Microbial Redox Regulator-Enabled Pulldown for Rapid Analysis of

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Microbial Redox Regulator-Enabled Pull-Down for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols Jin Oh Lee, Yoon-Mo Yang, Jae-Hoon Choi, Tae-Wuk Kim, Jin-Won Lee, and Young-Pil Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01991 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Analytical Chemistry

Microbial Redox Regulator-Enabled Pull-Down for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols

Jin Oh Lee,1,2,† Yoon-Mo Yang,1,† Jae-Hoon Choi,1,2 Tae-Wuk Kim,1,2 Jin-Won Lee,1,2* and Young-Pil Kim1,2,3*

1Department

2Research

of Life Science, Hanyang University, Seoul 04763, Republic of Korea

Institute for Natural Sciences and Research Institute for Convergence of Basic Sciences Hanyang University, Seoul 04763, Republic of Korea

3Institute

of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of Korea ACS Paragon Plus Environment

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Analytical Chemistry 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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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

†These

authors contributed equally to this work.

*To whom correspondence should be addressed: Phone: +82-2-2220-0952; +82-2-2220-2560, E-mail: [email protected]; [email protected]

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ABSTRACT

Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Here we report a newly designed, affinity pull-down platform using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA for rapid and cost-effective analyses of plasma LMW biothiols. In the presence of organic hydroperoxide, LMW biothiols triggered the rapid dissociation of FAM-labeled dsDNA from FLAG-tagged OhrRBS via S-thiolation of OhrRBS on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pull-down. This method was easily extended by using a reducing agent to detect free and total LMW biothiols simultaneously in mouse plasma. Unlike free plasma LMW biothiols, total plasma LMW biothiols were more elevated in LDLR mice than those in normal mice. Owing to the rapid dissociation of OhrR/dsDNA complexes in response to LMW biothiols, this pull-down platform is immediately suitable for monitoring rapid redox changes in plasma LMW biothiols as well as studying oxidative stress and diseases in blood.


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KEYWORDS. LMW thiol, Plasma thiol, Redox protein, OhrR, ROS, LDLR


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INTRODUCTION

Low-molecular-weight (LMW) biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play important roles in detoxification processes against reactive oxygen species (ROS) in prokaryotes and eukaryotes.1-2 They also serve as metabolic regulators in thiol-disulfide processes that maintain intracellular and extracellular redox homeostasis,3-5 because a thiol (SH) functional group, as a potent nucleophile, is susceptible to redox modifications such as reversible protein S-thiolation and LMW disulfide transformation.6 Unlike the intracellular environment in which approximately 90% of the LMW biothiol pool is reduced at several or several tens of millimolar concentrations (GSH is predominant),7-8 only 4% of LMW biothiols in human plasma are reduced at much lower micromolar concentrations (Cys is predominant).9 Increase in the oxidation stress of the extracellular environment is generally accompanied by the rapid transformation of LMW biothiols into thiolations or disulfides and the subsequently rapid influx of reduced Cys and Hcy from tissues into plasma, which maintains thiol-redox balance.10 It has been reported that the increase of total LMW biothiols (including reduced and oxidized forms) in plasma due to oxidative stress was highly correlated with the occurrence of age-related diseases such as atherosclerosis,11-12 Alzheimer’s disease,13-14 ACS Paragon Plus Environment

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cancers,15 and other diseases.16 However, despite the implications of rapid influx and oxidation of free LMW biothiols in plasma, there has been little focus on sequentially analyzing these biothiols. This task remains challenging due to the lack of appropriate analytical methods. Current methods, including high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are regarded as gold-standard tools for analyzing plasma LMW biothiols.17 However, these methods are neither sufficiently convenient nor rapid enough to detect free LMW biothiols because free LMW biothiols are susceptible to rapid oxidation during the labor-intensive processes and long operation times of these methods. Significant progress has been made using luminescent or colorimetric chemical probes18-20 or nanoparticles21-22; however, most have been used to detect intracellular free thiols. Moreover, these probes are complicatedly synthesized and indistinguishable between LMW and proteinous biothiols, and often induce significant background variation by interfering with other substances in plasma.23 In this regard, there still remains a limitation in measuring the content of plasma thiols. To study the implications and redox status of plasma LMW biothiols in human disease, a more indepth and rapid analytic method is required. Herein we report a novel method for the rapid detection of plasma LMW biothiols using a bacterial redox-sensing transcription repressor protein and its operator DNA element. Bacteria ACS Paragon Plus Environment

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have evolved self-defense systems using sensory and regulatory processes to protect against the extensive damages caused by ROS derived from host cells or environmental stimuli.24 Among many redox regulators in bacteria, we chose an organic hydroperoxide resistance regulator protein from Bacillus subtilis (OhrRBS). OhrRBS regulates the expression of the organic hydroperoxide resistance (ohr) gene that encodes a peroxiredoxin involved in the detoxification of organic hydroperoxides (OHPs).25-26 OhrRBS is a homodimeric transcriptional repressor that senses OHPs using the oxidation of Cys thiolate; it is a 1-Cys OhrR, which contains only a single conserved peroxidatic Cys residue (Cys15) per monomer, unlike 2-Cys OhrR such as Xanthomonas campestris OhrR (OhrRXC), which contains an additional resolving Cys residue required to form intersubunit disulfide bonds with peroxidatic Cys.27 The initial step of OHP sensing by OhrR involves the oxidation of a peroxidatic Cys residue to a Cys sulfenic acid (Cys-SOH). Further modification of this Cys-SOH derivative is required for the functional inactivation of OhrR. In 2-Cys OhrR such as OhrRXC, Cys-SOH forms an intersubunit disulfide bond with resolving Cys in the other monomer. However, Cys-SOH in OhrRBS forms disulfide bond with thiol groups in LMW biothiols because OhrRBS contains no resolving Cys residue. This mixed disulfide bond formation with LMW biothiols leads to the rapid decline of DNA binding competency of OhrRBS and thus allows the induction of ohr gene ACS Paragon Plus Environment

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that is normally repressed by active OhrRBS. The OHP-sensing mechanisms via disulfide bond formation with LMW biothiols allowed us to consider OhrRBS as a potent protein-based sensor for the detection of LMW biothiols. Since OhrRBS utilizes a highly reactive Cys-SOH functional group for the formation of disulfide bond, it exhibits higher sensitivity to LMW biothiols than other classical chemicals.26 In addition, OhrRBS is likely to selectively interact with LMW biothiols, presumably by steric hindrance of proteinous thiol binding at the Cys-SOH containing active site. In stark contrast, it is known that chemicals routinely used for the detection of biothiols cannot discriminate LMW biothiols from proteinous biothiols such as reduced Cys residue in bovine serum albumin. Furthermore, in contrast to other Cys-based peroxide sensors and Fe2+/histidine-based peroxide sensors, such as OxyR and PerR,28-29 which sense H2O2 and may easily be oxidized by H2O2, OhrRBS is refractory to H2O2- (and other oxidants, such as superoxide and nitroxide) mediated oxidation.30 Since it is known that the concentration of OHP is much lower than that of H2O2 in blood,31 the use of OhrRBS can efficiently eliminate signal responses caused by aberrant H2O2. These factors prompt us to explore the possibility of using OhrRBS as a sensor for LMW biothiols. To investigate this, we designed an affinity-based fluorescence assay of LMW biothiols based on the interaction of OhrRBS and a fluorophore (6-carboxyfluorescein, 6-FAM)-labeled DNA-binding element (FAMACS Paragon Plus Environment

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dsDNA). The affinity-tagged OhrRBS captured by antibody-coated beads can strongly bind FAM-dsDNA. The addition of LMW biothiols and OHP to the reaction mixture containing OhrRBS/bead and FAM-dsDNA can trigger a rapid dissociation of FAM-dsDNA, consequently leading to a strong fluorescence signal in the supernatant. With the use of reducing agent, this OhrRBS-facilitated fluorescence detection method can be used to quantitatively analyze both free and total LMW biothiols in mouse plasma in a short time, which has not yet been possible using conventional methods.


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Experimental Methods

Affinity pull-down fluorescence assay of LMW biothiols. To prepare OhrRBS-dsDNA complex, an ohrA operator DNA fragment was generated by annealing FAM (5-6F-TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3) or biotin-labeled ssDNA (5-biotin-TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3) and its unlabeled complement (5-TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3). The mixture of oligonucleotides was initially denatured at 95 °C for 10 min, and cooled at 4 °C. The annealed dsDNA was diluted in Tris-buffered saline (TBS) (50 mM Tris (pH 8.0) containing 100 mM NaCl and 50 mM KCl) prior to use. FLAG-tagged OhrRBS or His6-tagged OhrRBS was expressed in E. coli BL21(DE3) and purified using an affinitybased column (see the supporting information). His6-tagged OhrRBS To perform the affinity pull-down assay, FLAG (DYKDDDDK)-tagged OhrR (20 L at 50 μM) and an anti-FLAG antibody-conjugated bead (30 L with 4% agarose bead) were initially mixed at a final volume of 200 L for 1 h at 4 °C with mild rotation. The OhrR-bound microbead was washed thrice with TBS by centrifugation, and FAM-dsDNA (20 L at 2.5 μM) was added to the beadcontaining solution at a final volume of 200 L, followed by 1 h incubation at 4 °C with mild rotation. The OhrR/FAM-dsDNA-bound microbead was obtained by repetitive centrifugation ACS Paragon Plus Environment

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and washed with TBS to remove unbound FAM-dsDNA; it was then resuspended in TBS. To analyze LMW or HMW biothiols, the OhrR/dsDNA-conjugated bead was treated with first CHP and second biothiols unless otherwise stated. The bead was initially treated with CHP (20 μL at 100 μM) for 1 min at a final volume of 180 L with mild rotation. Next, 20 L of Cys, Hcy, or rBSA at varied concentrations was added to the solution, followed by 4 min incubation at room temperature (RT). To compare the effect of CHP on the thiolation, different reaction sequence was applied; the bead was initially treated with each biothiol, followed by the addition of CHP. After the bead solution was subjected to a short spin (10 sec) using a mini-centrifuge, the supernatant (190 L) was collected and transferred into a 96-well microplate. The fluorescence intensity of the supernatant was measured with excitation and emission at 485 nm and 518 nm using a VarioskanTM microplate reader (Thermo Fisher Scientific). Unless stated otherwise, the measurement of LMW biothiols in sera or plasma samples followed the above procedure except for sample preparation. The Cys-spiked sample was prepared by mixing free Cys with mouse plasma or sera for 30 min at RT. To remove albumin in mouse sera or plasma, a ProteoPrep® Immunoaffinity column was used according to the manufacturer’s protocol. For the measurement of free LMW biothiols, mouse sera or plasma (20 L) was directly added to with 180 L of the solution containing OhrR/FAM-dsDNAACS Paragon Plus Environment

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conjugated beads in the presence of CHP. For the measurement of total LMW biothiols, mouse sera or plasma were pretreated with 500 μM DTT and then incubated for 5 min RT, which was followed by immediate reaction with the CHP-treated bead solution.

Fluorescence anisotropy (FA). FA was measured with 100 nM 6-FAM-labeled duplex DNA and 100 nM OhrR (dimer) with varying concentrations of biothiols in 3 mL of 20 mM Tris (pH 8.0) containing 150 mM NaCl and 5% (v/v) glycerol. FA values were recorded automatically every 10 sec with an LS55 luminescence spectrometer (PerkinElmer, Wellesley, MA, USA) installed in an anaerobic chamber (ex=495 nm; slit width=15 nm, em=520 nm; slit width=20 nm, integration time=1 s). FA values were ﬁtted to a 1:1 binding model using a Table Curve 2D program.

Biolayer interferometry (BLI). Using BLI, protein binding affinities were measured using a BLItz instrument (ForteBio, Fremont, CA, USA). Prior to use, a streptavidin (SA)-coated optical probe (#18-5019, Fortebio, USA) was soaked in running buffer (20 mM Tris (pH 8.0) containing 150 mM KCl and 0.02% Tween 20) for at least 10 min. The SA-coated sensor was installed into the instrument and then equilibrated in the running buffer for 30 sec to establish ACS Paragon Plus Environment

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an initial baseline. Analytes were sequentially loaded in the following order: dsDNA (2.5 μM in the running buffer, 120 s), baseline (with running buffer, 30 s), OhrR (50 μM in the running buffer, (120s), and baseline (with running buffer, 30 s). OhrR was pretreated with LMW biothiols at different concentrations in the running buffer or diluted mouse serum containing 100 μM CHP. The mouse serum was diluted 50-fold in the running buffer, which confirmed a low concentration of free LMW biothiols after long-term oxidation. Dissociation constants (KD) between OhrR and dsDNA were generated by fitting the kinetic curves to a 1:1 binding model using a BLItz Pro software.

Plasma sample preparation. Ldlr−/− mice (C57BL6/J background) were obtained from Jackson Laboratory (Bar Harbor, ME). Western Diet feed (WD) (49.9% Carbohydrates, 17.4% protein, 20% fat, and 0.15% cholesterol) was obtained from Test Diet (cat. AIN-76b; St. Louis, MO).

Ldlr−/− mice were fed WD for 10 weeks. Blood samples were taken from the retro-orbital plexus using a heparinized capillary tube. Plasma samples were prepared from the collected blood by centrifugation at 14,500 g (Eppendorf) for 10 min at 4 ℃. Plasma samples were stored at 80 ℃ before use.


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RESULTS AND DISCUSSION

Scheme 1 depicts the principle of assaying LMW biothiols based on the interaction between OhrRBS (dimer of 17 kD monomer, termed OhrR hereafter) and an ohrA operator dsDNA fragment. The dsDNA is generated by annealing 5-TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3 and its complement. The dimeric OhrR strongly binds to the dsDNA fragment with high affinity (Kd = 5 nM)26. The reduced Cys residue (Cys15) at each OhrR monomer selectively responds to cumene hydroperoxide (CHP) (as a representative OHP) with high reactivity (k1 105 M1 s1),26 leading to the oxidation of reduced Cys (OhrRSH) to Cys sulfenic acid (OhrRSOH). Subsequent disulfide bond formation (OhrRSSX) between OhrR-SOH and LMW biothiol (X-SH) results in rapid dissociation (t1/2~0.5 min) of OhrR from the dsDNA element (Scheme 1A). This reaction has been well described by a previous report,26 in which the dissociation rate of OhrR from dsDNA was found to be approximately 30fold faster in the presence of both CHP and LMW biothiols than in CHP alone. An affinity pulldown method was employed to conduct a fluorescence assay of LMW biothiols based on the interaction between OhrR and dsDNA. FLAG (DYKDDDDK)-tagged OhrR (FLAG-OhrR) was initially conjugated with microbeads coated with anti-FLAG antibodies, and then bound to the ACS Paragon Plus Environment

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FAM-labeled dsDNA element (FAM-dsDNA) (Scheme 1B). After removing unbound FAMdsDNA by washing, the OhrR-bead complex was used to analyze free (reduced) and total (reduced and oxidized) LMW biothiols (Scheme 1C). In the absence of DTT (as a reducing agent), only reduced LMW biothiols in plasma can trigger the release of FAM-dsDNA, leading to a high fluorescence signal in the supernatant, whereas LMW biothiol-deficient or oxidized LMW biothiol-containing samples cannot generate a fluorescence signal within short period (less than 15 min), which is indicative of no FAM-dsDNA dissociation. In the presence of DTT, total LMW biothiols (the summation of reduced and oxidized LMW biothiols) in plasma can be detected by complete reduction of all LMW biothiols, leading to an increased fluorescence signal in the supernatant. Note that the two sulfhydryl groups of DTT result in intramolecular disulfide formation (cyclization) after reducing the oxidized LMW biothiols, and thus does not bind to Cys15 of OhrR. Therefore, the amounts of free and total LMW biothiols in plasma can be measured simultaneously in the same sample without and with DTT, respectively, where the concentration of LMW biothiols is proportional to the fluorescence intensity. To investigate whether this method is specific to free LMW biothiols, three representative LMW biothiols (Cys, Hcy, and GSH) and a proteinous high-molecular-weight (HMW) thiol (reduced bovine serum albumin (rBSA)) were examined in the buffer as a function of thiol ACS Paragon Plus Environment

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concentration (Fig. 1). The control set (without biothiols) was analyzed in the presence of CHP (defined as F0), and all reactions for free biothiols were performed in the presence of CHP (defined as F). When the relative fold change in fluorescence to the control set (F/F0) was measured, a sigmoidal or linear correlation between fluorescence change and free LMW biothiol concentration was observed in the range of 1 M to 1 mM with different degrees (Fig. S1). The signal changes in response to Cys and Hcy were comparable, but their signals were relatively higher than in response to GSH over the tested range (Fig. 1A). In contrast, no significant change was detected over the same range of rBSA concentration. It has been reported that native BSA has 17 intrachain disulfide bonds with one free thiol group at Cys34,32 but crude BSA isolated from plasma was found to have mixed disulfide bonds with LMW biothiols at multiple Cys residues.33 Therefore, we used the rBSA after the reducing column purification of BSA to remove BSA-bound LMW biothiols (BSASSLMW biothiols), which can also bind to Cys in OhrR via disulfide exchange. The detection limits at each biothiol were calculated based on three times the standard deviation (3) of fluorescence signals at the lowest concentration of each biothiol from the standard four-parameter logistic or linear equation, and these values were approximately 1.58.0-fold greater than that obtained by the commercialized chemical probe (Fig. S2 and Table S1). Although this method did not ACS Paragon Plus Environment

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discriminate different species of LMW biothiols, a number of reduced LMW biothiols, such as Cys, Hcy, GSH, cysteinylglycine, and -glutamylcysteine, can be collectively measured by this method, in which free Cys is a major target because it predominantly exists in plasma. Taking into consideration oxidized (186239 M) and reduced (712 M) concentration of Cys,9 this method can be performed within the clinical dynamic range of LMW biothiols. When the supernatants were compared by fluorescence imaging at concentrations of 100 M biothiols with different reaction sequences (CHPbiothiol (top) and biothiolCHP (middle), and biothiol without CHP (bottom)), stronger fluorescence intensities were observed at Cys and Hcy in both top and middle images than those at GSH, but no intensities were observed in bottom image (Figs. 1B and 1C). This result indicates that CHP is essential for the oxidative thiolation of OhrR with LMW biothiols and functions regardless of the order of reaction sequence in a short reaction period (less than 10 min). Most importantly, we found that this OhrR-based pulldown fluorescence assay was much more selective and sensitive to LMW biothiols than HMW biothiols (i.e. rBSA). This result also suggests that the molecular weight of free biothiols affects the reaction rate of OhrR with biothiols considering the observed fluorescence signal at a given time.


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Based on the interactions between OhrR and FAM-dsDNA, S-thiolation-driven kinetics of OhrR with different LMW biothiols was further monitored using FA (Fig. 2). While OhrR stably bound to the dsDNA for initial 300 s, the addition of CHP without free thiols led to a gradual decrease of DNA-binding activity as a function of time (at a zero concentration of LMW biothiol in Fig. 2A). In contrast, the addition of free biothiols (Cys, Hcy, and GSH) with CHP led to a significant concentration-dependent decrease in FA values. The half-maximal dissociation rate of OhrR (t1/2) was determined from time-lapse FA changes over the range of free biothiol concentration (from 1 to 64 M) (Fig. 2B). Compared to the value derived from the control (674 s), the value of t1/2 is reduced to 68.5% (462 s) at 1 M Cys and to 5.9 % (40 s) at 64 M Cys. Hcy exhibited similar dissociation rates with Cys over the same concentration range, whereas GSH showed relatively slower dissociation rate than the other two free biothiols, which reflected the data in Fig. 1. The second-order reaction rate of OhrR to free Cys was calculated to be approximately 4102 M1 s1, which is exceedingly faster than that of chemical probes (10310 M1 s1) to thiol groups20, 34 or that of LMW biothiol oxidation by peroxide such as H2O2 (0.22.9 M1 s1).35 This result indicates that free LMW biothiols triggered rapid dissociation of OhrR-dsDNA within several minutes. The ability of OhrR to bind to LMW biothiols in the absence of operator dsDNA was also validated by matrix-assisted laser ACS Paragon Plus Environment

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desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Fig. S3). S-thiolation of OhrR by Cys, Hcy, and GSH was monitored by observing the modification of T3 tryptic peptide containing Cys15. As expected T3 peptides were observed at m/z =1,776 for [OhrRSSCys]+, m/z = 1,790 for [OhrRSSHcy]+, and m/z = 1,962 for [OhrRSSGSH]+ after CHP treatment for the same duration (10 min). Furthermore, the respective peak intensity of S-thiolated OhrR showed a hyperbolic increase as the concentration of LMW biothiol increased over the range of 064 M. Among LMW biothiols, S-GSHylated OhrR showed relatively lower peak intensity than the other two S-thiolated OhrRs. These results are comparable with the data in Figs. 1 and 2, supporting that the S-thiolation of OhrR rapidly responses to free LMW biothiols in a semi-quantitative manner and the reaction rate is dependent on the molecular weight of LMW biothiols. To examine whether the interaction of OhrR-dsDNA can be modulated by LMW biothiols in complex media, the association and dissociation of OhrR with its operator dsDNA was measured in buffer and serum using BLI (Fig. 3). Unlike the FA-based measurement, which can be performed only in a buffer, the interferometric analysis provides information of DNAprotein interaction by flowing the serum over a fiber optic surface. In this analysis, the change in the BLI sensorgram between two reflective layers (target-binding and internal reference ACS Paragon Plus Environment

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layers), which is expressed as binding thickness, represents the binding amount of the target molecule. When biotinylated dsDNA was initially loaded onto SA-coated optical biosensors, the binding thickness increased up to approximately 2 nm at the equilibrium line. We then performed a binding analysis by using His6-tagged OhrR onto the operator dsDNA-coated layer in a buffer (Figs. 3A3B) and serum (Fig. 3C) with and without other components (CHP and/or LMW). Despite the variance in association and dissociation rate (slopes before and after injecting the OhrR), equilibrium binding constants (binding thickness) of OhrR alone (closed circle), OhrR with CHP (open circle), and OhrR with Cys (closed square) were similar as shown in Fig. 3A, indicating that OhrR strongly binds with dsDNA, and the addition of either CHP or Cys did not affect the binding thickness at a given binding time (2 min). In contrast, OhrR pretreated with both CHP and Cys (open square) exhibited a dramatic reduction in binding thickness as shown in Fig. 3A, indicating that OhrR readily dissociated from dsDNA via S-thiolation in the presence of CHP. Based on the binding thickness value, the equilibrium dissociation constant (KD) between OhrR and dsDNA was calculated to be 4.1 nM, which was similar to that reported in previous literature.26 Indeed, as observed in Fig. 2, the binding thickness of OhrR decreased as Cys concentration increased (from 1 M to 1 mM) in the presence of CHP (Fig. 3B). Importantly, the binding thickness of OhrR in serum without Cys ACS Paragon Plus Environment

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(closed circle in Fig. 3C) was notably similar to that of OhrR in the buffer (open circle in Fig. 3C) in the presence of CHP, whereas the binding thickness of OhrR in Cys-spiked serum (closed square in Fig. 3C) declined onto the dsDNA-modified surface. Considering the similar binding thickness of OhrR and dsDNA between the buffer (open square in Fig. 3A) and 100 M Cys spiked-serum (closed square in Fig. 3C), this result strongly indicates that the interaction between OhrR and dsDNA can be used for the detection of LMW biothiols in serum. Furthermore, to get insight into the effect of DTT on OhrR, we investigated the FA-based kinetic response and matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) of OhrR in the presence and absence of DTT (Fig. S4). Under conditions with DTT and without Cys, the addition of CHP led to the gradual dissociation of OhrR from FAM-dsDNA (black circle in Fig. S4A). In contrast, in the presence of DTT and Cys, the addition of CHP triggered the rapid dissociation of OhrR from dsDNA within 10 min followed by a gradual association of OhrR to dsDNA after 10 min (gray triangle in Fig. S4A), indicating that LMW biothiols can be detected by S-thiolation of OhrR in a short time period (less than 10 min) even in the presence of DTT, which functions as a strong reductant for oxidized biothiols in blood. This was further supported by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis as shown in Figs. S4B and S4C, in which the peak ACS Paragon Plus Environment

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intensities of S-cysteinylated OhrR and S-homocysteinylated OhrR were observed at 10 min after adding DTT to mouse serum containing OhrR and CHP. Plasma, instead of serum, is generally suggested as a blood sample to analyze LMW biothiols because plasma samples contain clotting factors in which some LMW biothiols are present within a diagnostic range36. Therefore, free (without DTT) and total (with DTT) LMW biothiols were measured in mouse plasma using the affinity pull-down fluorescence assay with the FLAG-OhrR/FAM-dsDNA complex (Fig. 4). To estimate the concentration of free and total biothiols on the basis of calibration data in Fig. 2, we determined the normalized fluorescence value (F/F0); the control set (without plasma and without DTT) in the presence of CHP was defined as F0, and the test set with plasma (DTT or not) in the presence of CHP was defined as F. Compared to that in the control set without plasma (the first black/white bars in Fig. 4A), the increased level of free LMW biothiols (the second black bar in Fig. 4A) and total LMW biothiols (the second white bar in Fig. 4A) was observed in mouse plasma, wherein total LMW biothiols was almost 1.8-fold higher than that in free LMW biothiols. The values observed in DTT-untreated (free) and treated (total) plasma corresponded to the concentrations of ~5 M and ~19 M, respectively, based on the Cys concentration. Both levels of free and total biothiol concentration also increased when free Cys (50 M) was added to the mouse plasma ACS Paragon Plus Environment

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(the third black/white bars in Fig. 4A). However, in this measurement, it is considered that the amount of total LMW biothiols in plasma could be released from oxidized albumins by DTT treatment, because albumin has multiple LMW biothiol-binding sites. The concentration of albumin was estimated to be approximately 50-fold higher than that of total LMW biothiols concentration in human plasma.9 It is also important to note that Hcy or Cys can be predominantly bound to albumin or other proteins in plasma under oxidative stress conditions.37 To investigate LMW biothiols under albumin-free condition, we removed albumin and immunoglobulin G (IgG) from mouse plasma using a commercialized immunoaffinity column and the albumin/IgG-free plasma was examined for free and total LMW biothiols under the same assay conditions (Fig. 4B). The level of free LMW biothiols (the second black bar in Fig. 4B) was similar but total LMW biothiols (the second white bar in Fig. 4B) was dramatically reduced, in comparison with those in Fig. 4A, indicating the loss of albumin-derived LMW biothiols. Interestingly, when free Cys (50 M) was added to albumin/IgG-deficient plasma, the fluorescence intensities from free and total LMW biothiols notably increased (the third black/white bars in Fig. 4B), compared to those in the third pairs in Fig. 4A. This result implies that additional Cys could be scavenged by albumin in Fig 4A. At a given DTT concentration (500 M), the oxidized LMW biothiols in plasma were not fully reduced, due to high ACS Paragon Plus Environment

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concentration of plasma albumin. Therefore, we removed albumin/IgG prior to LMW biothiol analysis to minimize the albumin-dependent interference on LMW biothiol pools in plasma. In an effort to investigate the relevance of LMW biothiols in mouse plasma to disease, the amounts of free and total LMW biothiols were measured in the plasma of normal and lowdensity lipoprotein receptor (LDLR) knock-out (Ldlr−/−) mice (Fig. 5). The knock-out mouse (LDLR) has been generally considered a model for atherosclerosis because LDLR is responsible for the clearance of LDL in blood38. When the affinity pull-down fluorescence assay was performed after a series of plasma sample preparation including the removal of albumin (Fig. 5A), the level of total LMW biothiols significantly increased compared to that of free LMW biothiols (p

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