Article pubs.acs.org/ac
Visualization of Phagosomal Hydrogen Peroxide Production by a Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling Masahiro Abo,† Reiko Minakami,†,‡ Kei Miyano,† Mako Kamiya,§ Tetsuo Nagano,∥ Yasuteru Urano,§,∥ and Hideki Sumimoto*,† †
Departments of Biochemistry and ‡Health Sciences, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan § Graduate Schools of Medicine and ∥Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan S Supporting Information *
ABSTRACT: Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O6benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.
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cytosolic NAPDH across the membrane, molecular oxygen (O2) is reduced to O2− on the inside of the phagosome.10−13 O2− immediately undergoes dismutation to H2O2, which is further converted into hypochlorous acid (HOCl), an agent of the highest microbicidal activity, by the phagocyte-specific enzyme myeloperoxidase (MPO) that is discharged from granules/lysosomes into the phagosome.14,15 The significance of the phagocyte NADPH oxidase in host defense is exemplified by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes genetically lack the Nox2-catalyzed ROS-producing activity.16 Importantly, Nox2 is required to be activated exclusively on the phagosomal membrane since ROS released on the outside of the cell are harmful to surrounding cells/ tissues, due to their high reactivity. In addition, H2O2 escaped from the phagosome into the cytoplasm appears to contribute to efficient phagosome maturation, a process that includes its fusion with granules/lysosomes.17−19
ntracellular production of reactive oxygen species (ROS) such as superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (·OH) is an inevitable consequence of aerobic metabolism and considered to be involved in pathogenesis of cancer, neurodegeneration, and cardiovascular diseases.1−3 However, it is currently realized that H2O2 functions as a physiological regulator of intracellular signaling pathways4−7 as well as a participant in killing of pathogenic microbes.8,9 Indeed there exist enzymes that purposefully produce H2O2 upon cell stimulation. Among them are evolutionarily conserved, membrane-integrated enzymes of the NADPH oxidase (Nox) family; human genome encodes seven members of the family, namely, Nox1 to Nox5, Duox1, and Duox2.10−13 Nox oxidases have wide tissue distribution and participate in various physiological events including hormone synthesis, in addition to the role in signal transduction and host defense. Nox2 (also known as gp91phox) is abundantly expressed in professional phagocytes such as neutrophils and macrophages. The phagocyte oxidase Nox2, dormant in resting cells, is activated during phagocytosis of invading microorganisms to primarily produce superoxide as a precursor of H2O2 and other microbicidal ROS. Because Nox2 transports electrons from © 2014 American Chemical Society
Received: March 22, 2014 Accepted: May 26, 2014 Published: May 26, 2014 5983
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Figure 1. Chemical properties of NBzF-BG. (A) Chemical structure of NBzF and NBzF-BG. (B) Fluorescence response of SNAP-NBzF with H2O2. H2O2 (1 mM) was added at 50 s to the solution of 1 μM NBzF-BG or 1 μM SNAP-NBzF in 100 mM sodium phosphate, pH 7.4. The reaction was performed at 25 °C with measuring fluorescence intensity at 520 nm with excitation at 500 nm. (C) Fluorescence spectral change of SNAP-NBzF through the reaction with H2O2. H2O2 (100 μM) was added to the solution of 0.5 μM SNAP-NBzF in 100 mM sodium phosphate, pH 7.4, and fluorescence spectra with excitation light at 508 nm were measured at the indicated time points after H2O2 addition. (D) Fluorescence response of SNAP-NBzF to H2O2. Various concentrations of H2O2 were added to the solution containing 1 μM SNAP-NBzF in 100 mM sodium phosphate, pH 7.4. Fluorescence at 520 nm (λ ex = 500 nm) were measured after incubation with H2O2 for 5 min at 25 °C. Error bars, SD (n = 3).
ment when SNAP-tag is expressed as a fusion with a specifically targetable protein domain. Chang and his colleagues have recently reported the synthesis of SNAP-tag substrates of the boronate-caged H2O2 fluorescent probe Peroxy Green, which are targeted to various subcellular regions via conjugation to specifically localized SNAP-tag proteins.32 Although they have also demonstrated that these targeted probes are capable of detecting H2O2 added exogenously to live cells,32 endogenously produced H2O2 has not been imaged with subcellular resolution. We have previously designed and synthesized NBzF, a highly H2O2-sensitive fluorescent probe with a high reaction rate,24 which is in contrast to the slow reaction rate with H2O2 of the boronate-caged fluorescent probes.22 Here we report the synthesis and application of a BG derivative of NBzF (NBzFBG) (see Figure 1); this novel fluorescent probe is suitable for detection of H2O2 and efficiently labels SNAP-tag in vitro. Labeling of plasma membrane-recruited SNAP-tag with NBzFBG in live macrophages enables us to visualize H2O2, which is endogenously produced during phagocytosis.
Various species of ROS each have unique chemical reactivity and likely contribute to distinct biological functions: H2O2 serves as a signal transducer as well as a precursor of microbicidal ROS,1−3 whereas microbial killing directly involves HOCl14,15 and possibly peroxynitrite (ONOO−),20,21 which is produced by the reaction of O2− with nitric oxide (NO) released from cells such as activated macrophages. For investigation of cell regulation by H2O2, fluorescence sensors for probing this reactive oxygen metabolite in living cells with high molecular specificity have been developed:22 the boronatecaged compounds Peroxy Green 1 and Peroxy Crimson 123 and the benzil-based probe 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF),24 which is designed and synthesized by utilizing the unique chemical reactivity of benzil and H2O225 together with a photoinduced electron transfer mechanism for control of the fluorescence.26 In addition to specific detection, it is also important to detect ROS with subcellular resolution because the function of ROS is largely dependent on the subcellular compartment where ROS are produced;27,28 e.g., ROS released into phagosomes play a crucial role in microbial killing, whereas ROS produced in mitochondria are assumed to be involved in DNA damage and aging. A recently developed strategy to target small sensor molecules to certain subcellular compartments and/or organelles utilizes the versatile SNAP-tag technology.29−31 SNAP-tag is a mutated human O6-alkylguanine-DNA alkyltransferase (hAGT) that reacts rapidly and specifically with O6 benzylguanine (BG) derivatives; thus, a BG-containing synthetic sensor that is covalently attached to SNAP-tag in living cells can be delivered to a specific subcellular compart-
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EXPERIMENTAL SECTION Materials. Chemicals used in the present study were of the highest purity commercially available from Tokyo Chemical Industries, Wako Pure Chemical, Daiichi Chemical, or Sigma− Aldrich, unless otherwise indicated. All solvents were used after appropriate distillation or purification. Plasmid Construction. SNAP-tag is a 182 amino-acid mutant protein of hAGT, in which the C-terminal 25 amino acids were deleted and 19 amino acid substitutions were 5984
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introduced.33 The cDNA for SNAP-tag was kindly provided from Dr. Kai Johnsson (École Polytechnique Fédérale de Lausanne, Switzerland);33 and pDisplay-SNAP, which encodes a protein containing the signal peptide of murine Ig κ-chain, SNAP-tag, and the transmembrane domain of platelet-derived growth factor receptor (PDGFR) in the mammalian expression vector pDisplay (Invitrogen), were kindly provided from Dr. Ryosuke Kojima (Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan). The SNAP-tag cDNA was also ligated to a modified pET-15b (Novagen), which was constructed for expression as a protein fused to an N-terminal hexahistidine-tag (His-tag) in Escherichia coli. All of the constructs were sequenced for confirmation of their identities. In Vitro SNAP-tag Labeling and In-Gel Fluorescence Analysis. His-tagged SNAP-tag (His-SNAP) was expressed in E. coli strain BL21 (DE3) and purified using HIS-Select Nickel Affinity Gel (Sigma−Aldrich), according to the manufacturer’s protocol. His-SNAP (10 μM) was incubated for 30 min at 25 °C with NBzF-BG at varying concentrations (0, 5, 10, 20, or 40 μM) in 100 mM sodium phosphate, pH 7.4. Samples were then analyzed by 13.2% SDS-PAGE and subsequent in-gel fluorescence scanning with LAS-1000 (FUJIFILM). For the preparation of the fluorophore−protein conjugate, NBzF-BG (20 μM) was incubated with His-SNAP (10 μM) for 30 min at 25 °C in 100 mM sodium phosphate, pH 7.4. To remove unconjugated NBzF-BG, the reaction mixture was applied to a PD-10 desalting column (GE Healthcare Life Sciences), and the purified conjugate was used for further spectroscopic evaluation. Spectrophotometric Analysis. UV−visible spectra were obtained on a JASCO V-550 or a Shimazu UVmini1240. Fluorescence spectroscopic studies were performed using a JASCO FP-6500 or a Hitachi F-2500. Relative fluorescence quantum efficiencies of dyes were obtained by comparing the area under the emission spectrum of the test sample excited at an absorption maximum with that of a solution of fluorescein in 0.1 N NaOH, which has a quantum efficiency of 0.85. Labeling with NBzF-BG of SNAP-tag-Expressing Cells and Microscopic Analysis. HEK293T cells34 and macrophage-like RAW264.7 cells35 were cultured in DMEM (Nissui Pharmaceutical) containing 10% fetal bovine serum (Nichirei Biosciences) in a 35 mm glass bottom dish, coated with poly-Dlysine (MatTek Corporation), and transfected with the SNAPtag cDNA using X-tremeGENE HP (Roche Applied Science). SNAP-tag on cells was labeled for 30 min at 37 °C with 5 or 10 μM NBzF-BG and 0.5, 1, or 2 μM SNAP-Surface Alexa Fluor 546 (New England Biolabs). Labeled cells were washed twice with DMEM and suspended in HEPES-buffered saline (HBS; 120 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, and 17 mM HEPES, pH 7.4). Confocal fluorescence imaging studies were performed with the laser-scanning confocal microscope LSM510 (Carl Zeiss) equipped with a 40× water-immersion objective lens. Excitation of NBzF-BG was carried out at 488 nm by an argon laser, and emission was collected by using an LP505 filter; SNAP-Surface Alexa Fluor 546 was excited at 543 nm by a helium−neon laser, and emission was collected by using an LP560 filter. Ratiometric images were constructed by a Ratio Plus plugin of ImageJ software (National Institutes of Health). Live Cell Imaging of Phagocytosis by RAW264.7 Cells. IgG-opsonized particles were prepared as follows: Polybead carboxylate microspheres (4.5 μm, Polysciences, Inc.) were incubated overnight at 25 °C with 10 mg of BSA (Nacalai
Tesque) in 1 mL phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4, pH 7.4), followed by incubation with anti-BSA polyclonal antibodies (Sigma−Aldrich). RAW264.7 cells labeled with NBzF-BG (5 μM) and SNAP-Surface Alexa Fluor 546 (0.5 μM) were incubated with IgG-opsonized particles for 10 min at 37 °C. After washing twice with PBS, cells were fixed in 3.7% formaldehyde for 10 min at 25 °C and analyzed with LSM510.
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RESULTS AND DISCUSSION Design and Synthesis of NBzF-BG. We have previously synthesized NBzF as a fluorescence probe for H2O2: NBzF is highly sensitive for detection of H2 O2 with excellent fluorescence activation and a reaction rate constant (k) of 4.4 × 10−3 s−1 with 1 mM H2O2 under a pseudo-first-order condition.24 To couple this sensitive H2O2 probe to SNAP-tag, we designed NBzF-BG (Figure 1A). NBzF-BG was synthesized from 5-iodofluorescein as a starting material via four steps including Sonogashira reaction, the conjugation with ethyl 4piperidinecarboxylate, oxidation by palladium(II) chloride and dimethyl sulfoxide (SDS), and the conjugation with O6-[4(aminomethyl)benzyl]guanine (for details, see Supporting Information Figure S1). In Vitro Evaluation of NBzF-BG for Optical Properties, Reactivity to H2O2, and Conjugation with Recombinant SNAP-tag Protein. NBzF-BG showed visible absorption centered at 505 nm and weak fluorescence with an emission maximum at 525 nm. The fluorescence quantum efficiency (Φfl) of NBzF-BG was calculated as 0.012 (Table 1). The Table 1. Spectroscopic Properties of NBzF, NBzF-BG, and SNAP-NBzFa compd
λex (nm)
ε (M−1 cm−1)
λem (nm)
Φfl
NBzF NBzF-BG SNAP-NBzF
495 505 508
68000 56000 66000
519 525 528
0.004 0.012 0.028
Dyes (0.33 μM) were dissolved in 100 mM sodium phosphate, pH 7.4. Fluorescence spectra were measured with excitation light at an absorption maximum. a
reaction of NBzF-BG with 1 mM H2O2 triggered approximately 30-fold fluorescence activation, confirming the response of NBzF-BG to H2O2 (Figure 1B). The kinetic analysis under a pseudo-first-order condition (1 μM NBzF-BG with 1 mM H2O2) gave a reaction rate constant (k) of 8.7 × 10−3 s−1. We next tested the ability of NBzF-BG to label a SNAP-tag protein in vitro. A recombinant His-tagged SNAP-tag (His-SNAP) was incubated with various concentrations of NBzF-BG, followed by in-gel fluorescence analysis. The analysis revealed that the conjugate SNAP-NBzF was quantitatively formed (Figure 2). Optical Properties and Reactivity to H2O2 of SNAPNBzF in Vitro. Spectroscopic analysis revealed that SNAPNBzF has an absorption maximum at 508 nm and an emission maximum at 528 nm, which is slightly red-shifted as compared with NBzF-BG (Table 1). The fluorescence quantum efficiency of SNAP-NBzF (Φfl = 0.028) is 2-fold higher than that of NBzF-BG (Table 1). The addition of H2O2 to SNAP-NBzF resulted in a marked increase of the emission with a peak at 525 nm (Figure 1C). The addition of H2O2 to SNAP-NBzF resulted in 9-fold fluorescence activation, and the reaction rate constant (k) of SNAP-NBzF with 1 mM H2O2 under a neutral 5985
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Figure 2. Analysis of the reaction of NBzF-BG with SNAP-tag. (A,B) Recombinant His-SNAP-tag (10 μM) was incubated with various concentrations of NBzF-BG (0, 5, 10, 20, and 40 μM; 0, 0.5, 1, 2, and 4 mol equiv to SNAP protein, respectively) for 30 min at 25 °C. Samples were applied to SDS-PAGE, followed by staining with Coomassie Brilliant Blue or by in-gel fluorescence scanning. (B) Quantification of SNAP-NBzF signals estimated by in-gel fluorescence scanning.
aqueous condition was calculated as 6.2 × 10−3 s−1 by fitting a pseudo-first-order model (Figure 1B). Because the increase in SNAP-NBzF fluorescence intensity is proportional to the concentration of H2O2 added (Figure 1D), the probe appears to function in a quantitative manner. The detection limit of H2O2 was calculated from Figure 1D to be 2.0 μM. The reaction rate constants of NBzF-BG (k = 8.7 × 10−3 s−1) and SNAP-NBzF (k = 6.2 × 10−3 s−1) are much higher than that of SPG2 (k = 1.0 × 10−3 s−1), which is a SNAP-tag substrate of the boronate-caged H2O2 fluorescent probe Peroxy Green.32 The fast reaction rate is crucial for sensitive detection of H2O2 in vivo because this oxidant is rapidly reduced in living organisms by glutathione and antioxidant proteins of the peroxiredoxin family6,36 and also can be removed by the catalysis of MPO in phagocytes, which leads to production of HOCl.14,15 In addition, NBzF functions as a probe more specific to H2O2 than boronate-caged probes do: NO is sensed by boronate-caged H2O2 probes23 but not by NBzF,24 and ONOO− reacts only weakly with NBzF24 but strongly with boronate-caged H2O2 probes.37−39 Detection of Exogenous H2O2 by NBzF-BG Presented on Living Cells. To use NBzF-BG for imaging H2O2 in living cells, we transfected HEK293T cells with pDisplay-SNAP encoding SNAP-PDGFR-TM, which fusion enables SNAP-tag to localize to the extracellular surface of the plasma membrane. SNAP-tag-expressing cells were incubated with both NBzF-BG (10 μM) and SNAP-Surface Alexa Fluor 546 (2 μM); the former exhibits green emission upon reaction with H2O2, whereas the latter is a SNAP-tag with red emission. The double labeling enables ratiometric measurements because solely NBzF-BG reacts with H2O2, thereby leading to an increase in green emission. The addition of H2O2 (100 μM) to doubly labeled HEK293T cells resulted in enhancement of green fluorescence on the cell surface (Figure 3A). The ratio of green and red fluorescence (F505/F560) rapidly increased up to 1.7fold by the addition of H2O2 (Figure 3B). Similar H2O2induced rapid increase in F505/F560 was observed on the cell surface, when macrophage-like RAW264.7 cells were used instead of HEK293T cells (Figure 4). Thus, NBzF-BG targeted to the cell surface functions as a probe for imaging H2O2. Imaging of H2O2 Produced during Phagocytosis by RAW264.7 Cells. It is known that, during phagocytosis of IgGopsonized particles, the phagocyte NADPH oxidase in RAW264.7 macrophages is activated to release superoxide as
Figure 3. Fluorescence detection of exogenous H2O2 in living HEK293T cells. (A) SNAP-tag-expressing HEK293T cells were labeled with NBzF-BG (10 μM) and SNAP-Surface Alexa Fluor 546 (2 μM). Fluorescence images were obtained before and after incubation for 10 min with or without 100 μM H2O2: differential interference contrast (DIC) image; emission from NBzF-BG (F505); emission from SNAP-Surface Alexa Fluor 546 (F560); and F505/F560 in a pseudocolor mode. Scale bars, 20 μm. (B) F505/F560 values were obtained every 2 min after the addition of 100 μM H2O2 to labeled HEK293T cells. Error bars, SD (n = 10).
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Figure 4. Fluorescence detection of exogenous H2O2 in living RAW264.7 cells. (A) Fluorescence images of SNAP-tag-expressing RAW264.7 cells which were labeled with NBzF-BG (10 μM) and SNAP-Surface Alexa Fluor 546 (2 μM). Fluorescence images were obtained after incubation for 10 min in the presence of 100 μM H2O2. Upper panels: differential interference contrast (DIC) microscopy; emission from NBzF-BG (F505); and emission from SNAP-Surface Alexa Fluor 546 (F560). Lower panels: ratiometric images in a pseudocolor mode (F505/F560). Scale bars, 10 μm. (B) Timelapse measurement of F505/F560 with the addition of 100 μM H2O2. Error bars, SD (n = 3).
a precursor of H2O2 into phagosomes.40 To visualize H2O2 production during phagocytosis, we expressed SNAP-tag on the surface of RAW264.7 cells and labeled it with NBzF-BG and SNAP-Surface Alexa Fluor 546. Doubly labeled RAW264.7 cells were subsequently incubated for 10 min with IgG-opsonized particles and fixed in 3.7% formaldehyde. As shown in Figure 5, the fluorescence ratio (F505/F560) in the phagosomal membrane was enhanced more efficiently than that in the plasma membrane, suggesting that H2O2 is produced mainly in the phagosomal membrane. To confirm that SNAP-NBzF reacts with H2O2 in the phagosome, we tested the effects of the H2O2
scavenger ebselen41 and diphenylene iodonium (DPI), an inhibitor of the phagocyte NADPH oxidase.42 Because it is generally known that phagosomes are heterogeneous,43−45 phagosomes observed in the present study were categorized into three groups on the basis of the ratio between the F505/F560 in the phagosomal membrane and the F505/F560 in the plasma membrane, i.e., Rphagosome/PM: group I (considered as high H2O2 production in the phagosome), Rphagosome/PM ≥ 1.3; group II (considered as moderate H2O2 production in the phagosome), 1.3 > Rphagosome/PM ≥ 1; and group III (considered as low H2O2 production in the phagosome), 1 > Rphagosome/PM. Because of difficulty to repeatedly load the two separate dyes into the cells in the same ratio in each experiment, F505/F560 varied to some extent among experiments; for rough comparison, the ratio Rphagosome/PM is used. Among 60 phagosomes tested in 4 independent experiments, 27%, 45%, and 28% of the phagosomes were classified into groups I, II, and III, respectively. The group III phagosomes were increased in cells treated with ebselen or DPI, whereas the group I phagosomes were decreased in these cells (Figure 6A,B). In addition, the average Rphagosome/PM value of all the phagosomes tested was reduced when cells were treated with ebselen or DPI (Figure 6C). Signal suppression by ebselen indicates that NBzF indeed detects H2O2 in phagosomes, and the result with DPI is consistent with the fact that the phagocyte oxidase Nox2 is responsible for H2O2 production in the phagosome.8−12 To the best of our knowledge, this is the first example of live cell imaging of endogenous H2O2 with subcellular resolution by a chemical sensor probe conjugated with a specifically localized protein. It is known that acidification of the phagosome occurs during phagocytosis46,47 and fluorescein decreases in fluorescence in acidic solution;48 indeed the fluorescence of the fluorescein derivative NBzF-BG was decreased at low pH (Supporting Information Figure S2), suggesting underestimation of H2O2 produced in phagosomes. In addition, it should be noted that the present probe NBzF-BG reacts with H2O2 in an irreversible manner. Whereas reversible H2O2 probes based on fluorescent proteins49,50 can be disturbed by cell fixation, NBzF-BG makes reliable detection possible even after fixation with formaldehyde (Figures 3−6), which would enable simultaneous staining analysis such as immunostaining. ROS produced by Nox2 during phagocytosis serve not only as microbicidal agents but also as signaling molecules for phagosome maturation. ROS are currently considered to participate in phagosomal recruitment of the autophagy protein
Figure 5. Molecular imaging of H2O2 during phagocytosis of RAW264.7 cells. SNAP-tag-expressing RAW264.7 cells were labeled with NBzF-BG (5 μM) and SNAP-Surface Alexa Fluor 546 (0.5 μM) and incubated with IgG-coated beads. Panels are representative images of cells containing phagosomes of group I, group II, and group III: differential interference contrast (DIC) image; emission from NBzFBG (F505); emission from SNAP-Surface Alexa Fluor 546 (F560); and F505/F560 in a pseudocolor mode. Scale bars, 5 μm. 5987
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phagosomes under similar conditions; it is known that phagosomes formed via the same receptors can find themselves in different chemical states even within the same macrophage.43−45 In cells other than phagocytes, it is also assumed that accumulation of ROS, especially H2O2, at a restricted region of the plasma membrane is involved in signal transduction. Nox-catalyzed ROS production that is confined to lamellipodia plays a crucial role in directed migration of endothelial cells.51,52 The H2O2 scavenger peroxiredoxin I (PrxI) associated with membranes is phosphorylated and thereby inactivated upon growth factor stimulation of various cells; the localized inactivation of PrxI allows the accumulation of H2O2 around membranes, where signaling components are concentrated.53 Effect of NO Synthase (NOS) or MPO Inhibition on Phagosomal Imaging of H2O2. We finally evaluated the effect of ROS other than H2O2 on the present imaging system because various ROS such as HOCl, NO, and ONOO− are known to be produced in phagosomes.14,20 As shown in Figure 7A, treatment of RAW264.7 cells with L-NG-nitroarginine methyl ester (L-NAME), a NOS inhibitor that blocks NO production and thus prevents formation of ONOO− from NO and O2−,54 did not affect the Rphagosome/PM value obtained 10 min after the addition of IgG-opsonized particles, indicating that NO and ONOO− do not exert effects on the present H2O2 imaging system. Thus, it seems likely that ONOO− is not produced in phagosomes under the present conditions since NBzF can react with ONOO−, albeit to a small extent.24 In addition, cell incubation for another 10 min resulted in a decrease in the Rphagosome/PM value. It seems possible that the fluorescence decay may be caused by ROS-mediated degradation of the fluorophore because fluorescein is known to be chlorinated and bleached by HOCl in activated neutrophils.55 ONOO− does not appear to contribute to the degradation because the decay was not influenced by L-NAME (Figure 7A). Treatment of RAW264.7 cells with 4-aminobenzoic hydrazide (ABAH), an MPO inhibitor that prevents formation of HOCl from H2O2 and Cl−,56 hardly affected phagosomal H2O2 imaging 10 min after the addition of IgG-opsonized particles (Figure 7B), consistent with the previous finding that NBzF does not act as a sensor for HOCl.24 However, when cells were
Figure 6. Effect of ebselen and DPI on phagosomal H2O2 production. SNAP-tag-expressing RAW264.7 cells were labeled with NBzF-BG (5 μM) and SNAP-Surface Alexa Fluor 546 (0.5 μM) and incubated for 10 min at 37 °C with IgG-opsonized particles in the presence or absence of ebselen (20 μM) or DPI (10 μM). (A) Panels are representative images of DIC and F505/F560 in a pseudocolor mode from the samples with 0.1% DMF as a vehicle, 20 μM ebselen, and 10 μM DPI. Scale bars, 5 μm. (B) Populations of phagosomes of group I, group II, and group III under the indicated conditions. (C) Graphs represent the average (Rphagosome/PM) ± SD (n = 60, with DMF; n = 36, with ebselen; and n = 37, with DPI). *P < 0.05; **P < 0.01.
LC3, which promotes phagosome maturation and microbial killing;17−19 however, only about 30% of phagosomes are LC3positive when RAW264.7 cells engulf IgG-opsonized particles.18 The heterogeneity of phagosomes is consistent with the present finding that Nox2 activity varies from phagosomes to
Figure 7. Effect of NOS and MPO inhibitors on phagosomal H2O2 production. (A,B) SNAP-tag-expressing RAW264.7 cells were labeled with NBzF-BG (5 μM) and SNAP-Surface Alexa Fluor 546 (0.5 μM) and incubated for 10 min at 37 °C with IgG-opsonized particles in the presence or absence of the NOS inhibitor L-NAME (1.0 mM) or the MPO inhibitor ABAH (1.0 or 2.0 mM). After IgG-opsonized particles were washed out, cells were incubated for further 10 min at 37 °C in the presence or absence of the inhibitor, followed by fixation in 3.7% formaldehyde. (A) Cells were incubated for 10 min in the presence (n = 52) or absence (n = 49) of L-NAME or for 20 min in the presence (n = 14) or absence (n = 12) of LNAME. The values represent the average (Rphagosome/PM) ± SD. (B) Cells were incubated for 10 min with (n = 42) or without (n = 42) 1.0 mM ABAH; or for 20 min with ABAH at 0 mM (n = 12), 1.0 mM (n = 9), or 2.0 mM (n = 19). The values represent the average (Rphagosome/PM) ± SD. *P < 0.05; **P < 0.01. 5988
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incubated with the particles for another 10 min, ABAH restored the Rphagosome/PM value in a dose-dependent manner (Figure 7B), raising the possibility that the decay may be caused by degradation of NBzF-BG by the highly reactive oxidant HOCl. The restoration might be also partly due to the blockade of H2O2 consumption by MPO. Thus, the usage of an MPO inhibitor in the present imaging system would help accurate estimation of H2O2 produced in the phagosome.
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CONCLUSIONS We have developed a novel localizable derivative of the H2O2specific fluorescent probe NBzF, namely, NBzF-BG. This probe is efficiently conjugated with the recombinant SNAP-tag in vitro and labels SNAP-tag that is expressed as a fusion with the PDGFR transmembrane domain and thus presented on the outside of the plasma membrane. Importantly, NBzF-BG possesses a high reaction rate with H2O2, which is crucial for sensitive detection in vivo because H2O2 is rapidly quenched by antioxidants in living organisms. These properties of NBzF-BG enable sensitive detection of H2O2 with subcellular resolution in the combination with localized SNAP-tag. Indeed, we demonstrate successful application of NBzF-BG to visualize endogenous H2O2 that is produced during phagocytosis in RAW264.7 macrophages. Thus, the present H2O2 imaging system using membrane-targeted NBzF-BG will be a useful tool for the investigation of redox regulation in a variety of cells.
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ASSOCIATED CONTENT
S Supporting Information *
Details of NBzF-BG synthesis and fluorescence spectra of NBzF-BG at various pH. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(H.S.) E-mail:
[email protected]. Tel: 81-92-6426096. Fax: 81-92-642-6103. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Kai Johnsson (École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) for providing the SNAP-tag cDNA and Dr. Ryosuke Kojima (Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan) for providing pDisplay-SNAP. This work was supported in part by Grants-in-Aid for Scientific Research from MEXT (the Ministry of Education, Culture, Sports, Science and Technology) and by Targeted Proteins Research Program (TPRP) from MEXT.
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