Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex
Ying Huang, … , Ulf Landmesser, Stanley L. Hazen
Ying Huang, … , Ulf Landmesser, Stanley L. Hazen
Published August 1, 2013
Citation Information: J Clin Invest. 2013;123(9):3815-3828. https://doi.org/10.1172/JCI67478.
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Research Article Cardiology

Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex

Abstract

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein–associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other’s function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

Authors

Ying Huang, Zhiping Wu, Meliana Riwanto, Shengqiang Gao, Bruce S. Levison, Xiaodong Gu, Xiaoming Fu, Matthew A. Wagner, Christian Besler, Gary Gerstenecker, Renliang Zhang, Xin-Min Li, Anthony J. DiDonato, Valentin Gogonea, W.H. Wilson Tang, Jonathan D. Smith, Edward F. Plow, Paul L. Fox, Diana M. Shih, Aldons J. Lusis, Edward A. Fisher, Joseph A. DiDonato, Ulf Landmesser, Stanley L. Hazen

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Figure 1

MPO and PON1 inhibit each other’s activity in vitro.

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MPO and PON1 inhibit each other’s activity in vitro. (A) PON1 (100 μg/ml...
(A) PON1 (100 μg/ml) was incubated with either HOCl or H2O2 and MPO (50 nM) in 50 mM Na[PO4] buffer (pH 7.0) supplemented with isolated human HDL (1 mg/ml) and 100 mM NaCl at 37°C for 60 minutes. This was followed by quantification of paraoxonase activity relative to no oxidant exposures. Also shown are the effects of varying levels of H2O2 alone (i.e., no MPO added) or with the addition of the HOCl scavenger methionine (Met) (10-fold molar excess relative to oxidant) to the MPO/H2O2 system. (B) Effect of PON1 on peroxidase activity by TMB assay of either MPO or HRP. (C) Effect by TMB assay of PON1 (650 nM), HDL (650 nM), or both on MPO (65 nM) peroxidase activity. (D and E) Human neutrophils isolated from healthy donors (PMNWT) or MPO-deficient subjects (PMNMPO–) were incubated at 37°C in 50% serum for 1 hour in the absence or presence of phorbol 12-myristrate 13-acetate (PMA) and MPO or PON1, as indicated. Endogenous serum arachidonic acid (AA), linoleic acid (LA), 9-HODE, and 9-HETE were then quantified by stable isotope dilution LC/MS/MS as described in Methods. (F) PON1, MPO/H2O2, neither, or both were added to 50% serum, incubated at 37°C for 1 hour, and then AA, LA, or the indicated oxidation products were quantified as described in Methods. Data shown represent the mean ± SD of triplicate determinations. *P < 0.05; **P < 0.001. NA, no additions; oxFA, oxidized fatty acids.