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. 2012 Jul;80(7):2528-37.
doi: 10.1128/IAI.06337-11. Epub 2012 Apr 23.

Microbicidal activity of vascular peroxidase 1 in human plasma via generation of hypochlorous acid

Hong Li  1 , Zehong Cao, D Ray Moore, Patricia L Jackson, Stephen Barnes, J David Lambeth, Victor J Thannickal, Guangjie Cheng
Affiliations

Microbicidal activity of vascular peroxidase 1 in human plasma via generation of hypochlorous acid

Hong Li et al. Infect Immun. 2012 Jul.

Abstract

Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H₂O₂ and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H₂O₂/Cl⁻ system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H₂O₂; killing is inhibited by peroxidase inhibitors and by the H₂O₂ scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.

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Figures

Fig 1
Fig 1
HOCl generation by VPO1. (A) Comparison of the peroxidase activities of MPO, bLPO, and rVPO1 determined using TMB as a substrate as described in Materials and Methods. A 110-μl reaction contained 100 μl of TMB, 10 nM/heme MPO, 50 nM/heme bLPO, or 50 nM/heme rVPO1. The reaction (n = 9) was carried out at 37°C for 30 min, and the absorbance at 650 nm was recorded. (B) Detection of Cl-tau generation by the A252. Cl-tau generation was carried out in a 100-μl reaction that contained 50 mM potassium phosphate buffer (pH 5.5), 5 mM taurine, 0.8 mM H2O2, 100 mM Cl−, and either 100 nM/heme MPO, 100 nM/heme bLPO, or 1 μM/heme rVPO1. Reactions without Cl− were also performed. The absorbance at 252 nm was recorded every 30 s for 15 min.
Fig 2
Fig 2
Detection of VPO1-mediated Cl-tau by MS. Cl-tau generation and LC-MS were carried out as described in Materials and Methods. Standard Cl-tau (Std) was prepared as follows. First, 20 mM HOCl in 100 mM potassium phosphate buffer (pH 7.4) was added in equal volumes of 100 mM taurine with vigorous stirring. Then, 10 μl of Cl-tau at 80 μM was analyzed. Heme peroxidase-mediated Cl-tau was generated in a 100-μl reaction containing 50 mM potassium phosphate buffer (pH 5.5), 5 mM taurine, 0.8 mM H2O2, 100 mM NaCl, and either 1,000 nM/heme VPO1, 100 nM/heme MPO, or 100 nM/heme LPO. The reaction was incubated at 37°C for 30 min. The injection volume for LC-MS was 10 μl. The ion spray voltage was 4,500 V, and nitrogen was the only gas used throughout the runs. The data are representative of at least two independent experiments.
Fig 3
Fig 3
Detection of VPO1-mediated HOCl by multiple approaches. (A) Taurine chlorination-TMB oxidation assay for reagent HOCl. Details of the procedures are described in Materials and Methods. The data are from three independent experiments. (B) Optimal pH for HOCl generation by heme peroxidases. Reactions (100 μl) containing 50 mM phosphate buffer (with the pH as indicated), 140 mM NaCl, 50 μM H2O2, 5 mM taurine, and either 10 nM/heme MPO, 100 nM/heme LPO, or 100 nM/heme rVPO1 were carried out at 37°C for 30 min. The reaction was stopped by adding catalase (25 μg/ml). The stopped reaction solution was then mixed with a developing agent. After 5 min, the absorbance at 650 mm was recorded. (C) Dose-dependent HOCl generation by VPO1 compared to that by MPO. The experiments were carried out as in panel B at pH 7.4. The concentration of NaCl is indicated. (D) Inhibition of HOCl generation by inhibitory reagents. Experiments were carried out as described in panel B with reaction solution (pH 7.4) containing 140 mM chloride, ABAH (200 μM), NaN3 (1 mM), catalase (25 μg/ml), or Met (0.5 mM) as indicated. The data are representative from ≥3 independent experiments. Error bars indicate the standard deviations (SD). (E) VPO1-mediated MCD chlorination. A 100-μl reaction containing 50 mM phosphate buffer (pH 5.0), 100 μM MCD, 140 mM NaCl, and either 500 nM VPO1 or 50 nM MPO was carried out at 37°C. H2O2 was added to start the reaction. The absorbance at 290 nm was immediately monitored. (a) pH-dependent MCD chlorination. (b) NaCl dose-dependent MCD chlorination. (c) VPO1-mediated MCD chlorination in presence or absence of GSH (0.1 mM) or Met (0.1 mM). (F) VPO1-mediated TNB oxidation. VPO1 at 500 nM was added to 50 mM phosphate buffer (pH 6.5) containing 100 μM TNB and 100 mM NaCl; 20 nM MPO or LPO was used as a positive or negative control. The reaction was initiated by adding 100 μM H2O2. The absorbance at 412 nm was recorded at 37°C every 30 s. (a) Comparison of VPO1-mediated TNB oxidation to that of MPO and LPO. (b) VPO1 dose-dependent TNB chlorination. The data are representative of three independent experiments.
Fig 4
Fig 4
Bactericidal activity of rVPO1. (A) Bactericidal activity of reagent HOCl. E. coli was incubated in 50 mM phosphate buffer (pH 6.2) and the indicated amount of reagent HOCl at 37°C for 30 min and 1 h, respectively. The cell mixture was plated on LB plates. In control experiments, no HOCl was present. The data are from three independent experiments. (B) E. coli was incubated in 50 mM phosphate buffer (pH 6.2) containing 140 mM NaCl, 10 μM H2O2, and the indicated amount of MPO, bLPO, or rVPO1 at 37°C for 1 h. Cell mixtures were plated on LB plates. In control experiments, only H2O2 (10 μM) or Cl− (140 mM) was present. The data are representative of ≥3 independent experiments. Error bars indicate the SD. (C) NaCl- and H2O2-dependent bactericidal activities by VPO1. Experiments were carried out as described for panel B with 200 nM VPO1. In the NaCl dose-dependent experiments (a), H2O2 was maintained at 10 μM and the NaCl concentrations are indicated, whereas in the H2O2-dose dependent experiments (b), NaCl was maintained at 140 mM and the H2O2 concentrations are indicated. MPO at 50 nM was used as a control. (D) VPO1 binds to E. coli. E. coli cells were added into VPO1 or MPO as indicated in 1× PBS (pH 7.4). The mixtures were incubated at 37°C for 1 h. E. coli cells were spun down at 5,000 rpm for 5 min. The cells were washed twice and subjected to immunoblotting with anti-VPO1 or anti-MPO antibody (a) and chemiluminescence assay with L-012 (b).
Fig 5
Fig 5
Bactericidal activity of plasma VPO1 and MPO. (A) Effect of dialysis on bactericidal activity of plasma. E. coli was incubated in 100 μl of 20 mM phosphate buffer (pH 7.2) containing 5 μl of plasma or dialyzed plasma at 37°C for 2 h. The cell mixtures were plated on LB plates. In control experiment B, the mixture contained only E. coli. The numbers are the plasma sample identifications. The data are representative of a minimum of three independent experiments for each group. (B) Bactericidal activity of plasma peroxidases. E. coli was incubated in 100 μl of 20 mM phosphate buffer (pH 7.2) containing 10 μl of dialyzed plasma sample each from 19 unrelated healthy individuals, H2O2 (10 μM), NaCl (140 mM), ABAH (500 μM), and 50 μg of catalase/ml as indicated at 37°C for 2 h. The cell mixtures were plated on LB plates and incubated at 37°C overnight. The colonies were counted, and the data are expressed as the percentages of colonies compared to the number seen with plasma plus E. coli alone. The data are representative of two independent experiments (n = 6) (means ± the SD). (C) Separation of plasma VPO1 from MPO. Portions (3 ml) of human plasma were loaded onto a Sephacryl S-300 column (1 by 110 cm) and eluted with phosphate buffer (pH 7.2). The eluent was collected (4 ml/tube), and the absorbance at 280 and 412 nm was monitored. The peroxidase activity of eluted fractions 10 to 20 was measured using TMB oxidation (inset). Fractions 10 to 20 were also subjected to analysis by SDS-PAGE and immunoblotting with antibodies against VPO1 and MPO (inset). These data are representative of at least three separations using different plasma samples. (D) Four human plasma samples were individually subjected to fractionation as described for panel C. The eluents with the strongest peroxidase activity (fractions 13 and 16 for VPO1 and MPO, respectively) were concentrated 10-fold to facilitate bacterial killing experiments. E. coli killing was carried out as described for panel B, except that 20 μl of fractionated plasma containing either VPO1 or MPO was added. The final concentrations of VPO1 and MPO were ca. 50% of that in native plasma. The numbers are plasma sample identifications. The data are representative of three independent experiments. Error bars indicate the SD. (E) VPO1-mediated bactericidal activity in the presence of RBCs. (a) Effects of VPO1 or MPO/H2O2/Cl− systems on RBCs. A total of 107 RBCs were incubated with 140 mM NaCl, 10 or 20 μM H2O2, and either 200 mM VPO1 or 50 nM MPO at 37°C for 1 h. Hemoglobin was determined by measuring the A415 of the supernatant and pellet, and the percentage of distribution was calculated using the A415 divided by the total A415 of the supernatant plus the pellet. (b) E. coli cells were incubated in 100 μl of 50 mM phosphate buffer (pH 6.2) containing 140 mM NaCl, the indicated amounts of H2O2, and either 200 nM rVPO1 or 50 nM MPO in the presence or absence of 107 RBCs at 37°C for 1 h. The cell mixtures were plated on LB agar plates, followed by incubation at 37°C overnight. The percentages of the CFU in the experimental group compared to the control group (E. coli only) were calculated.
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References

    1. Allen RC, Stephens JT., Jr 2011. Myeloperoxidase selectively binds and selectively kills microbes. Infect. Immun. 79:474–485 - PMC - PubMed
    1. Arnhold J, Flemmig J. 2010. Human myeloperoxidase in innate and acquired immunity. Arch. Biochem. Biophys. 500:92–106 - PubMed
    1. Chan JR, Hyduk SJ, Cybulsky MI. 2000. Alpha 4 beta 1 integrin/VCAM-1 interaction activates α-l-β2-integrin-mediated adhesion to ICAM-1 in human T cells. J. Immunol. 164:746–753 - PubMed
    1. Cheng G, et al. 2011. Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions. Free Radic. Biol. Med. 51:1445–1453 - PMC - PubMed
    1. Cheng G, Salerno JC, Cao Z, Pagano PJ, Lambeth JD. 2008. Identification and characterization of VPO1, a new animal heme-containing peroxidase. Free Radic. Biol. Med. 45:1682–1694 - PMC - PubMed

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