A myeloperoxidase precursor, pro-myeloperoxidase, is present in human plasma and elevated in cardiovascular disease patients

Abstract

Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease.

Fig 1. Halogenation activity of mature MPO and recombinant pro-MPO.

Initial rates of NADH oxidation were determined with various concentrations of A) bromide or B) chloride, 20 nM mature MPO or recombinant pro-MPO and 100 μM NADH in 50 mM phosphate buffer (pH 7.4) at 21°C. The reaction was started by adding 50 μM H₂O₂. The kinetics of the reaction of myeloperoxidase-generated hypobromous and hypochlorous acids with NADH were monitored by measuring the bromohydrin and chlorohydrin product at 275 nm. Data are representative of two or more experiments.

Fig 2. Detection of mature MPO and pro-MPO in plasma using affinity-purification and immunblot analysis.

A) Plasma was spiked with MPO standard (10 nM) and HL60 cell lysate (containing 10 nM MPO) and subjected to affinity purification. Purified samples along with MPO, pro-MPO standards and HL-60 lysates were separated by 10% SDS/PAGE, transferred to PVDF and probed with MPO-specific antibody. B) Neutrophils (5x10⁶ cells/ml) were added back into plasma and then stimulated with CytB and FMLP for 30 min at 37°C. Neutrophils were centrifuged and cell free plasma MPO was subjected to affinity purification and analyzed as described in A. After the ECL fluorescence of blots was developed, a photograph of the blot showing the molecular weight markers was taken and aligned with the fluorescence image as indicated by the black line.

Fig 3. Detection of mature MPO and pro-MPO in plasma from healthy controls and patients with myocardial infarction using affinity-purification and immunblot analysis.

Plasma from control subjects and from patients with myocardial infarct (MI) was subjected to affinity purification and analyzed by immunoblot as described Fig 1. This blot is representative of at least 10 plasma samples in each group. After the ECL fluorescence of blots was developed, a photograph of the blot showing the molecular weight markers was taken and aligned with the fluorescence image as indicated by the black line.

Fig 4. LC-MS/MS analysis of mutual, pro-MPO- and MPO- specific peptides.

A) Location of tryptic peptides used for LC-MS/MS analysis within the MPO sequence. The N-terminal signal peptide is shown in dark grey, the pro-peptide in bold light grey and the sequence of mature MPO in bold black. Highlighted by the black and grey boxes are tryptic peptides specific to pro-MPO or present in both mature MPO and pro-MPO, respectively. Twenty-five μg of recombinant pro-MPO or mature MPO were digested with trypsin and analyzed by LC-MS/MS. B), D) +F) Extracted ion chromatograms for SRM transitions specific for the mutual, pro-MPO and mature MPO-specific peptides (MS1/MS2 551.8/272.2, 1000.9/1033.5 and 648.8/935.5, respectively, MS1 = m/z for the doubly charged precursor species and MS2 = m/z for the singly charged y–ion fragment, c–carbamidomethyl-cysteine). C), E) +G) CID-MS/MS spectra confirming the sequence of the respective peptide. Representative chromatograms and spectra are shown.

Fig 5. LC-MS/MS detection of pro-MPO in affinity-purified plasma separated by SDS/PAGE.

Plasma was purified by affinity chromatography with an MPO-specific antibody and subjected to separation by SDS/PAGE. Bands with the molecular weight of pro-MPO (90 kDa) were subjected to in-gel tryptic digestion and analyzed by LC-MS/MS using SRM for a pro-MPO-specific peptide (1000.9->1033.5). (A) Extracted ion chromatogram for the SRM transition for the pro-MPO specific peptide. (B) CID-MS/MS spectrum confirming the sequence for the pro-MPO-specific peptide SSGcAYQDVGVTcPEQDK. Representative chromatograms and spectra are shown.

Fig 6. LC-MS/MS detection of pro-MPO and mature MPO in affinity-purified plasma from patients with myocardial infarction (MI).

Plasma was purified by affinity chromatography with an MPO-specific antibody, subjected to digestion with trypsin and analyzed by LC-MS/MS. A) Representative SRM- based extracted ion chromatogram for the pro-MPO specific peptide (1000.9->1033.5) and C) the mature MPO-specific peptide (648.8->935.5) measured in a MI plasma sample. B)+D) CID-MS/MS spectra were recorded simultaneously for m/z 1000.9 and 648.8 representing the SSGcAYQDVGVTcPEQDK and VTcPEQDKYR peptides, respectively.