrate peptide, the signal molecules can be much closer to the electrode surface for weakened electrostatic repulsion, resulting in obvious electrochemical responses. Consequently, PAD4 assay and subsequent inhibitor screening can be easily realized by tracing the electrochemical responses of [Ru(NH3 )5 Cl]2+ at the substrate peptide modified electrode.

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2. Experimental

assay, different concentrations of Cl-amidine were premixed with 5 nM PAD4 at 37 ◦ C for 20 min.

2.1. Materials and reagents Substrate peptides (GRGAGRGAC) were purchased from GL Biochem (Shanghai) Ltd. PAD4, alkaline phosphatase (ALP), bovine serum albumin (BSA), myoglobin, hemoglobin, mercaptohexanol (MCH), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), N2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) and pentaamminechlororuthenium (III) chloride ([Ru(NH3 )5 Cl]2+ ) were purchased from Sigma. Cl-amidine was purchased from Cayman Chemical. Enzymatic reaction buffer for PAD4: 50 mM Tris–HCl containing 1 mM CaCl2 (pH 7.6). Electrolytic solution for electrochemical measurements: 20 mM HEPES containing 50 mM [Ru(NH3 )5 Cl]2+ (pH 6.0). For all experiments, Milli-Q water (>18.0 M) was used, which was purified by a Milli-Q Plus 185 ultrapure water system (Millipore purification pack). 2.2. Preparation of substrate peptide monolayer modified electrode The gold electrode (3.0 mm diameter, Gaossunion Ltd.) was first polished to a mirror-like smooth with 1 ␮M, 0.3 ␮M, 0.05 ␮M alumina slurry, respectively. Then, the residual alumina powder was removed by sonicating the electrode sequentially in ethanol and double-distilled water. Afterward, the electrode was coated with piranha solution (concentrated H2 SO4 : 30% H2 O2 = 3:1) for 5 min and electrochemically cleaned in 0.5 M H2 SO4 by scanning from 0 V to 1.5 V. [27,28]. After drying in the nitrogen atmosphere, the cleaned electrode was dipped into a solution containing 50 ␮M substrate peptide (Fig. S1) and 10 mM TCEP for 18 h at 4 ◦ C, which was followed by thorough rinsing with double-distilled water. After treating with 1 mM MCH for 1 h, the substrate peptide monolayer modified electrode was prepared for further use. 2.3. PAD4 reaction with the substrate peptides on the electrode surface Enzymatic reaction buffer containing desired concentration of PAD4 was pre-activated at 37 ◦ C for 5 min. Then, the peptide monolayer modified electrode was dipped in the resulting solution for 2 h at 37 ◦ C (Fig. S2). Afterward, the electrode was thoroughly rinsed and ready for electrochemical measurements. In the inhibition

2.4. Electrochemical measurements Electrochemical measurements were performed on 660c Electrochemical Analyzer (CH Instruments). The three-electrode system consisted of the peptide monolayer modified electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. Before the electrochemical measurements, the electrolyte solutions should be thoroughly purged with high purity nitrogen for about 20 min to avoid the interference from the reduction of oxygen.

3. Results and discussion 3.1. The principle of our detection Fig. 1 may illustrate the principle of our method. The substrate peptide carrying two arginines can be self-assembled on the surface of gold electrode through the interaction between the sulfhydryl group of cysteine and gold surface. At pH 6.0, the peptide monolayer are electropositive because of the high isoelectric point (PI = 10.53). In this case, the positively charged peptide monolayer can prevent the signal molecules [Ru(NH3 )5 Cl]2+ from approaching to the electrode surface due to the strong electrostatic repulsion. In the presence of PAD4, PAD4 can catalyze the conversion of basic amino acid arginine to neutral amino acid citrulline, so [Ru(NH3 )5 Cl]2+ can be more accessible to the electrode surface for reduced positive charges on the peptide monolayer. As a result, an obvious electrochemical response can result from the electron transfer between the signal molecules and the electrode surface. As a comparison, only a low electrochemical response can be obtained after the incubation of PAD4 with the inhibitors Cl-amidine, which is ascribed to the high positive charges from the inhibition of PAD4-catalyzed citrullination on the electrode surface. Consequently, the assay of PAD4 activities and screening of PAD4 inhibitors can be realized by tracing the electrochemical responses of [Ru(NH3 )5 Cl]2+ at substrate peptide modified electrode.

Fig. 1. Schematic illustration of the electrochemical method for PAD 4 assay and inhibitor screening.

X. Chen et al. / Sensors and Actuators B 227 (2016) 43–47

Fig. 2. CV responses obtained at bare electrode (curve a), peptide monolayer modified electrode (curve b), and the modified electrode after the incubation with 50 nM PAD4 (curve c).

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Fig. 3. DPV responses obtained with different PAD4 concentrations (from a–j: 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 nM). Inset shows the linear relationship between the DPV peak current and the logarithm of PAD4 concentration in the range from 0.01 nM to 50 nM. Error bars demonstrates standard deviations for three measurements.

3.4. The studies of the specificity of our method 3.2. Electrochemical studies of our method by CV We have firstly studied our principle by monitoring the status of electrode surface with cyclic voltammetry (CV). As shown in Fig. 2, a pair of well-defined redox peaks can be observed at −0.24 mV (vs. SCE) at the bare electrode, which is ascribed to the electron transfer between [Ru(NH3 )5 Cl]2+ and the electrode surface (curve a). After the self-assembly of substrate peptide onto the electrode surface, the positive charges of the substrate peptide can prevent the signal molecules from approaching to the electrode surface. As a result, electrochemical response can be inhibited at the substrate peptide modified electrode (curve b). When the substrate peptide has been incubated with 50 nM PAD4, arginine within substrate peptide can be converted to citrulline by the catalysis of PAD4. Therefore, [Ru(NH3 )5 Cl]2+ can easily penetrate the peptide monolayer with the reduced positive charges to approach the electrode surface. Accordingly, a high electrochemical response can be obtained for the approaching electrochemical signal molecules, which is similar to that at the bare electrode (curve c). The CV results have clearly demonstrated the possibility of our method for PAD4 assay.

3.3. The studies of the sensitivity of PAD4 assay by DPV Then, we have employed a more sensitive electrochemical technique differential pulse voltammetry (DPV) to study the effect of PAD4 concentration. As shown in Fig. 3, the electrochemical response can increase along with the addition of PAD4 concentration, which is consistence with our expectation. The addition of PAD4 can accelerate the citrullination of arginine within the substrate peptide, thus reducing the positive charges on the surface of peptide monolayer modified electrode. Therefore, more signal molecules can approach to the electrode surface and promote the enhancement of the electrochemical responses. The inset of Fig. 3 has further shown a linear relationship between the peak current and PAD4 concentration in the range from 50 nM and 0.01 nM. The regression equation is I (␮A) = 0.572 + 0.233 lg CPAD4 (nM). The detection limit of the biosensor, defined as 3 (where  is the standard deviation of the zero standards), is calculated to be 3.5 pM (∼0.18 fmol in 50 ␮L), which is more sensitive than that in the previous reports [18,20,29]. Meanwhile, the average of the relative standard deviation for three times repeated measurements is 4.63%, which has confirmed the good reproducibility of our method.

The control experiments have been conducted to study the specificity of our method. As shown in Fig. 4, the peak current obtained with PAD4 is much higher than that with control protein (BSA, ALP, myoglobin or hemoglobin), even when the concentration of the control protein (1 ␮M) is much higher than that of PAD4 (50 nM). The control experiments have suggested that the changes of the positive charges on the electrode surface can only be resulted from PAD4-catalyzed citrullination of arginine within the substrate peptide, while the control protein have no obvious effect on the production of the electrochemical responses through unspecific adsorption. Therefore, the control experiments have fully demonstrated the high specificity of our method in PAD4 assay. Moreover, we have studied the application of our method by using the fetal calf serum as a contaminated example. The relative errors for the comparisons of the detected concentration and the given concentration are all within 10%, reconfirming the high selectivity of our detection as well as the great potential of our method for the clinical use (Table S1). 3.5. The inhibition studies of the potent inhibitors Furthermore, we have studied the application of our method in inhibitor screening by using the potent inactivator Cl-amidine as a model inhibitor. Cl-amidine is an irreversible PAD4 inhibitor,

Fig. 4. DPV responses obtained with (a) 50 nM PAD4, (b) 1 ␮M BSA, (c) 1 ␮M ALP, (d) 1 ␮M hemoglobin and (e) 1 ␮M myoglobin.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2015.12.050.

References

Fig. 5. DPV responses obtained with addition of different concentration of Clamidine (from a–f: 0, 0.05, 0.1, 0.5, 1 and 5 ␮M).

which can preferentially modify cys645 within PAD4 to induce both calcium- and substrate-dependent inactivation [29,30]. Fig. 5 has shown the peak currents obtained in the presence of different concentrations of Cl-amidine. The peak currents of [Ru(NH3 )5 Cl]2+ have been found to decrease with the addition of the inhibitors. Because Cl-amidine can inhibit PAD4-catalyzed citrullination of arginine within the peptide monolayer, the positive charges can remain on the surface of the peptide monolayer modified electrode, which can still strongly prevent [Ru(NH3 )5 Cl]2+ from approaching the electrode surface. As a result, restricted electron transfer between the signal molecules and the electrode surface can lead to a low electrochemical response, and the electrochemical response can also decrease with the increase of the inhibition ratio. Therefore, the inhibition studies has not only reconfirmed the high specificity of our method, but also indicated the great potential of our method for PAD4 inhibitor screening, which might provide support for the treatment of the related diseases in the future. 4. Conclusions In this paper, we have proposed a simple but effective electrochemical method to assay PAD4 and screen inhibitors. Electropositive arginine within substrate peptide can prevent the signal molecules approaching to the electrode surface and thus inhibit the produce of [Ru(NH3 )5 Cl]2+ -induced electrochemical responses. However, PAD4-catalyzed conversion of arginine to citrulline can reduce positive charges on the electrode surface, which may facilitate the access of [Ru(NH3 )5 Cl]2+ to the electrode surface. Therefore, the sensitive and specific assay of PAD4 can be realized by monitoring the electrochemical responses of [Ru(NH3 )5 Cl]2+ at the substrate peptide modified electrode. Meanwhile, since the addition of the potent inhibitor Cl-amidine can result in a decreased electrochemical response, our method may also have the potential for screening PAD4 inhibitors. Compared to the existing method, our method is simple to operate that can avoid the complex labeling or synthesis of signal molecules. Moreover, our work can be easily extended to assay more protease by designing the substrate peptide in the future, which might provide new insight into enzymatic analysis and potent technical support for the treatment of human diseases. Acknowledgements This work is supported by the National Natural Science Foundation of China (Grant No. 31200745) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026).

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Biographies Xixian Chen is a current master student in School of Life Sciences at Shanghai University in China. Her current research interests focus on the fabrication of biosensors for protein detection. Yun Lv is a current master student in School of Life Sciences at Shanghai University in China. Her current research interests focus on molecular recognition and biosensor fabrication. Yuanyuan Zhang is a current master student in Department of Obstetrics and Gynecology at The First Affiliated Hospital of Nanjing Medical University in China. Her current research interests focus on molecular recognition, disease diagnosis and biosensor fabrication. Jing Zhao is an associated professor in School of Life Sciences at Shanghai University in China. Her major research interests focus on the electrochemical signal amplification and biosensor fabrication. Lizhou Sun is a professor in Department of Obstetrics and Gynecology at The First Affiliated Hospital of Nanjing Medical University in China. Her major research interests focus on the diagnosis and treatment of breast tumors.