title: Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils.
abstract: Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Rachel P
authors.initials: RP
authors.lastname: Wilkie-Grantham
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Magon
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: D Tim
authors.initials: DT
authors.lastname: Harwood
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Margreet C
authors.initials: MC
authors.lastname: Vissers
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
authors.affiliation: From the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch 8140, New Zealand mark.hampton@otago.ac.nz.
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
mesh_terms: D000447:Aldehydes / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D040502:Calgranulin B / Q000378:metabolism; D002839:Chromans / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015151:Immunoblotting; D039841:Leukocyte L1 Antigen Complex / Q000378:metabolism; D015227:Lipid Peroxidation; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D010587:Phagocytosis; D050050:Protein Carbonylation / Q000187:drug effects; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D000447:Aldehydes; D040502:Calgranulin B; D002839:Chromans; D039841:Leukocyte L1 Antigen Complex; D020543:Proteome; D002084:Butylated Hydroxytoluene; D009195:Peroxidase; D019255:NADPH Oxidases; C027576:4-hydroxy-2-nonenal; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
keywords: 4-Hydroxynonenal; Lipid Peroxidation; Myeloperoxidase; NOX2; Neutrophil; Oxidative Burst; Oxidative Stress; Oxygen Radicals; Phagocytosis; Protein Carbonylation; S100 Proteins; calprotectin