Large-Scale Purification of Myeloperoxidase from HL60 Promyelocytic Cells: Characterization and Comparison to Human Neutrophil Myeloperoxidase

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4 -5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14-and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.

Section: Resultsmentioning

confidence: 99%

Synergistic Roles of Helicobacter pylori Methionine Sulfoxide Reductase and GroEL in Repairing Oxidant-damaged Catalase

Mahawar¹,

Tran

Sharp

et al. 2011

“…Myeloperoxidase (donor:hydrogen peroxide, oxidoreductase, EC 1.11.1.7) was isolated by lectin affinity and size exclusion chromatographies from human neutrophils (43,44) and stored at Ϫ20°C. Purified enzyme had an A 430 /A 280 ratio of 0.8 and was apparently homogeneous on SDS-PAGE analysis; its concentration was determined spectrophotometrically (⑀ 430 ϭ 0.17 M Ϫ1 cm Ϫ1 ) (45).…”

Section: Methodsmentioning

confidence: 99%

Human Atherosclerotic Intima and Blood of Patients with Established Coronary Artery Disease Contain High Density Lipoprotein Damaged by Reactive Nitrogen Species

Pennathur

Bergt

Shao

et al. 2004

255

232

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but itis not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 ؎ 178 mol/mol Tyr) than that in circulating HDL (104 ؎ 11 mol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

“…Myeloperoxidase (A 430 /A 280 ratio Ͼ 0.8) was purified from HL-60 cells by sequential lectin affinity, ion exchange, and size exclusion chromatographies (44,45 (47,48). HPLC Analysis of Cysteine Switch Peptide-Peptides were separated at a flow rate of 0.3 ml/min on a reverse-phase column (Vydac C18 MS, 2.1 ϫ 250 mm) using a Beckman HPLC system (Fullerton, CA) with UV detection at 214 nm.…”

Section: Methodsmentioning

confidence: 99%

Hypochlorous Acid Oxygenates the Cysteine Switch Domain of Pro-matrilysin (MMP-7)

Kassim

Parks

et al. 2001

424

143

Myeloperoxidase uses hydrogen peroxide (H 2 O 2 ) to generate hypochlorous acid (HOCl), a potent cytotoxic oxidant. We demonstrate that HOCl regulates the activity of matrix metalloproteinase-7 (MMP-7, matrilysin) in vitro, suggesting that this oxidant activates MMPs in the artery wall. Indeed, both MMP-7 and myeloperoxidase were colocalized to lipid-laden macrophages in human atherosclerotic lesions. A highly conserved domain called the cysteine switch has been proposed to regulate MMP activity. When we exposed a synthetic peptide that mimicked the cysteine switch to HOCl, HPLC analysis showed that the thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as sulfinic acid, sulfonic acid, and a dimer containing a disulfide bridge. In contrast, the peptide reacted slowly with H 2 O 2 , and the only product was the disulfide. Moreover, HOCl markedly activated pro-MMP-7, an MMP expressed at high levels in lipid-laden macrophages in vivo. Tandem mass spectrometric analysis of trypsin digests revealed that the thiol residue of the enzyme's cysteine switch domain had been converted to sulfinic acid. Thiol oxidation was associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. In contrast, H 2 O 2 failed to oxidize the thiol residue of the protein or activate the enzyme. Thus, HOCl activates pro-MMP-7 by converting the thiol residue of the cysteine switch to sulfinic acid. This activation mechanism is distinct from the well-studied proteolytic cleavage of MMP pro-enzymes. Our observations raise the possibility that HOCl generated by myeloperoxidase contributes to MMP activation, and therefore to plaque rupture, in the artery wall. HOCl and other oxidants might regulate MMP activity by the same mechanism in a variety of inflammatory conditions.Proteolysis of structural and adhesive matrix proteins by infiltrating cells is a key event in many destructive inflammatory conditions (1). The role of macrophage-derived matrix metalloproteinases (MMPs) 1 in mediating plaque rupture in atherosclerosis provides a physiologically relevant example of this process (2, 3). Formation of a thrombus over a ruptured atherosclerotic plaque precipitates most cases of acute myocardial infarction (4, 5). Atherosclerotic lesions resistant to rupture are stabilized by a fibrous cap, which separates an intensely procoagulant lipid core from the bloodstream. Because the extracellular matrix plays a key structural role in stabilizing this cap, matrix-degrading enzymes, and MMPs, in particular, might contribute to plaque instability (1-5). Indeed, the lipidladen macrophages that congregate in the shoulder regions of human atherosclerotic lesions express several MMPs: collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) (6 -8). In contrast, expression of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) is confined to lipidladen macrophages on the border of the acellular lipid core...