Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease

Abstract

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.

Figure 1

ApoA-I is a preferred target for nitration in serum. Serum samples (25 μg total protein per lane) from 3 healthy controls and 3 subjects with CVD were separated by SDS-PAGE. The gels (12.5%) were either stained for protein with Coomassie blue (A) or transferred and probed with a mAb specific for protein-bound NO₂Tyr (B). The apoA-I bands were identified by sequence analysis by tandem mass spectrometry (MS). The disproportionate recognition of the apoA-I band in the Western blot analysis is consistent with an enhanced NO₂Tyr content of that protein.

Figure 2

Confirmation of apoA-I as a nitrated protein by both 2D SDS-PAGE and anti-NO₂Tyr affinity chromatography coupled to tandem MS-based sequencing. Plasma from a subject with CVD was loaded onto an affinity matrix composed of immobilized affinity-purified rabbit anti-NO₂Tyr polyclonal antibodies, washed with high salt, and then eluted with addition of 5 mM free NO₂Tyr, as described in Methods. (A) Demonstration of apoA-I location on 2D SDS-PAGE. Identity of protein was established by tandem MS sequence analysis of peptides (>95% coverage). (B) The anti-NO₂Tyr eluent (5 mM NO₂Tyr) was subjected to 2D SDS-PAGE, and the presence of apoA-I was confirmed by Western blot analysis. Parallel studies using control nonimmune IgG as the affinity matrix failed to bind detectable levels of apoA-I (not shown).

Figure 3

Demonstration of apoA-I NO₂Tyr and MPO enrichment within HDL-like particles isolated from human atherosclerotic lesions. Left and center: Equal amounts of protein (40 μg per lane) from either HDL-like particles isolated from human atherosclerotic lesions (n = 12 subjects, pooled) or HDL isolated from pooled plasma from healthy donors were analyzed by SDS-PAGE (10–20% gradient gels), transferred onto PVDF membranes, and probed using antibodies to either anti-NO₂Tyr (left) or anti–apoA-I (center), and then visualized by brief chemiluminescence exposure, as described in Methods. Right: Isolated human MPO standard (std) or HDL-like particles isolated from human atherosclerotic lesions (40 μg total protein, n = 12 subjects, pooled) were analyzed by SDS-PAGE (10–20% gradient gels), transferred onto PVDF membranes, probed using antibodies to human MPO, and then visualized by brief chemiluminescence exposure, as described in Methods.

Figure 4

Coimmunoprecipitation of MPO and apoA-I in plasma. (A) ApoA-I immunoprecipitation (IP) of plasma, followed by MPO Western blot (10–20% SDS-PAGE). (B) MPO IP of plasma, followed by apoA-I Western blot (10–20% SDS-PAGE) (left) or Coomassie blue staining (right). The MPO Western blot (A) was probed with rat anti–human MPO and includes, in the first lane, isolated human MPO as standard. The anti-MPO antibody used predominantly recognizes the heavy chain of MPO and thus highlights both heavy chain and precursor protein forms of MPO. The apoA-I Western blot (B) was probed with goat anti–human apoA-I. For each Western blot, the lanes were loaded with 10 μg of protein from plasma, the IP supernatant (Supn), and the specific and control immune complexes recovered from the IP pellets, in the indicated sequential order.

Figure 5

Demonstration of an interaction between MPO and apoA-I using hydrogen/deuterium exchange tandem MS. Exchangeable protons on HDL and MPO were each deuterium-labeled by mixing in D₂O containing ND₄OAc, pD 7.0, at room temperature for 1 hour. The deuterated HDL was combined with either deuterated MPO or additional deuterium buffer and incubated for 1 hour at room temperature to allow binding. Samples were diluted 25-fold into NH₄OAc, pH 7.0, for 10 minutes for back (off) exchange before quenching by rapid cooling and addition of TFA to pH 2. Proteins were digested with immobilized pepsin, and then samples were immediately injected for analysis as described in Methods. The different spectra shown correspond to various stages in the hydrogen/deuterium exchange experiment. (A) ApoA-I isotopic clusters shown are for either the MPO-binding peptide A190–L203 (left) or a negative control peptide, L159–L170 (right). For each, spectra a and b contain peptide isotopic clusters before and after deuterium labeling, respectively. Spectra c and d contain deuterium-labeled peptide cluster after back (off) exchange with hydrogen in the absence and presence of MPO binding, respectively. The peptide isotopic cluster indicated by the asterisk represents a non–back-exchanged component of the A190–L203 isotope cluster due to inaccessibility of this region of apoA-I to solvent in the presence of MPO. Results shown are representative of 4 independent experiments. (B) Sequence confirmation of the identified peptic peptides was achieved by tandem MS. The collision-induced dissociation spectra and fragmentation analysis of the unlabeled and deuterium-labeled peptic peptide A190–L203 are illustrated.

Figure 6

MPO-generated reactive nitrogen species inhibit ABCA1-dependent cholesterol efflux functions of HDL. HDL (A) and apoA-I (B) were individually modified by ONOO− (100 μM), the MPO/H₂O₂/NO₂− system (100 μM H₂O₂), or the MPO/H₂O₂/Cl− system (100 μM H₂O₂), as described in Methods. Modified HDL or apoA-I preparations were then incubated with cholesterol-loaded murine macrophage RAW264.7 cells in the presence or absence of 8-Br-cAMP pretreatment, and ABCA1-dependent (top) and -independent (bottom) cholesterol efflux was quantified as described in Methods. Note that MPO-generated nitrating and chlorinating oxidants both selectively inhibited ABCA1-dependent cholesterol efflux. In marked contrast, exposure of HDL or apoA-I to ONOO− failed to influence HDL cholesterol efflux functions of the lipoprotein.

Figure 7

Correlation between functional impairment in ABCA1-dependent cholesterol efflux activity and apoA-I content of NO₂Tyr and ClTyr. HDL was immunoprecipitated from serum of consecutive Preventive Cardiology Clinic patients. ABCA1-specific cholesterol efflux activity in 5 μg apoA-I was then quantified using cholesterol-laden murine macrophages as described in Methods. In parallel, the content of apoA-I NO₂Tyr (A) and ClTyr (B) was determined by stable isotope dilution tandem MS as described in Methods.