Hypochlorite-modified high-density lipoprotein acts as a sink for myeloperoxidase in vitro

Abstract

Aims: Myeloperoxidase (MPO), a cardiovascular risk factor in humans, is an in vivo catalyst for lipoprotein modification via intermediate formation of reactive chlorinating species. Among the different lipoprotein classes, anti-atherogenic high-density lipoprotein (HDL) represents a major target for modification by hypochlorous acid (HOCl), generated from H2O2 by MPO in the presence of physiological chloride concentrations. As MPO was identified as an HDL-associated protein that could facilitate selective oxidative modification of its physiological carrier, the aim of the present study was to investigate whether and to what extent modification of HDL by HOCl affects the binding affinity of MPO in vitro.

Methods and results: We show that binding affinity of 125I-labelled MPO to HDL markedly increases as a function of increasing extent of HOCl modification of HDL. In contrast to native HDL, HOCl-HDL potently inhibits MPO binding/uptake by endothelial cells and effectively attenuates metabolism of MPO by macrophages. Reduction of HDL-associated chloramines with methionine strongly impaired binding affinity of MPO towards HOCl-HDL. This indicates that N-chloramines generated by HOCl are regulators of the high-affinity interaction between HOCl-HDL and positively charged MPO. Most importantly, the presence of HOCl-HDL is almost without effect on the halogenating activity of MPO.

Conclusion: We propose that MPO-dependent modification of HDL and concomitant increase in the binding affinity for MPO could generate a vicious cycle of MPO transport to and MPO-dependent modification at sites of chronic inflammation.

Figure 1. Myeloperoxidase binding to HOCl-modified lipoproteins.

(A) Albumin (Alb), apolipoprotein A-I (apoA-I), native high-density lipoprotein (HDL), and HOCl–HDL (oxidant:lipoprotein molar ratio of 50:1 and 200:1) were coated to microtitre plates. After washing and blocking of unspecific binding sites, ¹²⁵I-MPO (0.6, 3, and 15 μg/mL) was added (37°C, 1 h). Subsequently, the wells were washed and the radioactivity was counted. (B) High-density lipoprotein was immobilized onto wells of plastic dishes and modified by indicated concentrations of HOCl added as reagent or generated by the myeloperoxidase–H₂O₂–chloride system in vitro. H₂O₂, in the absence of myeloperoxidase and chloride, was used as a control. After washing and blocking of unspecific binding sites, ¹²⁵I-MPO (1 μg/mL) was added to coated wells at 37°C for 1 h. Subsequently, the wells were washed and the radioactivity was counted. (C) Dose–response curves of ¹²⁵I-MPO (1 μg/mL) binding competition to high-density lipoprotein-coated microtitre wells by high-density lipoprotein or HOCl–high-density lipoprotein (oxidant:lipoprotein molar ratio of 50:1, 100:1, and 200:1) at indicated concentrations. (D) One milligram high-density lipoprotein or HOCl–high-density lipoprotein (oxidant:lipoprotein molar ratio of 200:1) coupled to Sepharose-beads or Sepharose-beads alone (control) was incubated with ¹²⁵I-MPO (10 μg/mL) at 25°C. After 30 min, beads were spun down and radioactivity of free (supernatant) or bound (Sepharose-bound or Sepharose–lipoprotein-bound) ¹²⁵I-MPO was measured. Results represent mean ± SD (n = 3) of one experiment out of three.

Figure 2. Characterization of HOCl–high-density lipoprotein.

Data from Figure 1A (myeloperoxidase binding to native and modified high-density lipoprotein preparations at the highest myeloperoxidase concentrations) were plotted against relative electrophoretic mobility (REM) values (A) and the extent of lysine (B) and tyrosine modification (C) of native high-density lipo-protein and modified high-density lipoprotein (indicated oxidant:lipoprotein molar ratio). Relative electrophoretic mobility values and amino acid analyses were estimated as described in the Methods section.

Figure 3. Myeloperoxidase binding and competition experiments.

(A and B) Competition experiments of ¹²⁵I-MPO (1 μg/mL) binding to high-density lipoprotein-coated microtitre wells by high-density lipoprotein or HOCl–high-density lipoprotein (oxidant:lipoprotein molar ratio of 200:1) at indicated concentrations. Modification of high-density lipoprotein was performed at 0°C (1 h) or 37°C (12 h) in the absence or presence of methionine (Met) added at a five-fold molar excess over HOCl at the end of the incubation period. Results represent mean ± SD (n = 3) of one experiment out of three.

Figure 4. Measurement of lipoprotein-associated myeloperoxidase activity.

High-density lipoprotein or HOCl–high-density lipoprotein [prepared at indicated oxidant:lipoprotein molar ratio at 0 or 37°C, without or with methionine (Met) added at the end of incubation time] were co-incubated with myeloperoxidase. After 30 min, 100 μM H₂O₂ and 100 μM bromide were added and myeloperoxidase activity monitored continuously by measuring H₂O₂ consumption polarographically (see the Methods section). Values are given as percentage of activity of free myeloperoxidase (100% corresponds to 3.8 units). Results represent mean ± SD (n = 3) of one experiment out of three.

Figure 5. Cell association of ¹²⁵I-MPO by endothelial cells.

Human endothelial cells were incubated (1 h, 37°C) in DMEM (containing 50% foetal calf serum) with ¹²⁵I-MPO (200 ng/mL) in the absence or presence of indicated concentrations (20 and 100 μg/mL) of high-density lipoprotein or HOCl–high-density lipoprotein (oxidant:lipoprotein molar ratio of 200:1). Subsequently, the cells were washed and lysed, total cell-associated radioactivity was counted, and cell protein content was measured. Results represent mean ± SD (n = 3) of one experiment out of three.

Figure 6. Binding, internalization, and degradation of ¹²⁵I-MPO by macrophages.

RAW macrophages were incubated (2 h, 37°C) in DMEM (containing 50% foetal calf serum) with ¹²⁵I-MPO (200 ng/mL) in the absence or presence of indicated concentrations (20 and 100 mg/mL) of high-density lipoprotein or HOCl–high-density lipoprotein (oxidant:lipoprotein molar ratio of 200:1) to determine binding (A), internalization (B), and degradation of ¹²⁵I-MPO (C). After the incubation period, the cells were rinsed, and incubated with Dulbecco’s modified Eagle’s medium containing heparin (100 U/mL) for further 20 min to release cell surface-bound myeloperoxidase (heparin-releasable) (A). Then the cells were lysed and radioactivity counted to measure internalized myeloperoxidase (heparin-resistant) (B) and to measure protein content of the cells. Degradation of ¹²⁵I-MPO (C) was determined as described in the Methods section. Results represent mean ± SD (n = 3) of one experiment out of three.