Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living

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A Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living Cells Yiming Hu, Hongyu Li, Wen Shi, and Huimin Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03303 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 15, 2017

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Analytical Chemistry

A Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living Cells Yiming Hu,ab Hongyu Li,a Wen Shia* and Huimin Maab*

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: [email protected]; [email protected]

b

University of Chinese Academy of Sciences, Beijing 100049, China.

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ABSTRACT

Pantetheinase, which catalyzes the cleavage of pantetheine to pantothenic acid (vitamin B5) and cysteamine, is involved in the regulation of oxidative stress, pantothenate recycling and cell migration. However, further elucidating the cellular function of this enzyme is largely limited by the lack of a suitable fluorescence imaging probe. By conjugating pantothenic acid with cresyl violet, herein we develop a new fluorescence probe CV-PA for the assay of pantetheinase. The probe not only possesses long analytical wavelengths but also displays linear ratiometric (I628/582

nm)

fluorescence response to pantetheinase in the range of 5-400 ng/mL with a detection limit of 4.7 ng/mL. This probe has been used to evaluate the efficiency of different inhibitors and quantitatively detect pantetheinase in serum samples, revealing that pantetheinase in fetal bovine serum and new born calf serum is much higher than that in normal human serum. Notably, with the probe the ratiometric imaging and in situ quantitative comparison of pantetheinase in different living cells (LO2 and HK-2) have been achieved for the first time. It is found that the level of pantetheinase in LO2 cells is much larger than that in HK-2 cells, as further validated by western blot analysis. The proposed probe may be useful to better understand the specific function of pantetheinase in the pantetheinase-related pathophysiological processes.


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INTRODUCTION Pantetheinase, mainly known as vanin-1, is expressed at the extracellular membrane of epithelial cells via a glycosylphosphatidylinositol anchor at C-terminus.1-5 It has been reported that pantetheinase may play a role in oxidative stress,2-4,6,7 pantothenate recycling8 and cell migration.9 Functional studies preliminarily disclosed that pantetheinase expression was associated with a series of diseases such as acute kidney injury,10-13 diabetes,14-17 malaria,18-20 and influenza.21 However, the exact cellular mechanism of this enzyme remains poorly understood, largely due to the lack of a proper method to detect its distribution and expression in living cells. Therefore, developing an in situ analytical method for pantetheinase in biosystems is urgently needed. Early analytical methods for pantetheinase are mainly based on the quantification of the reaction product of cysteamine via radioactive isotope labelling coupled with paper chromatography.22 However, they require tedious procedures. Spectrophotometric assay of pantetheinase activity has been developed subsequently,23 but it is not suitable for in situ analysis. In 2010, Ruan et al. reported a fluorometric method,24 which, however, displayed a rather short excitation wavelength of 340 nm and is thus unfavourable for cell imaging. With these issues in mind, we have developed a new ratiometric fluorescence probe (CV-PA) with long analytical wavelengths (λex/em = 525/582 nm) by using pantothenic acid as a recognition moiety and cresyl violet (CV) as a fluorochrome. The probe was synthesized by linking the protected pantothenic acid to CV via an amide bond formed between the amino group and the carboxyl group, followed by deprotection in acetic acid (Scheme S1). Compared with CV, the resulting probe displays a blue shift in the absorption peak from 585 nm to 495 nm; however, reaction of CV-PA with pantetheinase leads to the removal of pantothenic acid 3 ACS Paragon Plus Environment


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(Scheme 1), accompanied by the decrease of fluorescence intensity at 582 nm and in the meantime the fluorescence increase at 628 nm. Such a fluorescence response provides the basis to establish a ratiometric (I628/582 nm) imaging method for pantetheinase in cells.

Scheme 1. Detection mechanism of pantetheinase by CV-PA

O

OH

OH

N H

pantetheinase

N

O N H cleavage site

O

NH2

CV-PA, λem = 582 nm

O

OH

OH

N H2N

O N H

O

NH2

OH CV, λem = 628 nm

EXPERIMENTAL SECTION Reagents. Calcium-d-pantothenate, trifluoroacetic acid (TFA), (1S)-(+)-10-camphorsulfonic acid (CSA),

anisaldehyde

triazolo[4,5-b]pyridinium

dimethyl 3-oxid

acetal,

1-[bis(dimethylamino)methylene]-1H-1,2,3-

hexafluorophosphate

(HATU),

N,N-diisopropylethylamine

(DIPEA), CV, 8-cyclopentyl-1,3-didropylxanthine (CPDX), roscovitine, β-lapachone, γ-glutamyl transpeptidase,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

bromide

(MTT),

monoamine oxidase A, monoamine oxidase B, pyroglutamate aminopeptidase, tyrosinase, dipeptidyl peptidase IV, and leucine aminopeptidase were purchased from Sigma-Aldrich. RR6 was obtained from MedChemExpress. Cell lines (LO2 and HK-2), Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from KeyGEN BioTECH Co., LTD, Nanjing, China. Lipofectamine™ 2000 transfection reagent, and fetal bovine serum (FBS) were obtained from Thermofisher. New born calf serum (NBCS) was purchased from Zhejiang Tianhang Biological Technology Co., Ltd. Mixed human 4 ACS Paragon Plus Environment

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serum (MHS) was purchased from Beijing GENIA Bioscience Technology Ltd, and an informed consent was obtained from each donor. Human Vanin-1 Gene ORF cDNA clone expression plasmid was purchased from Sino Biological Inc. Recombinant human vanin-1 was obtained from Bio-Techne China Co., Ltd. The stock solution (1 µg/mL) of pantetheinase was prepared in ultrapure water and stored in small aliquots at -70 °C to avoid repeated freeze-thaw cycles. Ultrapure water with resistivity over 18 MΩ·cm was obtained from a Milli-Q(Millipore) system. Apparatus. All of the fluorescence measurements and MTT analysis were made on microplate reader (Molecular Devices SpectraMax i3, USA). 1H and

13

C NMR spectra were obtained with a

Bruker Avance III HD 400, 500 or 600 spectrometers. High resolution electrospray ionization mass spectra (HR-ESI-MS) were performed on an APEX IV FTMS instrument (Bruker, Daltonics). UV-Vis absorption spectra were recorded with TU-1900 spectrophotometer (Beijing, China) in 1-cm quartz cells. Relative quantum yield (Φ) of the probe before and after reaction with pantetheinase was determined by using CV (Φ = 0.54 in methanol) as a standard. Fluorescence imaging was conducted on an FV 1200-IX83 confocal laser scanning microscope with the software FV10-ASW (Olympus, Tokyo, Japan). Synthesis of Fluorescence Probe CV-PA. CV-PA was prepared according to the route shown in Scheme S1 in the Supporting Information. The 3-step reaction is described briefly as follows. The protected pantothenic acid (PA) was synthesized according to the previous method.25 Calcium-d-pantothenate (1.9 g, 8.0 mmol) and CSA (187 mg, 0.8 mmol) were mixed in a 100 mL round-bottom flask, followed by addition of 15 mL TFA dropwise at 0 °C with vigorous stirring. After dissolving all the solid at the bottom, the excess TFA was evaporated under reduced pressure 5 ACS Paragon Plus Environment


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to give colorless oily d-pantothenic acid. d-Pantothenic acid was further dissolved in 20 mL CH2Cl2 and reacted with 4-methoxybenzaldehyde dimethyl acetal (4.5 g, 25 mmol) under stirring at room temperature for 8 h. Then, 20 mL of 1 M Na2CO3 was added. The precipitate was filtered and the aqueous phase filtrate was washed with CH2Cl2 (3× 100 mL). After acidification of the filtrate by acetic acid to pH 4.5, the product in the solution was extracted by CH2Cl2 and the separated CH2Cl2 phase was dried over anhydrous Na2SO4. The solvent was removed through evaporation to give PA as a white solid (1.3 g, 48%), which was used in the next step without further purification. PA was converted to CV-PA-PM (Scheme S1) through amidation reaction. PA (337 mg, 1 mmol), HATU (380 mg, 1 mmol) and DIPEA (400 µL, 2.3 mmol) were dissolved in anhydrous DMF (5 mL) with stirring at 0 °C for 40 min. Then, CV (268 mg, 0.8 mmol) in anhydrous DMF (5 mL) was introduced dropwise, and the reaction mixture was further stirred at room temperature for 6 h. The reaction solution was diluted with CH2Cl2 and washed with water. The organic phase was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography with CH2Cl2/CH3OH (v/v, 20:1) as eluent, affording CV-PA-PM (56 mg, yield 12%). 1H and 13C NMR spectra are shown in Figures S1 and S2. 1H NMR (500 MHz, CD3OD, 298 K): δ 8.68 (d, 1H, J = 8.5 Hz), 8.34 (d, 1H, J = 7 Hz), 7.78 - 7.74 (m, 3H), 7.62 (d, 1H, J = 9 Hz), 7.43 - 7.40 (m, 2H), 7.30 (d, 1H, J = 8.5 Hz), 6.88 - 6.86 (m, 2H), 6.52 (s, 1H), 5.53 (s, 1H), 4.16 (s, 1H), 3.74 (s, 3H), 3.73 - 3.65 (m, 2H), 3.59 (t, 2H, J = 6.5 Hz), 2.64 (t, 2H, J = 6.5 Hz), 1.10 (s, 3H), 1.04 (s, 3H).

13

C NMR (150 MHz, DMSO-d6, 298 K): δ 170.9, 168.8, 162.8, 160.0, 147.0, 146.2,

144.9, 142.2, 131.6, 131.3, 131.0, 130.1, 129.9, 129.1, 128.1, 127.5, 124.9, 124.7, 116.2, 113.8,


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105.6, 105.5, 100.8, 83.6, 77.8, 55.6, 36.2, 35.0, 33.0, 22.0, 19.5. HR-ESI-MS: m/z calcd. [C33H33N4O6]+, 581.2395; found, 581.2393. CV-PA-PM was subsequently treated with acetic acid solution for deprotection to afford probe CV-PA. CV-PA-PM (50 mg, 0.09 mmol) was treated with aqueous solution of 80% acetic acid at room temperature for 2.5 h. The solvent was removed by evaporation under reduced pressure and the residue solid was purified by silica gel chromatography eluted with CH2Cl2/CH3OH (v/v, 5:1), affording CV-PA as a red solid (35 mg, yield 84%). 1H and 13C NMR spectra are shown in Figures S3 and S4. 1H NMR (400 MHz, CD3OD, 298 K): δ 8.43 (d, 1H, J = 7.2 Hz), 8.14 (d, 1H, J = 7.2 Hz), 7.76 - 7.68 (m, 3H), 7.47 (s, 1H), 7.28 (d, 1H, J = 7.6 Hz), 6.54 (s, 1H), 3.97 (s, 1H), 3.66 - 3.56 (m, 3H), 3.49 (d, 1H, J = 11.2 Hz), 3.42 (d, 1H, J = 11.2 Hz), 3.35 (s, 1H), 2.65 (t, 2H, J = 5.6 Hz), 0.95 (s, 6H).

13

C NMR (100 MHz, CD3OD, 298 K): δ 177.0, 173.6, 167.5, 154.8, 147.4, 146.8, 144.3,

136.0, 134.0, 133.8, 133.3, 132.7, 127.8, 126.5, 126.3, 120.6, 107.5, 100.8, 78.2, 71.2, 41.2, 38.5, 36.8, 22.1, 21.7. HR-ESI-MS: m/z calcd. [C25H27N4O5]+, 463.1976; found, 463.1975 (Figure S5). General Procedure for Pantetheinase Detection. Unless otherwise noted, all the spectral measurements were performed in 20 mM phosphate buffer (pH 7.4) according to the following procedure. The stock solution (1.0 mM) of probe CV-PA was first prepared in DMSO. In a 96-well assay plate, 20 µL of 200 mM phosphate buffer (pH 7.4) was added to each well, followed by mixing 2 µL of 1.0 mM CV-PA stock solution, an appropriate volume of water and pantetheinase solution. The final volume was adjusted to 200 µL by ultrapure water. The assay plate was placed into microplate reader to measure fluorescence signal with the excitation wavelength of 525 nm at 37 °C. For absorbance measurements, 3 mL of the reaction solution was prepared and incubated at 37 °C for 7 ACS Paragon Plus Environment


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60 min in a thermostat. Then, the reaction solution was transferred to a quartz cell with 1-cm optical length for measurements. In the meantime, the blank solution without pantetheinase was also prepared and measured under the same conditions for comparison. Measurements of IC50 Values of Different Inhibitors. Pantetheinase solution (100 ng/mL, final concentration) was pipetted to the 96-well plate and mixed with varied concentrations of inhibitors in 20 mM phosphate buffer of pH 7.4 for 10 min. Then PA-CV (10 µM, final concentration) was added to the solution with incubation for another 2 h. Fluorescence data were obtained from the microplate reader with λex = 525 nm, and λem= 582 nm, 628 nm; the ratio (I628/I582) of fluorescence intensity at 628 nm and 582 nm was calculated. The inhibition ratio was calculated using the following equation: inhibition ratio = (R–Ri)/(R–R0) × 100%, where Ri and R are the fluorescence ratio (I628/I582) of the reaction system with and without inhibitor, respectively; R0 is the fluorescence ratio from only CV-PA without pantetheinase and inhibitor in the phosphate buffer (blank). Determination of Pantetheinase in Sera. Serum samples and pantetheinase were appropriately diluted with 20 mM phosphate buffer and incubated with 10 µM CV-PA. The reaction progress was monitored by fluorescence signal of I628/I582. Cytotoxicity Assay. The cytotoxicity of CV-PA to LO2 and HK-2 cells was examined by standard MTT assay according to the previous report.26 Cell Culture and Fluorescence Imaging. HK-2 cells were plated on glass-bottom culture dishes (MatTek

Co.)

in

DMEM/F12

supplemented

with

10%

(v/v)

FBS

and

1%

(v/v)

penicillin-streptomycin at 37 °C in a humidified 5% CO2 incubator. LO2 cells were cultured as described before.27 For pantetheinase imaging, HK-2 cells were incubated with CV-PA (10 µM) in 8 ACS Paragon Plus Environment

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DMEM/F12 at room temperature for 1 h and then washed by DMEM/F12. Such method was also applied to LO2 cells except that DMEM was used instead of DMEM/F12. Then the cell imaging was conducted with the excitation wavelength of 515 nm, the fluorescence emission was collected between 530-595 nm (green) and 615-675 nm (red) through a 40 × 0.95 NA objective. The pixel intensity of living cells in each fluorescence image was measured from at least 10 cells. Cell Transfection. HK-2 cells were plated at approximately 70% confluence in confocal dish with 2 µg plasmid DNA and Lipofectamine™ 2000 transfection reagent according to the manufacturer’s instructions.

RESULTS AND DISCUSSION Fluorescent Response of CV-PA to Pantetheinase. The spectroscopic properties of CV-PA towards pantetheinase were investigated in the phosphate buffer (20 mM) of pH 7.4. As shown in Figure S6, probe CV-PA displays a maximum absorption at 495 nm. Addition of pantetheinase causes a large bathochromic shift of the absorption peak to 585 nm with an isosbestic point of 525 nm. Using 525 nm as the excitation, CV-PA itself (Φ = 0.51 in 20 mM phosphate buffer) exhibits a maximum emission at 582 nm (Figure 1A). However, reaction of CV-PA with pantetheinase leads to a great decrease of the emission peak, concomitant with the appearance of a new emission peak at 628 nm (Φ = 0.27 in 20 mM phosphate buffer), which provides the basis for achieving a ratiometric signal output (I628/I582). Moreover, the absorption and fluorescence spectra of the reaction system are in accordance with those of the fluorochrome CV (Figure S7), implying the production of CV during the reaction. Such observation is further confirmed by HR-ESI-MS analysis. As depicted in Figure 9 ACS Paragon Plus Environment


S8, the mass peak at m/z = 262.0974 is the characteristic molecular ion peak of CV in the reaction solution. In the meantime, the spectroscopic properties of intermediate CV-PA-PM as a control compound were also investigated, revealing that although the control compound showed a similar absorption spectrum (Figure S7) to CV-PA, but it produced nearly no absorbance and fluorescence change upon reaction with pantetheinase (Figure S9), suggesting that the two hydroxyl groups in

3x10

7

2

1

A

10

1 2

B 8

2x10

7

1x10

7

I628/I582

Fluorescence Intensity

CV-PA are vital for the pantetheinase recognition.

6 4 2

0 560

600 640 Wavelength (nm)

0

680

0

20

40

60

80

100

120

Time (min)

0.3

9

D

0.2

I628/I582

-1

)

C ∆R/∆t (min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.1

6 3

0.0 0

100

200

300

0

400

Species 1-28

Pantetheinase (ng/mL)

Figure 1. (A) Fluorescence spectra of CV-PA (10 µM) before (1) and after (2) reaction with pantetheinase (400 ng/mL). The inset shows the color change of the fluorescence reaction. (B) Fluorescence kinetic curves of CV-PA (10 µM) in the presence of pantetheinase at varied concentrations (from bottom to top): 0 (control), 20, 50, 100, 300, 400 ng/mL. (C) Linear fitting curve of fluorescence intensity ratio I628/I582 toward the concentration of pantetheinase from 5–400 ng/mL. (D) Fluorescence responses of CV-PA (10 µM) to different species 1-28 (from left to right): (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) vitamin C (1 mM); (10) glutathione (5 10 ACS Paragon Plus Environment

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mM); (11) cysteine (1 mM); (12) lysine (1 mM); (13) glutamic acid (1 mM); (14) aspartic acid (1 mM); (15) BSA(100 µM); (16) carboxylesterase (1 U/mL); (17) H2O2 (100 µM); (18) O2·– (100 µM); (19) OCl– (100 µM); (20) monoamine oxidase A (2 µg/mL); (21) monoamine oxidase B (2 µg/mL); (22) tyrosinase (100 U/L); (23) dipeptidyl peptidase IV (2 µg/mL); (24) γ-glutamyltransferase (2 µg/mL); (25) pyroglutamate aminopeptidase (2 µg/mL); (26) leucine aminopeptidase (2 µg/mL); (27) calcium pantothenate (1 mM); (28) pantetheinase (400 ng/mL). λex = 525 nm.

Fluorescence kinetic curves of CV-PA reacting with pantetheinase are depicted in Figure 1B. CV-PA was gradually hydrolyzed by pantetheinase over a period of 2 h, and when the concentration of pantetheinase is up to 300 ng/mL, the ratiometric fluorescence response I628/I582 reached a plateau at 60 min. On the other hand, within a short period of time such as 10 min, the ratiometric response of CV-PA to various pantetheinase displays a good linearity. Hence, the initial rate (∆R/∆t) of the ratiometric response in the early 10 min was adopted for quantifying pantetheinase hereafter, unless otherwise noted. Furthermore, no significant fluorescence change was observed in the probe without incubation with pantetheinase, indicating the excellent stability of CV-PA. The effects of pH from 4.5 to 9.5, and temperature between 25 to 42 °C were evaluated. As shown in Figure S10, the fluorescence ratio I628/I582 of CV-PA is not significantly affected by the change of pH from 7.0 to 8.5 in the phosphate buffer and temperature from 25 to 42 oC, suggesting the good applicability of CV-PA under physiological conditions. Under the optimized conditions, the fluorescence response of CV-PA to pantetheinase at varied concentrations was investigated, obtaining a good linear equation of ∆R/∆t = (10.3 + 5.85 [C]) ×10-4 with a correlation coefficient of 0.997 in the range of 5–400 ng/mL pantetheinase (Figure 1C). The limit of detection was determined to be 4.7 ng/mL (k = 3). Moreover, the initial rates for the 11 ACS Paragon Plus Environment


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enzyme-catalyzed reaction were obtained at various concentrations of CV-PA (5-50 µM) and the Michaelis constant was determined to be 78 µM (Figure S11). The selectivity of CV-PA was examined for pantetheinase over other potential interfering species, such as inorganic salts (KCl, MgCl2, CaCl2, ZnCl2, CuCl2, and FeCl3), glucose, vitamin C, glutathione, BSA, calcium pantothenate, amino acids (cysteine, lysine, glutamic acid, and aspartic acid), reactive oxygen species (O2·–, OCl– and H2O2) and some enzymes (carboxylesterase, monoamine

oxidase

A,

monoamine

oxidase

B,

tyrosinase,

dipeptidyl

peptidase

IV,

γ-glutamyltransferase, pyroglutamate aminopeptidase, leucine aminopeptidase). As shown in Figure 1D, the probe displays a high selectivity for pantetheinase over the other species tested. Evaluation of Inhibitors. Since pantetheinase is a potential biomarker for early diagnosis of renal injury,5,10-12 developing its specific inhibitors has attracted much attention in drug study. Considering its good properties, CV-PA may serve as a useful tool to examine the efficiency of different inhibitors in vitro. Here, four commercial available inhibitors (CPDX, roscovitine, β-lapachone, and RR6) were evaluated by the proposed probe. Prior to evaluation, the effects of all the inhibitors were studied on the fluorescence of CV-PA and the corresponding fluorochrome CV, revealing no influence (Figure S12). As depicted in Figure 2, each dose-response plot shows a sigmoid fitting curve, and the corresponding IC50 values of the inhibitors are summarized in Table S1. It is found that the order of the inhibition effect is: β-lapachone > RR6 > CPDX > roscovitine, which is in agreement with the previous observation24 except RR6 that has not yet been compared under the same condition. Interestingly, RR6 exhibits a comparable inhibition (IC50 = 0.63 µM) with


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β-lapachone (IC50 = 0.45 µM). In addition, RR6 is a more specific inhibitor of pantetheinase,28 therefore this inhibitor was used in the following serum and cell experiments.

100 100 B

Inhibition Ratio (%)


A 75 50 25 0 0.01

0.1

1 10 CPDX (µM)

75 50 25 0

100

0.01

0.1

1

10

100

Roscovitine (µM)

100 100 D


C Inhibition Ratio (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


75 50 25 0

0.01 0.1 1 10 β-Lapachone (µM)

75 50 25 0.01

0.1

1

10

100

RR6 (µM)

Figure 2. Inhibition of pantetheinase (100 ng/mL) by (A) CPDX, (B) roscovitine, (C) β-lapachone, and (D) RR6.

Detection of Pantetheinase in Sera. Serum is believed to be a natural and physiological source of pantetheinase including its secreted form.28 However, quantification of pantetheinase in serum has not been reported previously. On the basis of its excellent anti-interference performance, CV-PA was also attempted to assay pantetheinase in different sera such as FBS, NBCS and MHS. In these experiments, FBS, NBCS and MHS were diluted 1000, 200 and 2.5 times with 20 mM phosphate buffer of pH 7.4. The diluted serum samples were treated with 10 µM CV-PA, and the time-dependent fluorescence signals of the reaction solutions were recorded (Figure S13). The specific fluorescence response of CV-PA to serum pantetheinase was further validated through the introduction of 10 µM RR6 (inhibitor) to the reaction systems (Figure S13). Then, the relative 13 ACS Paragon Plus Environment


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reaction rates (∆R/∆t) were calculated and converted to the level of pantetheinase in the undiluted sera. As shown in Table 1, the concentration of pantetheinase in FBS, NBCS, and MHS was determined to be 12 ± 0.6 µg/mL, 5.0 ± 0.4 µg/mL and 0.15 ± 0.01 µg/mL, respectively; FBS contains the highest level of pantetheinase. Moreover, different amounts of pantetheinase were spiked into MHS containing the lowest level of the enzyme. The recovery of pantetheinase in the spiked samples was calculated, and ranged from 96 to 108%, demonstrating that probe CV-PA may be useful for the assay of pantetheinase in serum samples. Table 1. Determination of pantetheinase in serum samples Sample

Pantetheinase added (µg/mL)

Pantetheinase found by our methoda (µg/mL)

recoverya (%)

FBS

0

12 ± 0.6

-

NBCS

0

5.0 ± 0.4

-

MHS

0

0.15 ± 0.01

-

MHS

0.25

0.41 ± 0.03

104 ± 12

MHS

0.50

0.69 ± 0.03

108 ± 6

MHS

0.75

0.87 ± 0.03

96 ± 4

a

Mean of three determinations ± standard deviation

Cytotoxicity of CV-PA. The toxicity of the probe was evaluated with LO2 cells and HK-2 cells by standard MTT assay. As shown in Figure S14, cell viability of both types was not significantly affected by no more than 10 µM CV-PA with incubation at 37 °C for 24 h, indicating a good biocompatibility of the probe. Fluorescence Imaging of Pantetheinase in Living Cells. It is reported that pantetheinase is highly expressed in liver.1,3,28,29 Probe CV-PA was thus explored to image pantetheinase in normal 14 ACS Paragon Plus Environment

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liver cell line LO2. As depicted in Figure 3A, LO2 cells themselves display no fluorescence under the excitation wavelength of 515 nm (column 1), whereas CV-PA loaded cells exhibit strong fluorescence (column 2). Moreover, such ratiometric fluorescence signal can be decreased via introducing the specific inhibitor of RR6 (columns 3 and 4), indicating that the intracellular fluorescence response is indeed aroused by pantetheinase, and the probe is capable of imaging the presence of pantetheinase in cells.

A

1

2

3

B

4

Green 0.8 0.6

Red Ratio

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0.4 0.2

DIC

0.0

1

2

3

4

Ratio

Figure 3. (A) Confocal fluorescence images of LO2 cells. (1) Cells only (control); (2) cells incubated with CV-PA (10 µM) for 1 h; (3) cells pretreated with RR6 (5 µM) for 2 h, followed by incubation with CV-PA (10 µM) for 1 h; (4) cells pretreated with RR6 (50 µM) for 2 h, followed by incubation with CV-PA (10 µM) for 1 h. The first and second rows are the green and red channel of CV-PA. The third row is the differential interference contrast (DIC) image, and the fourth is the ratio (Red/Green) image. The color strip in the ratio image represents the pseudocolor change. Scale bar: 50 µm. (B) The relative ratio value of the corresponding ratio images in panel A. The results are the mean ± standard deviation of the measurements of all the cells (> 30 cells) in the image.



Pantetheinase has also been discovered to be a potential biomarker for acute kidney injury as mentioned above, thus the fluorescence imaging of LO2 and human proximal tubular cell line HK-2 (human kidney 2) is compared under the same conditions. As shown in Figure 4, the ratiometric fluorescence in LO2 cells is much stronger than that in HK-2 cells, suggesting a higher expression of pantetheinase in LO2 cells. To verify this observation, western blot analysis was performed, and the results (Figure S15) also disclosed a higher level of pantetheinase in LO2 cells. Furthermore, transient transfection of HK-2 cells displayed a higher fluorescence ratio value compared to untransfected HK-2 cells (Figure S16). All the above data validate our imaging result that LO2 cells have a higher expression level of pantetheinase than HK-2 cells. A

Green

Red

DIC

Ratio

B 0.6

HK-2

Ratio

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0.4 0.2

LO2 0.0

HK-2

LO2

Figure 4. (A) Confocal fluorescence images of HK-2 and LO2 cells. Scale bar: 50 µm. (B) The relative ratio value of the corresponding ratio images in panel A.

CONCLUSIONS In summary, by combining pantothenic acid with cresyl violet, we have developed a new long-wavelength ratiometric fluorescent probe for the assay of pantetheinase. The probe shows high selectivity and sensitivity with a detection limit of 4.7 ng/mL pantetheinase, and has been applied to evaluating the efficiency of different inhibitors and detecting pantetheinase in serum samples. 16 ACS Paragon Plus Environment

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Moreover, using the probe the ratiometric imaging of pantetheinase in different living cells has been achieved, disclosing a higher level of pantetheinase in LO2 cells than HK-2 cells, as further evidenced by western blot analysis. The probe may find a wide use in detecting pantetheinase in some complex biosystems.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx. The synthetic route of CV-PA, 1H and 13C NMR spectra and HR-ESI-MS of the probe, additional spectroscopic data, IC50 values, cytotoxicity assay, western blot analysis, and fluorescence imaging of transfected HK-2 cells.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]; [email protected].

ACKNOWLEDGMENT We are grateful to the financial support from the NSF of China (Nos. 21535009, 21675159, 21435007 and 21621062), the 973 Program (No. 2015CB932001), the Chinese Academy of Science (XDB14030102), and Youth Innovation Promotion Association of CAS (2016027). 17 ACS Paragon Plus Environment


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For TOC only Fluorescence

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pantetheinase

O

N

O

N OH OH H

N H

O

CV-PA

CV

NH2

560

CV-PA

600 640 680 Wavelength (nm)

N H2N

O

CV

NH2

HK-2 LO2 less pantetheinase more pantetheinase


Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living

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