Oxidative Modification of Calprotectin During Inflammation: Development as a Biomarker
Magon, Nicholas James
Calprotectin is a heterodimer of S100A8 (A8) and S100A9 (A9) proteins present in the cytosol of neutrophils. It is released from activated neutrophils and is also excreted as a result of cell disruption or death. Calprotectin levels increase in inflamed tissues and faecal levels are used as a biomarker in inflammatory bowel disease (IBD). Activated neutrophils generate large amounts of reactive oxygen species, and recent work has suggested that calprotectin is oxidatively modified during neutrophil activation. The purpose of this thesis was to characterise the modifications produced on calprotectin after exposure to compounds generated during the oxidative burst of activated neutrophils, and determine if these modified products can be developed as biomarkers of neutrophil activation. Existing purification protocols were modified to obtain sufficiently pure calprotectin from human neutrophils for use in this study. Previous work in our laboratory demonstrated that A9 is carbonylated in the cytosol of neutrophils following phagocytosis of Staphylococcus aureus, and preliminary evidence suggested that the lipid peroxidation product 4-hydroxynonenal (HNE) contributed to carbonylation. To further investigate these observations, calprotectin was exposed to HNE and the resulting modifications were characterised. Cys2 of A9 was determined to be particularly susceptible to modification readily forming HNE Michael adducts as well as intra- and inter-molecular crosslinks with lysine residues. An intermolecular cysteine-HNE-lysine calprotectin crosslink was determined to occur between Cys2 of A9 and Lys49 of A8, representing the first characterisation of a cysteine-HNE-lysine crosslink. A sensitive LC/ESI-MS assay was developed for the detection and quantification of the A9 N-terminal tryptic peptide TCK and the TCK-HNE Michael adduct. However, no evidence of HNE adduct formation was observed on calprotectin isolated from the cytosol of stimulated neutrophils. Neutrophil myeloperoxidase uses hydrogen peroxide to oxidise chloride to the powerful oxidant hypochlorous acid (HOCl). Treatment of calprotectin with HOCl resulted primarily in the oxidation of cysteine, methionine and tryptophan residues leading to multiple oxidation products. Cys2 of A9 was determined to be particularly susceptible to modification by HOCl and was involved in the formation of stable dithiothreitol-resistant intra- and inter-molecular sulfur-nitrogen calprotectin crosslinks. Further analysis suggested that the major intermolecular crosslink was formed between Cys2 of A9 and Lys84 of A8. Dithiothreitol-resistant calprotectin crosslinks were also detected in the cytosol of stimulated neutrophils, in faecal extracts from patients with inflammatory bowel disease, and bronchoalveolar lavage fluid (BALF) from children with cystic fibrosis. The stable nature and high abundance of these crosslinks makes them ideal candidates for biomarkers of inflammation. Dehydromethionine, a stable cyclic product formed on free and N-terminal methionine, was identified as a novel modification of A8 formed following the activation of neutrophils and subsequent production of HOCl. A sensitive LC/ESI-MS assay was developed for the detection and quantification of the A8 N-terminal tryptic peptide MLTELEK, the diastereoisomers of dehydro-MLTELEK, and MLTELEK sulfoxide. Dehydro-MLTELEK concentration was significantly higher (p = 0.036) in the BALF of children with cystic fibrosis (n = 18) than disease controls (n = 10), and was significantly associated with myeloperoxidase activity (rs = 0.873, p < 0.0001) and glutathione sulfonamide concentration (rs = 0.771, p < 0.0001) in BALF from children with cystic fibrosis (n = 18). This is the first time dehydromethionine has been detected in clinical samples, thereby demonstrating that it is a relatively stable and biologically relevant modification that may be useful as a biomarker of neutrophil myeloperoxidase activity.
Advisor: Hampton, Mark B.; Kettle, Anthony J.; Gearry, Richard B.
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: calprotectin; S100A8; S100A9; neutrophil; myeloperoxidase; hypochlorous acid; 4-hydroxynonenal; dehydromethionine; biomarker; inflammatory bowel disease; cystic fibrosis
Research Type: Thesis