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A Bioluminescent Probe for Detection of Starvation-induced Pantetheinase Upregulation Yuxing Lin, Yuqi Gao, Zhao Ma, Zhenzhen Li, Chunchao Tang, Xiaojun Qin, Zheng Zhang, Guankai Wang, Lupei Du, and Minyong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02266 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018
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Analytical Chemistry
A Bioluminescent Probe for Detection of Starvation-induced Pantetheinase Upregulation Yuxing Lin, † Yuqi Gao, † Zhao Ma, † Zhenzhen Li, † Chunchao Tang, † Xiaojun Qin, † Zheng Zhang, † Guankai Wang, † Lupei Du, † Minyong Li†,‡,* †
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China. Tel./fax: +86-531-8838-2076; E-mail:
[email protected] ‡
State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China
ABSTRACT: Pantetheinase, a glycosylphosphatidylinositol (GPI) anchored enzyme, overexpresses in intestine, liver and kidney with various biological functions such as its linkage to the inflammation and some metabolic diseases. It can hydrolysis pantetheine to cysteamine, an antioxidant, and pantothenic acid (Vitamin B5) that is an essential component of coenzyme A (CoA). Until now, very few analytic methods were developed for this enzyme so that hampered the further investigation of its biological functions. In this article, we report the design, synthesis, and biological examination of a highly sensitive bioluminogenic probe for pantetheinase with a limit of detection (LOD) of 1.14 ng/mL. Furthermore, animal experiments validated that our probe can be applied to detect the endogenous pantetheinase activity. To the best of our knowledge, this is the first bioluminogenic probe achieving the detection of pantetheinase level in vivo.
Introduction Pantetheinase, a glycosylphosphatidylinositol (GPI) anchored enzyme, can hydrolysis pantetheine into cysteamine and pantothenic acid.1 Pantothenic acid is an essential material for the biosynthesis of coenzyme A (CoA), and CoA is an indispensable cofactor associated with tricarboxylic acid cycle and fatty acid metabolism.2 Cysteamine can inhibit the γ-glutamylcysteine synthetase and thereby decrease the glutathione pool, which may promote the inflammatory reaction.3 It was also reported as the antagonist of transglutaminase.4 Functional investigation of this enzyme indicated its link with the hepatic lipid metabolism, gluconeogenesis, and inflammation.5-7 Previous analytical techniques for pantetheinase such as the quantification of cysteamine via radioactive isotope labeling and spectrophotometric assay did not fit for the in situ examination of its biological activity.8,9 In 2010, Ruan et al. discovered a fluorescent probe, pantothenate– AMC (pantothenate–7-amino-4-methyl coumarin), for the screening of pantetheinase vanin-1 inhibitors,10 which was often employed for the detection of pantetheinase level in plasma and tissue homogenates.6 7 11 Recently, Ma et al. reported a fluorescent probe, which possessed a long-wavelength ratiometric fluorescence, high selectivity and sensitivity for the pantetheinase, for evaluating the efficiency of different inhibitors in vitro and cell imaging.12 However, all of these fluorometric methods could not be utilized for detecting pantetheinase level in vivo.
Compared to the fluorescence, bioluminescence does not require an excitation light in the presence of the ideal signal to noise ratio. Firefly luciferin-luciferase assay was commonly applied in the medicinal and analytical fields by employing luciferin or aminoluciferin as the substrate in the presence of ATP, Mg2+, and O2. 6’-Amino (or 6’hydroxyl) group of aminoluciferin (or luciferin) is the critical point for recognition with luciferase. Therefore, caging these groups could quench the bioluminescence, which granted luciferins various modification possibility for the off-on switch of bioluminescence.13 In this way, due to that the bioluminescence of luciferins could penetrate tissue of living animal, numerous probes were exploited for the in vivo imaging of different analytes.13-22 In this work, we developed a bioluminogenic probe for pantetheinase by coupling pantothenic acid with aminoluciferin. In such a case, pantetheinase can cleave the amide bond to release aminoluciferin, which can be subsequently recognized by firefly luciferase to emit bioluminescence signal (Scheme 1).
Scheme 1. Design strategy for the probe of pantetheinase
Experimental Section Synthesis. Synthesis of probe 1 was completed within few steps as depicted in Scheme S1. The detailed synthesis was described in the Supporting Information.
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Reagents. All reagents and solvents available from commercial sources were used without further purified unless otherwise noted. Calcium D-pantothenate, trifluoroacetic acid (TFA), (1S)-(+)-10-camphor sulfonic acid, anisaldehyde dimethyl acetal, 1-[bis (dimethylamino)methylene]1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate (HATU), were obtained from Energy Chemical Co., China. RR6 ((R)-2,4-dihydroxy-3,3-dimethyl-N-(3-oxo-4phenylbutyl)butanamide), a specific inhibitor of pantetheinase, was synthesized by ourselves according to the previous article.23 γ-Glutamyl transpeptidase (GGT), aminopeptidase N (APN) and tyrosinase (TYR) were purchased from Sigma-Aldrich. Recombinant human vanin-1 was obtained from Biotech China Co., Ltd. Human Vanin1 Gene with C terminal OFPSpark tag (excitation/emission maximum is 549 and 566 nm, respectively) cDNA clone expression plasmid was purchased from Sino Biological Inc. Water used for all biological studies was doubly distilled and further purified with a Milli-Q filtration system. Bioluminescence Assay Instrumentation. Buffer solution used in the bioluminescent assay was the Tris-HCl buffer (10 mM, pH 7.4) comprising 10 mM MgCl2. The IVIS Kinetic imaging system (Caliper Life Sciences, Hopkinton, Massachusetts, U.S.A.) equipped with a cooled charge-coupled device (CCD) camera was manipulated for the bioluminescent imaging. Firefly luciferase was purchased from Promega company. Circular ROIs were drawn over the areas and quantified using Living Image software (Caliper Life Sciences, Hopkinton, Massachusetts, U.S.A.). Kinetic Analysis. 50 μL of pantetheinase in Tris buffer (pH 7.4, 20 U/L) was added to 50 μL of probe 1 with different concentration (1-60 μM) in the 96-well black plate. After incubation at 37 oC in a shaker incubator, the fluorescence intensity at λem=520 nm (λex=355 nm) was recorded by the microplate reader. Vmax and Km values were measured by the procedure demonstrated in this article and calculated by Lineweaver−Burk plot.24 The fluorescence intensity at λ= 520 nm was recorded by a POLARstar Omega Microplate Reader (Thermo Scientific). Fluorescence intensity changes of probe 1 at different incubation times. Probe 1 was modified to 20 μM, and pantetheinase was adjusted to 15.625 ng/mL, 31.25 ng/mL and 125 ng/mL with Tris-HCl buffer. Then, 50 μL of probe 1 was mixed with the pantetheinase solution at a varied concentration in the 96-well black plate. The fluorescence intensity at λ= 520 nm was recorded by microplate reader at different time interval. In vitro bioluminescence assay. 50 μL of probe 1 (20 μM) was mixed up with the pantetheinase solution in different concentration incubated at 37 °C in a shaker incubator for 1.5 h. Then 50 μL of luciferase with 2 mM ATP was added each well, the bioluminescence was observed by an IVIS Kinetic imaging system (Caliper Life Science).
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Bioluminescence selectivity of probe 1. 100 μM solutions of FeCl3, MgCl2, BaCl2, MnSO4, CaCl2, ZnCl2, AlCl3, PbCl2, CuSO4, reduced glutathione (GSH), D-cysteine, glucose, sodium ascorbate (VcNa), L-valine and homocysteine (Hcy) were selected to examine the selectivity of our probe 1. APN (50 U/L), GGT (50 U/L) and TYR (100 ng/mL) were also tested in this study. The bioluminescence was obtained by an IVIS Kinetic imaging system (Caliper Life Sciences, Hopkinton, Massachusetts, U.S.A.). Cell transfection. ES-2-Fluc cells were plated in the confocal dish. After about 70% confluence, 2 μg plasmid DNA (Human Vanin-1 Gene with OFPSpark tag cDNA clone expression plasmid) and Lipofectamine 2000 were added in light of the manufacturer’s instructions. The transfection efficiency was confirmed by the fluorescence imaging of the OFPSpark. Fluorescence imaging was performed by the Zeiss Axio Observer A1 microscopy. Cytotoxicity assay. The cytotoxicity of probe 1 was performed on ES-2 cells expressing firefly luciferase (ES-2Fluc) with a standard CCK8 method.25 Compound RR6 and probe 1 were tested at various concentration and incubated for 6 h. Bioluminescence imaging in cellulo. ES-2 cells expressing firefly luciferase (ES-2-Fluc) were cultivated in RPMI 1640 (10% fetal bovine serum at 37 °C. 100 μL of cell suspension with 4×105 cells per mL was added to the 96-well plate (Corning, 3603) and cultured for 24 h. 50 μL of RR6 (100 μM) was added each well and incubated for 30 min. Then, the medium was removed, and 50 μL of probe 1 (20 μM) was added. The bioluminescence intensity was measured by an IVIS Kinetic imaging system (Caliper Life Sciences, Hopkinton, Massachusetts, U.S.A.). ES-2-Fluc cells and the transfected ES-2-Fluc cells were plated in 96-well plate (4×105) and cultured for 24h. After the removal of medium, probe 1 (20 μM) was added, and bioluminescence intensity was measured. Bioluminescence imaging in vivo. All animal studies were approved by the Ethics Committee and IACUC of Cheeloo College of Medicine, Shandong University, and were conducted in compliance with European guidelines for the care and use of laboratory animals. Pathogen-free luciferase-expressing transgenic mice (FVB- Tg(CAG-luc,GFP)L2G85Chco/FathJ17) were obtained from the Jackson Laboratory. FVB-luc+ mice were divided into two groups. They were orally administrated with RR6 in saline (30 mg/kg) or equal amount of vehicle, respectively. After 40 minutes, all mice were intraperitoneally injected with probe 1 (1 mM, 200 μL). The bioluminescence images of each group were measured at the different time within 35 min. FVB-luc+ mice fasted for 24 hours as the starvation model and the control group was fed with normal diet. Then both groups were intraperitoneally injected with probe 1 (1 mM, 200 μL). The bioluminescence images of
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Analytical Chemistry each group were captured at the different time within 35 minutes. Pantetheinase tissue quantification. Mouse tissues were collected from C57Bl/6 mice and homogenized in potassium phosphate buffer with 0.1% Triton X-100 and 0.4% proteasome. 10 μg total protein of tissue homogenates was used for the test of pantetheinase activity, which was determined by BCA protein assay. 50 μL of probe 1 (20 μM) was mixed up with the solution and incubated at 37 °C in a shaker incubator for 1.5 h.
2A-B). It also showed ideal linearity (Y = 3.887X-1.449, R2 = 0.9906) at the range from 0 to 31.25 ng/mL (Figure 2C). The detection limit of probe 1 was 1.14 ng/mL (S/N=3). Next, the Michaelis constant was measured at 36.89 μM by the Lineweaver−Burk plot, which demonstrated the high reactivity of probe 1 with pantetheinase (Figure S3).
Mice were fasted for 24 hours as the starvation model, and the control group was fed with normal diet. The mouse plasma was diluted 100 times with Tris-HCl buffer. 50 μL of probe 1 (20 μM) was mixed up with the solution and incubated at 37 °C in a shaker incubator for 1.5 h. Then, 50 μL of luciferase with 2 mM ATP was added each well, the bioluminescence was observed by an IVIS Kinetic imaging system.
Result and Discussion HPLC analysis. To verify the mechanism of the off-on switch of our probe, an HPLC analysis was performed by a 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA). The retention time of aminoluciferin and probe 1 was 12.150 and 10.112 min, respectively (Figures 1AB). As a result, probe 1 can release aminoluciferin via the reaction with pantetheinase (Figures 1C-D) and the identity of the product peak was confirmed by ESI-MS (Figure S1).
Figure 1. HPLC analysis of the reaction between compound 1 and pantetheinase: (A) aminoluciferin (0.2 mM); (B) probe 1 (0.2 mM); (C) probe 1 (0.2 mM,1 mL) with the addition of pantetheinase (500 ng/mL, 1 mL) and incubated for 5 min; (D) probe 1 (0.2 mM, 1 mL) with the addition of pantetheinase (500 ng/mL, 1 mL) and incubated for 30 min. Bioluminescence imaging in vitro. Initially, we test the time-dependence fluorescence enhancement of our probe at 520 nm with several concentrations of pantetheinase (15.625, 31.25 and 125 ng/mL) in Figure S2. Then, we applied it to the bioluminescence assay. It was incubated with diverse concentrations of pantetheinase at physiological condition (37 °C and pH=7.4). The bioluminescence augmentation at a concentration of 62.5 ng/mL was about 195-fold increasing compared to the blank (Figure
Figure 2. (A) Relative total photo flux change of probe 1 (20 μM) with different concentration pantetheinase after incubation 90 min; (B) Bioluminescence imaging of part A; (C) Linear fitting curve between the relative bioluminescence intensity of the probe and the concentration of pantetheinase (R2= 0.9906).
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Solution of FeCl3, MgCl2, BaCl2, MnSO4, CaCl2, ZnCl2, AlCl3, PbCl2, CuSO4, these inorganic salts all displayed little bioluminescent response (Figures 3A-B). Reduced glutathione (GSH), D-cysteine, glucose, sodium ascorbate (VcNa), L-valine, homocysteine (Hcy), APN, GGT and TYR also showed negligible disturbance to pantetheinase measurement in vitro because of the specific cleavage of the pantothenic acid moiety (Figures 3C-D). Hence, these data indicated that probe 1 was sensitive and specific enough to monitor the level of pantetheinase activity in vitro.
negligible influence on the bioluminescence of aminoluciferin in cellulo (Figure S6). Furthermore, we also found the transient transfected ES-2-Fluc cells released stronger bioluminescence than the untransfected one (Figures 4C-D). The transfection efficiency was validated by the fluorescence cell imaging (Figure S7). All these data confirmed the bioluminescence enhancement of ES-2-Fluc cells was induced by pantetheinase.
Figure 4 (A) Bioluminescence imaging of ES-2-Fluc cells, the wells below were incubated with 100 μM RR6 for 30 min before the imaging and the wells above were the control group; (B) Quantification of bioluminescence intensity of part A; (C) Bioluminescence imaging of 4×104 ES-2-Fluc cells (left) and ES-2-Fluc cells transfected with human vanin-1 gene (right); (D) Quantification of bioluminescence intensity of part C. (n3; *p
